EXTREMOphiles_2012

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on extremophiles

Sevilla, 10-13 September 2012

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Edited by Antonio Ventosa and Cristina Sánchez-Porro 2012 Dept Microbiology and Parasitology University of Sevilla, Spain Printed by «Maquetando» All rights reserved Deposito Legal: SE3812-2012 http://www.congreso.us.es/extremophiles

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table of contents

Welcome address 7

Acknowledgements 9

Congress Committees 11

International Organizing Committee 13

Scientific Advisory Committee 13

National Network Advisory Board 15

Local Organizing Committee 16

Young scientists grants 17

General information 19

Scientific programme 27

Posters sessions 39

Abstracts 59

Index of authors 335

List of participants 343

Notes 371 table of contents table

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Welcome address

Dear colleagues,

On behalf of the Organizing Committee for Extremophiles 2012 and the International Society for Extremophiles (ISE) it is our great pleasure and honour to welcome you to the 9th International Congress on Extremophiles. Following the tradition of previous congresses the programme includes a very broad range of topics covering different areas of research on extremophilic microorganisms. The programme was prepared on the basis of the proposals and suggestions of the International Organizing Committee and the Scientific Advisory Committee. We hope you agree that the selected topics and speakers are of great interest and exceptional quality. We maintained the same registration fees than in the previous congress, including a reduced registration fee for students in order to encourage their participation to the meeting. Besides, we obtained financial support from FEMS and ISE for young scientific grants and a total of 26 young researchers will receive financial support in order to attend the congress. The meeting programme includes six main symposia plus the opening and closing keynote lectures and eight offered oral parallel sessions. Besides, two poster sessions will group the almost 200 poster presentations. We encourage all delegates to participate actively on all these activities. Besides, several attractive social activities have been programmed. We would like to thank all institutions and companies that contributed to the organization of this congress, very specially to the University of Sevilla, the Federation of European Microbiological Societies and the International Society for Extremophiles. We hope that all delegates will enjoy during their stay in Sevilla and will found an excellent atmosphere to discuss about scientific matters, new collaborations and future directions in the field of extremophilic microorganisms and their impact on industrial, health, energy, astrobiology and several other areas. We also hope that you discover the hospitality of the people of Sevilla and have some free time for visiting this beautiful city. Welcome to Sevilla!

Antonio Ventosa Chairman of the Organizing Committee welcome address welcome

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acknowledgements

The following institutions and companies are acknowledged for their generous support to this Congress:

Vicerrectorado de Relaciones Institucionales, Universidad de Sevilla Vicerrectorado de Investigación, Universidad de Sevilla Servicio de Medios Audiovisuales (SAV), Universidad de Sevilla International Society for Extremophiles (ISE) Federation of European Microbiological Societies (FEMS) Sociedad Española de Microbiología (SEM) Consejería de Economía, Innovación y Ciencia, Junta de Andalucía Turismo de Sevilla, Ayuntamiento de Sevilla Red Nacional de Microorganismos Extremófilos (REDEX) Society for General Microbiology (SGM), UK Kyokuto Pharmaceutical Industrial Co., Ltd Springer Eppendorf Roche acknowledgements

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congress committees

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INTERNATIONAL ORGANIZING COMMITTEE

Michael Adams, University of Georgia, USA Garabed Antranikian, Hamburg University of Technology, Germany Don Cowan, University of Pretoria, South Africa Koki Horikoshi, JAMSTEC, Japan Mosè Rossi, Institute of Protein Biochemistry, Naples, Italy Helena Santos, ITQB, New University of Lisbon, Portugal

SCIENTIFIC ADVISORY COMMITTEE

Michael Adams, University of Georgia, USA Ricardo Amils, Centro de Astrobiología, Spain Mohammad A. Amoozegar, University of Tehran, Iran Garabed Antranikian, Hamburg University of Technology, Germany Haruyuki Atomi, University of Kyoto, Japan Douglas Bartlett,Scripps Institution of Oceanography, USA Simonetta Bartolucci, Università degli Studi di Napoli Federico II, Italy Victoria Béjar, University of Granada, Spain José Berenguer, Universidad Autónoma de Madrid, Spain John Bowman, University of Tasmania, Australia Stephanie Burton, Cape Peninsula University of Technology, South Africa Angela Corcelli, University of Bari, Italy Milton da Costa, University of Coimbra, Portugal Don Cowan, University of Pretoria, South Africa Michael Danson, University of Bath, UK Arnold Driessen, University of Groningen, The Netherlands Georges Feller, University of Liège, Belgium Patrick Forterre, Université Paris Sud, France William D. Grant, University of Leicester, UK Peter Gogarten, University of Connecticut, USA Anne Godfroy, IFREMER, France Ralf Grote, Hamburg University of Technology, Germany Shaun Heaphy, University of Leicester, UK

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Koki Horikoshi, JAMSTEC, Japan Li Huang, Chinese Academy of Sciences, China Harald Huber, University of Regensburg, Germany Masahiro Ito, Toyo University, Japan Brian Jones, Genencor International, The Netherlands Masahiro Kamekura, Halophiles Research Institute, Japan Chiaki Kato, JAMSTEC, Japan Yanhe Ma, Chinese Academy of Sciences, China Dominique Madern, Institut de Biologie Structurale, France Marco Moracci, Institute of Protein Biochemistry, Naples, Italy Volker Müller, Johan Wolfgang Goethe-University of Frankfurt, Germany Aharon Oren, Hebrew University of Jerusalem, Israel Thane Papke, University of Connecticut at Storrs, USA Emilia Quesada, University of Granada, Spain Fred Rainey, University of Alaska Anchorage, USA John Reeve, Ohio State University, USA Frank Robb, University of Maryland Biotechnology Institute, USA Mosè Rossi, Institute of Protein Biochemistry, Naples, Italy Francisco Rodríguez-Valera, Universidad Miguel Hernández, Alicante, Spain Helena Santos, ITQB, New University of Lisbon, Portugal Peter Schönheit, Christian-Albrechts University Kiel, Germany Karl O. Stetter, University of Regensburg, Germany Michael Thomm, University of Regensburg, Germany John van der Oost, Wageningen University, The Netherlands Juergen Wiegel, University of Georgia, USA William B. Witman, University of Georgia, USA Coordinator: Antonio Ventosa, University of Sevilla, Spain

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NATIONAL NETWORK ADVISORY BOARD

Ricardo Amils, Centro de Astrobiología, Madrid, Spain Josefa Antón, University of Alicante, Spain Victoria Béjar, University of Granada, Spain José Berenguer, Universidad Autónoma de Madrid, Spain Mª José Bonete, University of Alicante, Spain Emilio Casamayor, Centro de Estudios Avanzados de Blanes, Spain Fernando de la Calle, Pharmamar, Spain Asunción de los Ríos, Museo Nacional de Ciencias Naturales (CSIC), Spain Caterina Gómez, Arquebio, Spain Felipe Gómez, Centro de Astrobiología, Madrid, Spain Juan M. González, Instituto de Recursos Naturales y Agrobiología (CSIC), Spain José M. Guisán, Instituto de Catálisis y Petroquímica (CSIC), Spain Irma Marín, Universidad Autónoma de Madrid, Spain Carlos Pedrós-Alió, Centro Mediterráneo de Investigaciones Marinas y Ambientales (CSIC), Spain Emilia Quesada, University of Granada, Spain Francisco Rodríguez-Valera, Universidad Miguel Hernández, Spain Ramón Rosselló-Mora, Instituto Mediterráneo de Estudios Avanzados (CSIC-UIB), Spain José Luis Sanz, Universidad Autónoma de Madrid, Spain

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LOCAL ORGANIZING COMMITTEE

Chairman: Antonio Ventosa, University of Sevilla Secretary: Cristina Sánchez-Porro, University of Sevilla Treasurer: Rafael R. de la Haba, University of Sevilla Members: Montserrat Argandoña, University of Sevilla Paulina Corral, University of Sevilla Constanza Espinosa, University of Sevilla Rosario Fernández-Castillo, University of Sevilla Ana B. Fernández, University of Sevilla Mª Teresa García, University of Sevilla Juan M. González, Institute of Natural Resources and Agrobiology, CSIC, Sevilla Mª Carmen Gutiérrez, University of Sevilla Laura Herrera, University of Sevilla Carmen Infante, University of Sevilla Mª José León, University of Sevilla Clara López-Hermoso, University of Sevilla Mª Carmen Márquez, University of Sevilla Manuel Megías, University of Sevilla Encarnación Mellado, University of Sevilla Francisco Merchán, University of Sevilla Mª de Lourdes Moreno, University of Sevilla Joaquín J. Nieto, University of Sevilla Dolores Pérez, University of Sevilla Patricia Prieto, University of Sevilla Francine Puibeli, University of Sevilla Mercedes Reina, University of Sevilla Manuel Salvador, University of Sevilla Carmen Vargas, University of Sevilla Blanca Vera, University of Sevilla Joaquin Vera, University of Sevilla

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YOUNG SCIENTISTS GRANTS

FEMS Young Scientists Meeting Grants

Sana Alavi, University of Tehran, Iran Riku Aono, Kyoto University, Japan Pinar Çaglayan, Marmara University, Istanbul, Turkey Anaïs Cario, Ecole Normale Supérieure de Lyon, France Ana Maria da Silva Esteves, Instituto de Tecnologia Quimica e Biologica, Oeiras, Portugal Sergey Gavrilov, Winogradsky Institute of Microbiology, Moscow, Russia Zahra Khomarbaghi, University of Tehran, Iran Anniina Kivistö, Tampere University of Technology, Finland Julia Kort, University of Duisburg-Essen, Germany Ilya V. Kublanov, Winogradsky Institute of Microbiology, Moscow, Russia Nahid Oueriaghli, University of Granada, Spain Rocío Peralta, Universidad de Santiago de Chile, Chile Olga Podosokorskaya, Winogradsky Institute of Microbiology, Moscow, Russia Bernadette Rauch,University of Duisburg-Essen, Germany Marta V. Rodrigues, Instituto de Tecnologia Quimica e Biologica, Oeiras, Portugal Ralf Salzer, Goethe University Frankfurt, Germany Irene Sánchez-Andrea, Universidad Autónoma de Madrid, Spain Andrea Strazzulli, Institute of Protein Biochemistry, Naples, Italy Ali Tahrioui, University of Granada, Spain Sato Tsubasa, Okayama University, Japan Katarzyna Tych, University of Leeds, UK

International Society for Extremophiles Grants

Diya Alsafadi, University College Dublin, Ireland Maria Carmina Ferrara, Institute of Protein Biochemistry, Naples, Italy Roberto González, University of Vigo, Spain Leonardo H. Pinto, Rio de Janeiro Federal University, Brazil Felipe Sarmiento, University of Georgia Athens, USA

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general information

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general informatión

Venue The meeting will be held in the Hotel NH Central Convenciones, Avenida Diego Martinez Barrios 8, 41013 Sevilla, Spain; phone: +34 954 548 500; fax: +34 954 426 450; e-mail: nhcentralconvenciones@ nh-hotels.com http://www.nh-hoteles.es/nh/es/hoteles/espana/sevilla/nh-central-convenciones.html The scientific sessions will be at Salón Andalucía (plenary sessions and parallel sessions A) and Salón Avenida (parallel sessions B), and the posters sessions at Salón Marbella. The coffee-breaks will be held at the convention hall on the entrance of the meeting rooms, and the lunches at the hotel restaurant (first floor).

Language English is the official language of the congress.

Registration and information desk The registration will he held on 10 September from 12:00 to 18:00 h. and during the meeting period at the desk located on the convention hall of the hotel. Information about the congress, social activities, etc. can also be obtained at the registration desk during the meeting. The official travel agency of the congress, Viajes El Corte Inglés will be available at the registration desk for accommodation, touristic and travel information, etc.

Badge policy Delegates are requested to exhibit the congress badges all the time during the scientific and social events.

Lunch and coffee-breaks The lunch on 11, 12 and 13 September will be held at the hotel restaurant (included on the registration fee for meeting participants). Accompanying persons can also have lunch at the restaurant (ticket should be purchased in advance on the registration desk). The coffee-breaks will be held at the convention hall on the entrance of the meeting and poster rooms.

Oral sessions Plenary sessions will be held in the Salón Andalucía. Parallel sessions will be held in the Salón Andalucía (sessions A) and Salón Avenida (sessions B) as indicated on the programme of the congress.

Information for speakers The keynote speakers have 25 minutes for their presentations plus 5 minute for questions (30 minutes total), while the offered oral communications will be presented during 15 minutes plus 5 minutes for questions (20 minutes total). We encourage speakers to prepare their presentations

abstracts book 21 EXTREMOphiles_2012 according to the time assigned. Session Chairs have been asked to ensure that speakers restrict themselves to the allotted time; they have been advised to adhere most strictly to this policy. The meeting rooms are prepared with a PC (Powerpoint 2007) and projector. Macintosh is not available. Please note that we are unable to allow the use of personal laptops. Please bring your presentation in a memory stick (using the USB port in the computer) and load it on the meeting computer. Congress staff will be prepared to assist you. All presentations must be loaded preferably the day before or at least before the start of the previous session.

Poster sessions Poster boards are located at Salón Marbella on the convention area of the hotel. Posters should be mounted on 10 September, preferably before 18:00 h. and removed on 13 September at 18:30 h. The organization will provide the materials necessary for mounting the posters on the boards. The dimensions of the posters are: 90 cm (width) by 120 cm (height). Please see the scientific programme for the board number on which you should display your poster. All posters will be displayed during the four days of the congress and they can be viewed during the coffee-breaks. Official poster sessions will be held on Tuesday 11 September and Thursday 13 September from 15:00 to 16:00 h. Authors are requested to stand by their posters on the day indicated (Tuesday for even poster numbers and Thursday for odd poster numbers).

The posters are grouped by topic area as follows: Diversity and ecology P1-P55 Adaptation to extreme environments P56-P75 Physiology and metabolism P76-P91 Proteins and enzymes P92-P141 Genetics and gene expression P142-P163 Genomics and metagenomics P164-P179 Applications P180-P195

Best poster awards The International Society for Extremophiles (ISE) will honour three young researchers with the Best Poster Award. Candidates to this award (maximum age 31 years) must inform the Organizing Committee on Monday 10 September, at the registration desk (or by e-mail to the congress secretariat: [email protected]). The laureates will be selected by a jury based on the quality and originality of the work and clarity of the poster presentation. The award consists of a certificate plus 100 euro and a book. The award ceremony will take place during the Congress Dinner on 13 September at restaurant Abades.

Social programme The social programme includes the welcome cocktail, visit to the Bodegas Gonzalez-Byass and Congress dinner. These events are included in the registration fee for congress participants and accompanying persons. Non-registered accompanying persons may purchase tickets in advance for those events at the registration desk. Please, remember to bring the invitations, they will be requested by the organization.

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Welcome cocktail The welcome cocktail will be served at 20:00 h. on Monday 10 September, following the opening ceremony and opening lecture, at the hall of the convention area.

Visit to Bodegas Gonzalez-Byass The visit to the Bodegas González-Byass in Jerez de la Frontera will be held on Wednesday 12 September. Buses will leave from the hotel NH Central Convenciones (in front of main entrance) at 16:45 h. Please be on time. Invitations will be required at the bus. Wine tasting and meal will be provided at Salón Los Apóstoles in the Bodegas González-Byass.

Congress dinner The congress dinner will take place on Thursday 13 September at 20:30 h. at Restaurante Abades (Calle Betis 69; phone 954 286 459; http://www.abadestriana.com). The restaurant is at walking distance from the hotel (about 30-35 minutes); alternatively you can go by taxi. Invitations will be requested at the entrance. The winners of the Best Poster Awards and the ISE Award for Lifetime Achievements will be announced at the end of the dinner.

Accompanying persons The registration as accompanying participants includes the welcome cocktail, visit to Bodegas González-Byass, congress dinner at restaurant Abades and a guided visit to the Real Fabrica de Tabacos (on Tuesday at 11:00 h.).

Dietary Participants with specific dietary needs such as vegetarians should contact in advance with the congress organization in order to prepare alternative menu for the lunches and congress dinner.

Internet connection Wireless internet service will be available in the congress venue.

Liability and insurance The organizers are not able to take responsibility whatsoever for injury or damage involving persons and property during the congress.

Mobile phones Participants are requested to keep their mobile phones switched off in the session rooms.

Time Sevilla time is the Central European time zone (Greenwich Mean Time + 2:00), i.e. one hour more than in Lisbon or London.

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Climate and clothing Sevilla enjoys a typically Mediterranean climate throughout the year. September in Sevilla is not as hot as August but is still extremely warm. The average daily temperature may reaches 32°C (about 90°F) at noon and 18°C (64°F) at night. The average daily sunshine is 9 hours a day with average rainfall being just 24 mm over 2 days. This means that the chance of rain is extremely unlikely, and you can still bring light clothing.

Environment Hotel NH Central Convenciones includes this congress on its ECO-Meeting programme, which includes efficient use of water and energy, environmentally friendly materials, low fair-trade coffee and voluntary commitment of CO2 emissions.

Travel agency The official travel agency is Viajes El Corte Inglés (Srta. Rocío Fernández), Calle Teniente Borges 5, 41002 Sevilla, Spain; phone: +34 954 506 605; fax: +34 954 223 512; e-mail: sevillacongresos1@ viajeseci.es

Contact details The address of the organization is: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Calle Profesor Garcia Gonzalez 2, 41012 Sevilla, Spain; phone: +34 954 556 768; fax: +34 954 628 162; e-mail: [email protected]

http://www.congreso.us.es/extremophiles

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scientific programme scientific programme scientific abstracts book 27 EXTREMOphiles_2012

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MONDAY, 10 September

12:00-18:00 h. Registration

18:00 h. Opening ceremony (Salón Andalucía)

Antonio Ventosa, Chairman of the Organizing Committee Milton da Costa, Federation of European Microbiological Societies (FEMS) Helena Santos, President of the International Society of Extremophiles (ISE) Antonio Ramírez de Arellano, Rector of the University of Sevilla

OL1. 19:00 h. Opening lecture (Salón Andalucía)

Chairperson: Antonio Ventosa

Using genomes to track the evolution of life on Earth James A. Lake, University of California at Los Angeles, USA

20:00 h. Welcome cocktail

TUESDAY, 11 September

Symposium 1. Astrobiology and analogs of other planets (Salón Andalucía)

Chairperson: W. D. Grant

S1.1. 9:00-9:30 h. Life in extreme environments–what does it tell us about the prospects for life elsewhere? Charles Cockell, University of Edinburg, UK

S1.2. 9:30-10:00 h. Subsurface geomicrobiology of the Iberian Pyrite Belt Ricardo Amils, Centro de Astrobiología (CSIC-INTA) and Universidad Autónoma de Madrid, Spain

S1.3. 10:00-10:30 h. Atacama desert–a Mars analog inhabited by polyextremophiles Fred A. Rainey, University of Alaska Anchorage, USA

S1.4. 10:30- 11:00 h. Evolutionary relationships between membrane vesicles, plasmids and viruses Patrick Forterre, University Paris-Sud and Institute Pasteur, Paris, France

11:00-11:30 h. Coffee-break and poster viewing

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Symposium 2. Genetics in model extremophiles (Salón ndalucía)A

Chairperson: Bruno Franzetti S2.1. 11:30- 12:00 h. Genetic tools and their optimization for the crenarchaeote Sulfolobus acidocaldarius Sonja V. Albers, MPI, Marburg, Germany

S2.2. 12:00-12:30 h. The genetic toolbox for Thermus thermophilus Jose Berenguer, Universidad Autónoma de Madrid, Spain

S2.3. 12:30-13:00 h. One stator that couples to two different ions: flagellar stator and motility of alkaliphilic Bacillus species Masahiro Ito, Toyo University, Japan

S2.4. 13:00-13:30 h. Virus-host relationships in hyperthermophiles David Prangishvili, Institute Pasteur, Paris, France

13:30-15:00 h. LUNCH

15:00-16:00 h. POSTER SESSION I (Salón Marbella) Authors with even poster numbers will be present

ORAL PARALLEL SESSIONS (16:00-18:50 h.)

Salón Andalucía:

Session 1A. Astrobiology and life in other planets

Chairperson: Milton da Costa

O1. 16:00-16:20 h. Martian Habitability Index studies for Mars Science Laboratory (MSL) in four extreme environments (Earth analogues) and laboratory atmospheres simulation facilities Felipe Gómez, J. A. Rodríguez-Manfredi, J. Gómez-Elvira and REMS team Department of Planetary Geology and Habitability, Centro de Astrobiología (INTA-CSIC), Torrejón de Ardoz, Madrid, Spain

O2. 16:20-16:40 h. Probing the limits of extremophilic life in extraterrestrial environment simulated experiments Claudia Lage, Gabriel Dalmaso, Lia Teixeira, Amanda Bendia, Ivan Paulino-Lima, Douglas Galante, Eduardo Janot-Pacheco, Ximena Abrevaya, Armando Azúa-Bustos, Vivian Pelizzari and Alexandre Rosado

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Laboratório de Radiações em Biologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil

O3. 16:40-17:00 h. Archipelago of endolithic microbial life in the hyper arid core of the Atacama Desert Jacek Wierzchos, Alfonso F. Davila, Jocelyne DiRuggiero, Asunción de los Ríos, Sergio Valea, Beatriz Cámara, Octavio Artieda, Mariela Speranza, Courtney Robinson, Tiffany Souterre, Virginia Souza- Egipsy and Carmen Ascaso Museo Nacional de Ciencias Naturales, MNCN-CSIC, Madrid, Spain

Salón Avenida:

Session 1B. Physiology and Metabolism

Chairperson: Haruyuki Atomi

O4. 16:00-16:20 h. The energy sulfur metabolism of the hyperthermophilic bacterium Aquifex aeolicus Marianne Guiral, Clément Aussignargues, Laurence Prunetti, Pascale Infossi, Marianne Ilbert and Marie-Thérèse Giudici-Orticoni IMM-CNR-AMU, Marseille, France

O5. 16:20-16:40 h. Genetic and functional analysis of CdvB paralogs inSulfolobus acidocaldarius Nuan Yang and Arnold J.M. Driessen Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands

O6. 16:40-17:00 h. Enzymes and regulation involved in archaeal coenzyme A biosynthesis Hiroya Tomita, Yuusuke Yokooji, Takuya Ishibashi, Tadayuki Imanaka and Haruyuki Atomi Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan

17:00-17:30 h. Coffee-break and poster viewing

Salón Andalucía:

Session 2A. Diversity and Ecology

Chairpersons: Anne Godfroy and Mohammad A. Amoozegar

O7. 17:30-17:50 h. Prokaryotic diversity in hydrothermal springs of the Azores analysed by a network of 16S rRNA gene based methods

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Kerstin Sahm, Patrick John, Heiko Nacke, Ralf Grote, Rolf Daniel and Garabed Antranikian Institute of Technical Microbiology, Hamburg University of Technology, Germany

O8. 17:50-18:10 h. Continuous enrichment cultures using diluted hydrothermal fluid as medium: new insights into active microbial diversity inhabiting active deep-sea vent chimney of Guaymas Basin Nolwenn Callac, Olivier Rouxel, Françoise Lesongeur, Carole Decker, Céline Liorzou, Claire Bassoullet, Karine Estève, Patricia Pignet, Sandrine Cheron, Yves Fouquet, Céline Rommevaux-Jestin and Anne Godfroy Université Européenne de Bretagne, Brest, and Ifremer, Laboratoire de Microbiologie des Environnements Extrêmes, Plouzané, France

O9. 18:10-18:30 h. Evidence of species recruitment and development of hypolithic communities along an aridity gradient in the Namib Desert Marla Tuffin, Francesca Stomeo, Thulani Makhalanyane, Angel Valverde, Chris McKay, Kimberley Warren-Rhodes, Donna Lacap, Stephen Pointing and Don Cowan Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Cape Town, South Africa

O10. 18:30-18:50 h. New thermophilic Planctomycetes isolated from terrestrial hot springs and deep subsurface environments Galina Slobodkina, Margarita Miroshnichenko, Olga Kovaleva, Alexander Slobodkin, Esta van Heerden, Alexander Lebedinsky and Elizaveta Bonch-Osmolovskaya Winogradsky Institute of Microbiology RAS, Prospekt 60-letiya Oktyabrya, Moscow, Russia

Salón Avenida:

Session 2B. Proteins and Enzymes

Chairpersons: Mosè Rossi and Peter L. Bergquist

O11. 17:30-17:50 h. Developing novel biophysical tools to explore protein adaptation in stressful environments Toni Hoffmann, Katarzyna Tych, David J. Brockwell and Lorna Dougan School of Physics and Astronomy, University of Leeds, UK

O12. 17:50-18:10 h. Dehalogenase enzymes from extremophilic organisms Jennifer Littlechild, Halina Novak, Christopher Sayer and Misha Isupov Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, UK

O13. 18:10-18:30 h. Protein methylation in crenarchaea Yindi Chu, Zhenfeng Zhang and Li Huang

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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

O14. 18:30-18:50 h. The DNA-alkyltransferase of Sulfolobus solfataricus: in vivo and in vitro studies on a conserved protein involved in repair of DNA alkylation damage Giuseppe Perugino, Antonella Vettone, Anna Valenti, Mosè Rossi and Maria Ciaramella Institute of Protein Biochemistry, National Council of Research of Italy, Naples, Italy

WEDNESDAY, 12 September

Symposium 3. Genomics/Metagenomics (Salón Andalucía)

Chairperson: Brian Jones

S3.1. 9:00-9:30 h. Understanding complexity and processes in Antarctic niche habitats Don Cowan, University of Pretoria, South Africa

S3.2. 9:30-10:00 h. Assembly-driven metagenomics of a hypersaline microbial ecosystem Eric E. Allen, Scripps Institution of Oceanography, University of California San Diego, USA

S3.3. 10:00-10:30 h. Genomic diversity of a Haloquadratum walsbyi population in a single saltern-pond Francisco Rodríguez-Valera, University Miguel Hernandez, Alicante, Spain

S3.4. 10:30-11:00 h. Biased gene flow in haloarchaea: can it account for the appearance and maintenance of phylogenetic clusters? R. Thane Papke, University of Connecticut at Storrs, USA

11:00-11:30 h. Coffee-break and poster viewing

Symposium 4. Molecular evolution and adaptation of proteins (Salón Andalucía)

Chairperson: Michael J. Danson

S4.1. 11:30-12:00 h. Unraveling protein adaptation to high salt concentration using resurrected ancestral-halophilic archaeal enzymes Dominique Madern, Institute of Structural Biology, Grenoble, France

S4.2. 12:00-12:30 h. From non-homogeneous evolutionary models to molecular thermometers Manolo Gouy, University of Lyon, France

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S4.3. 12:30-13:00 h. Function and evolution of proteins encoded by interrupted genes in Archaea Marco Moracci, Institute of Protein Biochemistry-CNR, Naples, Italy

S4.4. 13:00-13:30 h. Hyperstability in resurrected Precambrian proteins Jose M. Sanchez-Ruiz, University of Granada, Spain

13:30-15:00 h. LUNCH

ORAL PARALLEL SESSIONS (15:00-16:40 h.)

Salón Andalucía:

Session 3A. Adaptation to Extreme Conditions

Chairpersons: Koki Horikoshi and Angela Corcelli

O15. 15:00-15:20 h. The proteomic response of an extreme acidophile, Acidithiobacillus ferrooxidans, to increased chloride concentrations Timothy J. McCredden, Robert F.L. Steuart, Christopher G. Bryan and Elizabeth L.J. Watkin School of Biomedical Sciences, Curtin University, Perth, Western Australia, Australia

O16. 15:20-15:40 h. Mutational loss of the membrane carotenoid of an alkaliphilic Bacillus results in changes in membrane fluidity, cell shape, growth and oxidative stress responses Terry A. Krulwich, Oliver Fackelmayer, Amyeo Jereen, Amy LaFountain, Harry Frank, Ronald Gordon, Anthony W. Kingston, John Helmann and David B. Hicks Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York NY, USA

O17. 15:40-16:00 h. Evolutionary analysis of the biosynthesis of glyceryl-glucosides Luís G. Gonçalves, Nuno Borges, Pedro Fernandes, Hernán Dopazo and Helena Santos Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal

O18. 16:00-16:20 h. Microbial diversity and genome characteristics of thermo-piezophiles Mohamed Jebbar, Pauline Vannier, Viggo Marteinsson, Olafur Fridjonsson, Phil Oger, Xiang Xiao, Xu Jun, Jean Louis Birrien and Daniel Prieur Université Bretagne Occidentale (UBO), CNRS, Ifremer, IUEM (Institut Universitaire Européen de la mer), Technopole Brest-Iroise, Plouzané, France

O19. 16:20-16:40 h.

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Membrane homeoviscous adaptation in the archaeon Thermococcus barophilus requires the regulation of unsaturation of accessory, but not core, lipids Anaïs Cario, Vincent Grossi, Philippe Schaeffer and Philippe Oger Laboratoire de Géologie de Lyon, CNRS UMR 5276, Ecole Normale Supérieure de Lyon, Université Lyon 1, Lyon, France

Salón Avenida:

Session 3B. Genomics and Metagenomics

Chairpersons: Beate Averhoff and Chiaki Kato

O20. 15:00-15:20 h. Genomic and metagenomic studies of the abundance of acidic proteins in halophilic microorganisms and hypersaline environments Aharon Oren and Rahel Elevi Bardavid Department of Plant and Environmental Sciences, The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel

O21. 15:20-15:40 h. Comparison of viral assemblages from different hypersaline environments Fernando Santos, Manuel Martínez-García, Judith Villamor, Víctor Parro, Ramon Rosselló-Móra and Josefa Antón Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Spain

O22. 15:40-16:00 h. Genomic characterization of a novel Nanoarchaeota system from Obsidian Pool, Yellowstone National Park Mircea Podar, Kira S. Makarova, David E. Graham, Eugene V. Koonin and Anna-Louise Reysenbach Microbiology Department, University of Tennessee, Knoxville and Biosciences Division, Oak Ridge National Laboratory, Oak Ridge TN, USA

O23. 16:00-16:20 h. Microbial ecology of endolithic life in the Atacama Desert Courtney Robinson, Tiffany Souterre, Jacek Wierzchos, Jacques Ravel, Octavio Artieda, Carmen Ascaso and Jocelyne DiRuggiero Department of Biology, The Johns Hopkins University, Baltimore MD, USA

O24. 16:20-16:40 h. New tools for functional analysis of genes and meta/genomes David Mead, Phil Brumm, Tom Schoenfeld, Colleen Drinkwater, Jan Deneke, Ronald Godiska, Eric Steinmetz Lucigen Corp., Middleton WI, USA

16:45 h. Departure to visit to Bodegas González-Byass (Jerez de la Frontera)

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THURSDAY, 13 September

Symposium 5. Non-conventional extreme environments (Salón Andalucía)

Chairperson: Helena Santos

S5.1. 9:00-9:30 h. Microbial community development in and beneath deep-sea hydrothermal vents in the Earth, and probably in the Enceladus Ken Takai, JAMSTEC, Japan

S5.2. 9:30-10:00 h. Extremophiles in Antarctica: insight into adaptation, evolution and ecosystem function of cold aquatic systems using metagenomics and metaproteomics Ricardo Cavicchioli, University of New South Wales, Australia

S5.3. 10:00-10:30 h. The benefits of James Cameron’s Deepsea Challenge Expedition to Hadal Microbiology Douglas H. Bartlett, Scripps Institution of Oceanography, University of California San Diego, USA

S5.4. 10:30-11:00 h. Radiation- and desiccation resistance of hyperthermophilic microorganisms Petra Rettberg, DLR, Köln, Germany

11:00-11:30 h. Coffee-break and poster viewing

Symposium 6. Physiology/Metabolism (Salón Andalucía) (Costas Drainas Tribute Session)

Chairperson: Michael Thomm

S6.1. 11:30-12:00 h. One-carbon metabolism in a Thermococcus strain, and potential for hydrogen production Sung Gyun Kang, Korea Ocean Research & Development Institute, Korea

S6.2. 12:00-12:30 h. Biosynthesis, evolution and role of unique compatible solutes in heat stress adaptation of marine hyperthermophiles Nuno Borges, ITQB, New University of Lisbon, Portugal

S6.3. 12:30-13:00 h. Microbial life under extreme energy limitation: how to make a living by producing acetate from carbon dioxide and hydrogen Volker Müller, Institute of Molecular Biosciences, University of Frankfurt, Germany

S6.4. 13:00-13:30 h. Novel insights into sugar metabolism of extremophilic Archaea Peter Schönheit, IFAM, University of Kiel, Germany

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13:30-15:00 h. LUNCH

14:30-15:00 h. International Society for Extremophiles (ISE) meeting (Salón Andalucía)

15:00-16:00 h. POSTER SESSION II (Salón Marbella) Authors with odd poster numbers will be present

ORAL PARALLEL SESSIONS (16:00-17:00 h.)

Salón Andalucía:

Session 4A. Genetics and Gene Expression

Chairperson: Asunción de los Ríos

O25. 16:00-16:20 h. Heterologous gene expressions in Geobacillus kaustophilus HTA426 for generating functional thermophiles Hirokazu Suzuki, Toshihisa Ohshima and Yutaka Kawarabayasi Faculty of Agriculture, Kyushu University, Fukuoka, Japan

O26. 16:20-16:40 h. Mannosylglucosylglycerate biosynthesis in Rhodopirellula baltica,a representative of the ancient phylum Planctomycetes Sofia Cunha, Filipa d’Avó, Ana Mingote, Pedro Lamosa, Milton S. da Costa and Joana Costa Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Portugal

O27. 16:40-17:00 h. Genetic and phenotypic analysis of a two-component regulatory system in the moderately halophilic bacterium Halomonas anticariensis Ali Tahrioui, Inmaculada Llamas and Emilia Quesada Department of Microbiology, Faculty of Pharmacy, University of Granada, Spain

Salón Avenida:

Session 4B. Applications

Chairperson: Garo Antranikian

O28. 16:00-16:20 h. Production of rhamnolipid biosurfactants by thermophilic bacteria cultivated on sunflower oil and glycerol Lucie Siříšťová, Věra Šalplachtová and Tomáš Řezanka Department of Biotechnology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Prague, Czech Republic

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O29. 16:20-16:40 h. Secondary metabolites from deep sea bacteria: an efficient route for biodiscovery from extreme marine habitats? Antje Labes, Jan Oesterwalbesloe and Johannes F. Imhoff Kieler Wirkstoff-Zentrum at GEOMAR – Helmholtz Centre for Ocean Research, Kiel, Germany

O30. 16:40-17:00 h. Methods to improve productivity and applicability of anin vitro translation system using the lysate of Thermococcus kodakarensis Tamotsu Kanai, Yuki Sakai, Daijiro Ikegami, Takashi Endoh, Tadayuki Imanaka and Haruyuki Atomi Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan

17:00-17:30 h. Coffee-break and poster viewing

17:30-18:30 h. Closing ceremony (Salón Andalucía)

CL1. 17:30 h. Closing lecture

Chairperson: Garo Antranikian

Basic and applied studies of life near 100°C Michael W. W. Adams, University of Georgia, Athens, USA

18:10 h. Closing ceremony

20:30 h. Congress dinner at Restaurante ABADES Announcement of ISE Awards

38 abstracts book EXTREMOphiles_2012

posters sessions Salón Marbella posters sessions posters abstracts book 39 EXTREMOphiles_2012

40 abstracts book EXTREMOphiles_2012

SESSION 1. Diversity and ecology

P1. Characterization and identification of two hyperthermophilic bacteria isolated from a hot spring in Thailand: a Thermotoga sp. strain FC 1002 and a Fervidobacterium sp. strain FC 2004 Wirojne Kanoksilapatham, Porranee Keawram, Patlada Pasomsup, Maria C. Portillo and Juan M. González

P2. Characterization of moderately and extremely halophilic microorganisms from salt-pack cured hides Pinar Çaglayan, Cristina Sanchez-Porro, Antonio Ventosa and Meral Birbir

P3. Polyphasic characterization of a new extremely halophilic bacterium isolated from an Iranian hypersaline lake Seyed Abolhassan Shahzadeh Fazeli, Mohammad Ali Amoozegar, Ali Makhdoumi-Kakhki, Mohadeseh Ramezani, Peter Schumann and Antonio Ventosa

P4. Thermosulfurimonas dismutans gen. nov., sp. nov., a novel extremely thermophilic sulfur- disproportionating bacterium from a deep-sea hydrothermal vent Alexander Slobodkin, Anna-Louise Reysenbach, Galina Slobodkina, Roman Baslerov, Nadezhda Kostrikina, Icaak Wagner and Elizaveta Bonch-Osmolovskaya

P5. Deferrisoma camini gen. nov., sp. nov., a novel moderately thermophilic dissimilatory Fe(III)- reducing bacterium from a deep-sea hydrothermal vent that forms a distinct phylogenetic branch in Deltaproteobacteria Galina Slobodkina, Anna-Louise Reysenbach, Angela Panteleeva, Nadezhda Kostrikina, Isaac Wagner, Elizaveta Bonch-Osmolovskaya and Alexander Slobodkin

P6. Natrinema salaciae sp. nov., a halophilic archaeon isolated from the deep, hypersaline anoxic Lake Medee in the Eastern Mediterranean Sea Luciana Albuquerque, Marco Tabortda, Violetta La Cono, Michail Yakimov and Milton S. da Costa

P7. Characterization of novelNatronococcus strains and their polar lipidomic analysis Paulina Corral, Roberto Angelini, Patrizia Lopalco, Carmen Gutiérrez, R. Thane Papke, Angela Corcelli and Antonio Ventosa

P8. Characterization of an extremely thermophilic bacterium, Coprothermobacter sp. PM9-2, isolated from an undersea petroleum reservoir Wataru Urushibata, Tetsu Funayama and Masaaki Morikawa

P9. High genetic diversity and novelty of planktonic microeukaryotes inhabiting inland and coastal hyperhaline waters Xavier Triadó-Margarit and Emilio O. Casamayor

P10. Genus Salinivibrio: biodiversity in aquatic hypersaline habitats Clara López-Hermoso, Cristina Sánchez-Porro, Rafael R. de la Haba, R. Thane Papke and Antonio Ventosa

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P11. Isolation and identification of polyextremophilic bacteria from Gomishan Wetland, an alkaline thalassosaline lake in North of Iran Mohammad Ali Amoozegar, Azadeh Shahinpei, Seyed Abolhassan Shahzedeh Fazeli and Abbase Akhavan-Sepahi

P12. Eukaryotic diversity in the deep sedimentary rock from Horonobe, Japan. Yoshimoto Saitoh, Toru Nagaoka and Yuki Amano

P13. New species of the genus Marinobacter isolated from hypersaline habitats. Cristina Sánchez-Porro, María Jose León, Carmen Infante, Clara López-Hermoso and Antonio Ventosa

P14. Ornatilinea apprima gen. nov., sp. nov., a first cellulolytic representative of the class Anaerolineae Olga Podosokorskaya, Elizaveta Bonch-Osmolovskaya, Andrey Novikov, Tatyana Kolganova and Ilya Kublanov

P15. Detection of putatively thermophilic methanotrophic archaea by using new primer systems Alexander Y. Merkel, Julie A. Huber and Alexander V. Lebedinsky

P16. Isolation of new groups of halophilic microorganisms based on metagenomic studies of a marine saltern Carmen Infante, Mª José León, Clara López-Hermoso, Ana B. Fernández, Cristina Sánchez-Porro, Francisco Rodríguez-Valera and Antonio Ventosa

P17. Spatial distribution of microbial communities along salinity gradient in six saline and hypersaline lakes from Transylvania (Central Romania) Andreea Baricz, Stefan Adrian Andrei, Vasile Muntean, Cristian Coman, Zsolt Gyula Keresztes, Mircea Alexe and Horia Banciu

P18. Prokaryotic community structure and diversity at a shallow hydrothermal vent off Panarea Island (Italy) by Illumina high-throughput sequencing Valeria Lentini, Concetta Gugliandolo, Boyke Bunk, Jorg Overmann and Teresa L. Maugeri

P19. Isolation and characterization of a new arsenate-reducing bacterium, which produces an arsenic sulfide ore from the Salar de Ascotán, Chile:Fusibacter sp. 3D3 Lorena V. Escudero, Cinthya Tebes, Jonathan Bijman, Olga Encalada and Cecilia Demergasso

P20. Analysis of extremophilic bacterial community from Kamchatka hot oil field Igor Babkin, Konstantin Kutashev, Vera Morozova, Alla Bryanskaya, Elena Lazareva, Sergei Zhmodik, Irina Babkina, Andrei Matveev and Nina Tikunova

P21. Comparison of bacterial communities from microbial mats accumulating germanium in high and low concentration, Barguzin hot springs of Baikal Rift Zone Nina Tikunova, Alla Bryanskaya, Elena Lazareva, Vera Morozova, Igor Babkin, Aleksandr Kurilshikov, Artem Tikunov, Irina Babkina, Andrei Matveev and Sergei Zhmodik

P22. Diversity of microorganisms isolated from saline lakes of Novosibirsk region Vera Morozova, Irina Saranina, Vladimir Repin, Igor Babkin and Nina Tikunova

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P23. An update of the Multilocus Sequence Analysis (MLSA) of the Halobacteriales Rafael R. de la Haba, Andrea Makkay, Kamisha Byas, Chelsea Gifford, Antonio Ventosa and R. Thane Papke

P24. «Melioribacter roseus» gen. nov., sp. nov., a novel facultatively anaerobic moderately thermophilic cellulolytic bacterium and proposal of «Ignavibacteriae» phyl. nov. Ilya V. Kublanov, Olga Podosokorskaya, Vitaly Kadnikov, Sergey Gavrilov, Andrey Mardanov, Alexander Merkel, Olga Karnachuk, Nikolay Ravin and Elizaveta Bonch-Osmolovskaya

P25. Biodiversity analysis of archaeal and bacterial communities from two hot springs in Galicia (northwestern Spain) Roberto González, Olalla López-López, Clara Fuciños, Natalia Estévez, A. Cristina Rodrigues, Martín Míguez, Pablo Fuciños and M. Luisa Rúa

P26. Survey of microbial diversity, abundance and activity in anaerobic sediments of Tinto river: a natural acid and high heavy metals content environment Irene Sánchez-Andrea, Nuria Rodriguez, Ricardo Amils and Jose Luis Sanz

P27. Roles of an extreme thermophile, Calditerricola satsumensis, in high-temperature compost Takahiro Yoshii, Takafumi Sugihara, Risa Yamabi, Yoshihiro Furui, Hiroko Yano, Toshiyuki Moriya and Tairo Oshima

P28. Investigation of bacterial community dynamics in PAH-contaminated soil by PCR-DGGE Zahra Khomarbaghi, Mohammad Ali Amoozegar, Mahmoud Shavandi, Seyed Mohammad Mehdi Dastgheyb and Hasan Tirandaz

P29. The geochemistry of the deep Iberian Pyrite Belt (IBP) subsurface supports multiple microbial metabolisms Victor Parro, Fernando Puente-Sánchez, Miriam García-Villadangos, Mónica Sánchez-Román, Mercedes Moreno-Paz, Patricia Cruz-Gil, Pablo Fernández, Luis A. Rivas, Yolanda Blanco, Francisco López de Saro, Monike Oggerin, Enoma Omoregie, Nuria Rodríguez, Antonio Molina, Sagrario Arias- Rivas, José Antonio Rodríguez-Mafredi, Kenneth Timmis, David Fernández-Remolar and Ricardo Amils

P30. The development of cyanobacteria on volcanic ashes L.M. Gerasimenko, G.A. Karpov, V.K. Orleansky and G.T. Ushatinskaya

P31. Iron-tolerant cyanobacteria from thermal spring in Volcanic Complex of Furnas (Sao Miguel Island, Azores) Olga Samylina and Liudmila Gerasimenko

P32. Halocin-producing archaea are persistent in Algerian hypersaline ecosystems Alyssa Carré-Mlouka, Manon Vandervennet, Nacéra Imadalou-Idres, HouaYahiaoui, Said Benallaoua, Jean Peduzzi and Sylvie Rebuffat

P33. Phylogenetic diversity of subseafloor aerobic microorganisms isolated from Suruga Bay in Japan Takeshi Miura, Hiroaki Minegishi, Tohru Kobayashi and Koki Horikoshi

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P34. Taxonomic diversity of culturable and unculturable moderately halophilic and halotolerant bacteria in Urmia Lake, second biggest hypersaline lake in the world Maliheh Mehrshad, Mohammad Ali Amoozegar, Bagher Yakhchali and Seyed Abolhassan Shahzedeh Fazeli

P35. Environmental antibody microarray immunoassays reveal metabolic versatility of South African deep-mine biofilms Luis A. Rivas, Yolanda Blanco, Antonio García-Moyano, Jacobo Aguirre, Patricia Cruz-Gil, Arantxa Palacín, Esta Van Heerden and Víctor Parro

P36. Microbial diversity of lithic microbial ecosystems in Miers and Garwood Valleys (McMurdo Dry Valleys, Continental Antarctica) Asunción de los Ríos, Sergio Pérez-Ortega, Rüdiger Ortiz, Steve Pointing, Alan Green and Carmen Ascaso

P37. Polyphasic characterization of Bacillus persicus, a novel slightly halophilic bacterium from Aran-Bidgol salt lake, Iran Maryam Didari, Mohammad Ali Amoozegar, Maryam Bagheri, Maliheh Mehrshad, Peter Schumann, Cathrin Spröer, Cristina Sánchez-Porro and Antonio Ventosa

P38. Changing the microbial community at the Japan Trench, after the East Japan Earthquake of March 11th, 2011 Chiaki Kato, Takayoshi Sekiguchi, Sayumi Kinoshita, Chinatsu Zama, Norio Miyamoto, Hiromi Watanabe, Masahiro Ito, Makiko Enoki and Katsunori Fujikura

P39. Microbial diversity and enzyme characteristics of the unique ikaite columns; a permanently cold and alkaline environment in SW Greenland Mikkel A. Glaring, Jan K. Vester, Jeanette Lylloff, Waleed A. Al-Soud, Søren J. Sørensen and Peter Stougaard

P40. Seasonal variability in microbial mat composition along a temperature gradient in Northern Patagonia geothermal systems Roy Mackenzie, Beatríz Díez and Carlos Pedrós-Alió

P41. Isolation of halophilic and halotolerant microorganisms from core samples of North Kanto area in Japan Tomonori Takashina, Atsuro Watanabe, Takeshi Miura and Akira Inoue

P42. Influence of environmental variables on bacterial community composition in Rambla Salada, a hypersaline environment in south-eastern Spain Nahid Oueriaghli, Victoria Béjar, Emilia Quesada and Fernando Martínez-Checa

P43. Halophile diversity of the Darling Salt Pans of the Western Cape, South Africa Brian E. Jones, William D. Grant, Shaun Bond, Marla Tuffin and Don Cowan

P44. Microbial diversity of Namib Desert Salt Pans Melissa Du Plessis, Angel Valverde, Marla Tuffin and Don Cowan

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P45. Characterisation of halotolerant and halophilic Actinobacteria: phylogeny and enzymatic activities Gabriela Scarlett Alonso Carmona, Félix Aguirre, Horacio Sandoval Trujillo, Ninfa Ramírez Durán and Hugo Ramírez Saad

P46. Comparative analysis of yellow microbial communities growing on the walls of geographically distinct caves indicates a common core of microorganisms involved in their formation Estefania Porca, Valme Jurado, Darja Žgur-Bertok, Cesareo Saiz-Jimenez and Lejla Pašić

P47. Diversity and activity of the denitrification population ofHalomonas in Rambla Salada Carmen María González-Domenech, Nahid Oueriaghli, Fernando Martínez-Checa, Emilia Quesada, Melanie Mormile and Victoria Bejar

P48. Partial characterization and activity of extremely halophilic microorganisms isolated from an Algerian salt lake (Chott el Beida) Houa Yahiaoui, Samy Brahimi, Nabil Bouali and Said Benallaoua

P49. Microbial community in a Zinc impacted mining site Hugh Morgan and Lin Tan

P50. Roseivivax salinus and Roseovarius halophilus, two new species from a multi-pond saltern Mª José León, Ana B. Fernández, Cristina Sánchez-Porro, Francisco Rodríguez-Valera and Antonio Ventosa

P51. The effect of carbon input on the evolution of the soil microbial community in the central hyper-arid Namib Desert Alacia Armstrong, Jean-Baptiste Ramond, Marla I. Tuffin and Don A. Cowan

P52. Sulfolobus mongibelli sp. nov., an acidophilic hyperthermophilic archaeon isolated from Etna fumarole Tatyana Sokolova, Michail Yakimov, Nikolai Chernyh, Alexander Lebedinsky and Elizaveta Bonch- Osmolovskaya

P53. Microstructure and prokaryotic diversity of microbialites formed around a man-made geothermal spring from Romania Cristian Coman, Bogdan Drugã, Adriana Hegedus, Lucian Barbu-Tudoran, Nicolae Dragos and Cosmin Sicora

P54. Diversity of an hydrolytic bacterial community isolated from heavy-metal-contaminated soils of the Atacama Desert María de Lourdes Moreno, Francine Piubeli, Maricy R.L. Bonfá, María Teresa García, Lucia R. Durrant and Encarnación Mellado

P55. Phylogenetic diversity of xylan-degrading thermophilic bacteria from hot springs in Benguet, Philippines Socorro Martha Meg-ay V. Daupan, Phuc Thi Ha, In-Seop Chang and Windell L. Rivera

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SESSION 2. Adaptation to extreme environments

P56. Improvement in organic solvent-tolerance by double mutations of acrR and marR genes in Escherichia coli Rei Watanabe and Noriyuki Doukyu

P57. Genetic analysis of the genes involved in archaeosine biosynthesis of hyperthermophilic archaeon Thermococcus kodakarensis Hiroki Higashibata and Tadayuki Imanaka

P58. Elemental and intact polar lipid compositions of the hyperthermophilic archaeonThermococcus kodakarensis grown under control and phosphate-reduced conditions Travis B. Meador, Emma J. Gagen, Michael Loscar, Michael Thomm and Kai-Uwe Hinrichs

P59. Morphology of alkaliphilic Bacillus halodurans C-125 Shun Fujinami and Masahiro Ito

P60. Evidence of arsenate reducing bacterium in sediment from Salar de Gorbea, an acidic salt flat in northern Chile Lorena V. Escudero, J. Bijman, O. Encalada, G. Chong, J. Pueyo and C. Demergasso

P61. Specific jarosite biomineralization by acidophilic fungi from Río Tinto Monike Oggerin, Nuria Rodríguez, Catalina del Moral, Mónica Sánchez-Román and Ricardo Amils

P62. Extreme microbial sulfur isotope fractionation in a Mars analogue environment at Rio Tinto, SW Spain Esther Velasco, Paul Mason, Pieter Vroon, Wilfred Röling, Ricardo Amils and Gareth Davies

P63. Microbialites from hyper alkaline Lake Van: insights into astrobiological application Yasemin Gulecal and Yildirim Dilek

P64. Osmoadaptative accumulation of moderately halophilic bacteria in subsurface Quaternary sediments of the Qaidam basin Kai Jiang, Yanfen Xue, Xiaowei Wang and Yanhe Ma

P65. Raman spectroscopy in halophile research: an additional tool for direct and fast identification of compatible solutes and pigments Jan Jehlicka, Aharon Oren, Howell G.M. Edwards and Petr Vitek

P66. Halotolerant and halophilic fungi isolated from an athalassohaline environment Irma Marín, Ana Morato and José P. Abad

P67. A clue to longevity - interplay of cell entombment, potassium supply, water activity and membrane potential in Halobacterium salinarum Dorthe Kixmüller and Jörg-Christian Greie

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P68. Octaheme nitrite reductases from haloalkaliphilic bacteria of the genus Thioalkalivibrio: adaptation to extreme environment Tamara Tikhonova, Alexey Tikhonov and Vladimir Popov

P69. From sequencing to gene discovery to function: desiccation tolerance in an Antarctic metagenome Dominique Anderson, Mark Taylor, Marla Tuffin, Don Cowan and Craig Cary

P70. Mannosylglycerate protects staphylococcal nuclease with restriction of backbone andβ -sheet motions Tiago M. Pais, Pedro Lamosa, Manolis Matzapetakis, David L. Turner and Helena Santos

P71. Microorganisms isolated from Antarctica Wakao Fukuda, Kozo Yamada, Tomomi Kimura, Shigeo Araki, Yohzo Chino, Yuki Miyoshi, Yuka Kondo, Hirokazu Okuno, Tamotsu Kanai, Haruyuki Atomi and Tadayuki Imanaka

P72. Genome-wide functional screening to identify nickel-resistance determinants in the extreme nickel-resistant Acidiphilium sp. PM Patxi San Martin-Uriz, Salvador Mirete, Manuel J. Gómez, Pedro J. Alcolea, Ricardo Amils and Eduardo Gonzalez-Pastor

P73. Niche specific oxidative stress response in extreme acidophiles Juan Pablo Cárdenas, Mauricio Arenas, Raquel Quatrini and David S. Holmes

P74. High hydrostatic pressure alters Thermococcus barophilus metabolism Anaïs Cario, Florence Lormières and Philippe Oger

P75. Accumulation of compatible solutes in a member of the deep-branching phylum Planctomycetes-Rhodopirellula baltica Filipa d’Avó, Sofia Cunha, Ana Mingote, Pedro Lamosa, Milton S. da Costa and Joana Costa

SESSION 3. Physiology and Metabolism

P76. Genome-wide dissection of the key enzymes and distinct pathways for PHBV production in Haloferax mediterranei Jing Han, Jing Hou, Fan Zhang, Shuangfeng Cai, Bo Feng, Qiuhe Lu, Songnian Hu and Hua Xiang

P77. The physiological intricacies in Caldicellulosiruptor saccharolyticus Sudhanshu Pawar and Ed W.J. van Niel

P78. Thermodynamic challenges of the formate-oxidizing metabolism and implications for hydrogen production Hyun Sook Lee, Jae Kyu Lim, Seung Seob Bae, Tae Wan Kim, Jung-Hyun Lee and Sung Gyun Kang

P79. Analysis of the DNA polymerase B disruptant of hyperthermophilic archaeon Thermococcus kodakarensis Takashi Kushida, Issay Narumi, Shinsuke Fujiwara, Tadayuki Imanaka and Hiroki Higashibata abstracts book 47 EXTREMOphiles_2012

P80. Metabolic versatility of «Melioribacter roseus» gives insights into the evolution and deep subsurface origin of a novel phylum «Ignavibacteriae» Sergey Gavrilov, Olga Podosokorskaya, Ilya Kublanov, Alexander Merkel, Vitaly Kadnikov, Andrey Mardanov, Nikolay Ravin, Yulia Frank, Olga Karnachuk and Elizaveta Bonch-Osmolovskaya

P81. Mutants of Pyrococcus furiosus as a means to study the roles of mannosylglycerate and di- myo-inositol phosphate in stress adaptation Ana M. Esteves, Sanjeev K. Chandrayan, Patrick M. McTernan, Michael W. Adams, Nuno Borges and Helena Santos

P82. The biosynthesis of glycero-phospho-inositol: do Archaea «like» to be different? Marta V. Rodrigues, Rita Ventura, Christopher Maycock, Nuno Borges and Helena Santos

P83. Caloramator boliviensis a thermoanaerobe with interesting metabolic properties Carla Crespo, Anselm Moshi and Bo Mattiasson

P84. Anoxybacillus–thermophilic facultative anaerobic bacteria with potential biorefining properties Emanuel Ron, Carla Crespo, Bo Mattiasson and Eva Nordberg Karlsson

P85. Sugar degradation in the haloarchaeon Haloferax volcanii Ulrike Johnsen, Moritz Sutter, Andreas Pickl and Peter Schönheit

P86. Identification of a global regulator of glucose metabolism in Sulfolobus solfataricus Olha V. Rohulya, Sonja-Verena Albers and Arnold J.M. Driessen

P87. Dissimilatory sulfate reduction in the crenarchaeote «Vulcanisaeta moutnovskia» Nikolai A. Chernyh, Margarita L. Miroshnichenko, Olga L. Kovaleva, Maria I. Prokofeva, Nikolai V. Pimenov, Alexander V. Lebedinsky and Elizaveta A. Bonch-Osmolovskaya

P88. Physiological characterization ofHalomonas pacifica and Halomonas campisalis isolated from saline alkaline lakes in the Libyan Sahara Abdolkader A. Mohmmed and D. James Gilmour

P89. C14-AHL signalling molecules enhances biofilm formation on metal sulfides by the extremophilic bacterium Acidithiobacillus ferrooxidans Alex González, Sören Bellenberg, Sigde Mamani, Lina Ruiz, Alex Echeverría, Laurent Soulère, Alain Doutheau, Cecilia Demergasso, Wolfgang Sand, Yves Queneau, Mario Vera and Nicolas Guiliani

P90. Ferritin distribution in organisms developing in an extreme acidic environment, Río Tinto Ricardo Amils, Lourdes Rufo, Nuria Rodríguez and Vicenta de la Fuente

P91. Molecular dynamics response of Halobacterium salinarum to environmental stress V. Marty, E. Fabiani, F. Gabel, G. Zaccai and B. Franzetti

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SESSION 4. Proteins and enzymes

P92. Functional and structural characterization of Dye-linked L-proline dehydrogenase from hyperthermophiles Takenori Satomura, Ryushi Kawakami, Shin-ichiro Suye, Haruhiko Sakuraba and Ohshima Toshihisa

P93. Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii Diya Alsafadi and Francesca Paradisi

P94. Slow unfolding pathway of a hyperthermophilic protein, Tk-RNase H2, examined by pulse proteolysis using a super-stable protease, Tk-subtilisin Kazufumi Takano, Jun Okada, Yuichi Koga and Shigenori Kanaya

P95. Acylpeptide hydrolases inSulfolobus solfataricus: an enzyme system with «surprising» features M. Gogliettino, M. Balestrieri, E. Cocca, M. Rossi and G. Palmieri

P96. Construction of bifunctional biomass-degrading enzymes from thermophiles Skander Elleuche, Mazen Rizk and Garabed Antranikian

P97. Structural insight into the evolution and broad substrate specificity of the fourth type of tRNA splicing endonuclease Akira Hirata, Kosuke Fujishima, Ryota Yamagami, Takuya Kawamura, Jillian F. Banfield, Akio Kanai and Hiroyuki Hori

P98. Transfer RNA recognition mechanism ofThermus thermophilus folate/FAD-dependent tRNA methyltransferase (TrmFO) Ryota Yamagami, Koki Yamashita, Hiroshi Nishimasu, Chie Tomikawa, Anna Ochi, Chikako Iwashita, Akira Hirata, Ryuichiro Ishitani, Osamu Nureki and Hiroyuki Hori

P99. Transfer RNA modifications in thermophilic bacteria Takuya Kawamura, Chie Tomikawa, Takayuki Ohira, Yasushi Inoue, Akihiko Yamagishi, Tsutomu Suzuki and Hiroyuki Hori

P100. Prediction of novel phosphor-sugar metabolic pathway by functional genomics of acidothermophilic archaeon, Sulfolobus tokodaii Yutaka Kawarabayasi

P101. Creation of a thermostable NADP-dependent D-amino acid dehydrogenase fromUreibacillus thermosphaericus meso-diaminopimelate dehydrogenase by site-directed mutagenesis Hironaga Akita, Katsumi Doi, Yutaka Kawarabayashi and Toshihisa Ohshima

P102. Study of novel recombinant proline dipeptidase from anaerobic organotrophic archaeon Thermococcus sibiricus Elvira Slutskaya, Ekaterina Bezsudnova, Anton Trofimov, Dmitry A. Korzhenevsky, Vadim Gumerov and Vladimir Popov

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P103. Characterization of recombinant thermostable L-asparaginase from Thermococcus kodakaraensis KOD1 Sung-Jun Hong, Yun-Ha Lee and Jae-Ho Shin

P104. The natural transformation machinery inThermus thermophilus HB27: a pilus-independent DNA transporter comprising unique motor ATPase and secretin complexes R. Salzer, J. Burkhardt, J. Vonck and B. Averhoff

P105. Purification and characterization of a thermophilic feather-degrading enzyme from a moderate thermophile Bacillus sp. FN60 Mio Kojima and Akira Inoue

P106. Molecular basis for polysaccharides degradation capabilities ofesulfurococcus D fermentans, a hyperthermophilic crenarchaeon Anna Perevalova, Ida Steen, Runar Stokke, Dwi Susanti, Elizaveta Bonch-Osmolovskaya and Biswarup Mukhopadhyay

P107. A new chemo-enzymatic tool for the GOS synthesis using a mutant of the thermophilic b-galactosidase from Alicyclobacillus acidocaldarius Andrea Strazzulli, Beatrice Cobucci-Ponzano, Perugino Giuseppe, Maria Michela Corsaro, Emiliano Bedini, Mosè Rossi and Marco Moracci

P108. New groups of potentially novel enzyme specificities in the extremophilic glycoside hydrolase family GH57 Karol Blesak and Stefan Janecek

P109. A relatedness between the CAZy alpha-amylase families GH57 and GH119 - a novel clan? Stefan Janecek, Karol Blesak and Andrea Kuchtova

P110. Archaeal glycosylation: deciphering the machinery in Sulfolobus solfataricus Maria Carmina Ferrara, Beatrice Cobucci-Ponzano, Andrea Carpentieri, Angela Amoresano and Marco Moracci

P111. Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway Riku Aono, Takaaki Sato, Ayumu Yano, Shosuke Yoshida, Yuichi Nishitani, Kunio Miki, Tadayuki Imanaka and Haruyuki Atomi

P112. Dynamic, ligand-dependent conformational change triggers the reaction of ribose-1,5-bisphosphate isomerase from Thermococcus kodakarensis Takaaki Sato, Akira Nakamura, Riku Aono, Masahiro Fujihashi, Yosuke Nishiba, Shosuke Yoshida, Ayumu Yano, Tadayuki Imanaka, Kunio Miki and Haruyuki Atomi

P113. Single molecule force spectroscopy study of cold shock protein from the hyperthermophile Thermotoga maritima Katarzyna Tych, Toni Hoffmann, David J. Brockwell and Lorna Dougan

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P114. A novel alkaline xylanase from alkaliphilic Bacillus sp. SN5 Wenqin Bai, Yanfen Xue, Cheng Zhou and Yanhe Ma

P115. Polimeryzing laccase-like activity in antarctic psychrophiles and thermophiles R. Peralta, L. Blamey, J. Pereira and J. M. Blamey

P116. Tetrathionate hydrolase from the marine acidophilic sulfur-oxidizing bacterium Acidithiobacillus thiooxidans strain SH Tadayoshi Kanao, Yuki Hashimoto, Tatsuya Toge, Hiroshi Kikukawa and Kazuo Kamimura

P117. Characterization of the extremophile homologs of mitoNEET Emi Hagiuda, Yoshiharu Miyajima-Nakano, Daijiro Ohmori, Teruo Kusano, Shinichi Matsushita, Sergei A. Dikanov, Takashi Kumasaka and Toshio Iwasaki

P118. Structural basis for metallosulfur recognition of archaeal Rieske protein scaffold Toshio Iwasaki and Takashi Kumasaka

P119. Selective isotope labelling of extremophile metalloproteins Risako Fukazawa, Myat T. Lin, Yoshiharu Miyajima-Nakano, Amgalanbaatar Baldansuren, Shinichi Matsushita, Sylvia K. Choi, Sergei A. Dikanov, Robert B. Gennis and Toshio Iwasaki

P120. Active site structure of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) Kazuya Hasegawa, Shinichi Matsushita, Asako Konosu, Amgalanbaatar Baldansuren, Risako Fukazawa, Sergei A. Dikanov, Toshio Iwasaki and Takashi Kumasaka

P121. Engineering the enzyme LipBL from Marinobacter lipolyticus to identify the residues involved in the hydrolytic activity Dolores Pérez, Filip Kovačić, Susanne Wilhelm, Karl-Erich Jaeger, María Teresa García, Antonio Ventosa and Encarnación Mellado

P122. Optimization of tellurite reducing enzyme from moderately halophilic bacterium,Salinicoccus iraniensis Sana Alavi, Mohammad Ali Amoozegar and Khosro Khajeh

P123. The intriguing inositol-1-phosphate cytidylyltransferase activity ofRhodothermus marinus Carla D. Jorge, Nuno Borges and Helena Santos

P124. Bacterial subfamily II isocitrate dehydrogenase: catalytic and structural adaptations to low and high temperatures Anita-Elin Fedøy, Hanna-Kirsti S Leiros, Ida H Steen and Nils-Kåre Birkeland

P125. Mineral respiration under extreme acidic conditions: from a supramolecular organization to a copper helper protein Magali Roger, Cindy Castelle, Frédéric Biaso, Marie-Thérèse Giudici-Orticoni and Marianne Ilbert

P126. Extracellular α-amylase from an isolated extreme halophilic archaeon Halorubrum xinjiangense Mohammad Ali Faramarzi and Mahsa Moshfegh

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P127. A new thermophilic microorganism from Deception Island, Antarctica: isolation and characterization of a microorganism with biotechnological potential Patricio A. Flores and Jenny M. Blamey

P128. A heterotrimeric PCNA from Metallosphaera sedula Fumiya Iwata, Hidehiko Hirakawa and Teruyuki Nagamune

P129. Extremophilic arsenite-oxidizing and arsenate-reducing bacteria L. Blamey, F. Sarmiento, J. Pereira, C. Gaete and J. M. Blamey

P130. Properties of 3-isopropylmalate dehydrogenase from the deep-sea and non deep-sea Shewanella strains Yuki Hamajima, Takayuki Nagae, Nobuhisa Watanabe, Chiaki Kato, Yasuyuki Kato-Yamada and Takeo Imai

P131. Analysis of lipolytic enzyme genes from the bio-plastic degrading piezophilic Moritella sp. strain JT01 isolated from the Japan Trench Chiaki Kato, Takayoshi Sekiguchi, Chinatsu Zama, Mona Windmeisser, Yuki Hamajima, Makiko Enoki and Haruyuki Kanehiro

P132. Screening of yeast from the semi-arid region of Bahia, with sucrase activity in aiming to use them as adjuvant therapy in sucrose intolerance Patricia Morais Lopes Pereira, Sandra Aparecida de Assis and Elinalva Maciel Paulo

P133. Molecular cloning and characterization of Anoxybacillus flavithermusT1 esterase/lipase Laura Chiş, Monica Hriscu, Gergely Rona, Beata G. Vértessy and Florin Dan Irimie

P134. Physiological role of eicosapentaenoic acid in the cold-adaptation mechanism of an Antarctic bacterium, Shewanella livingstonensis Ac10 Jun Kawamoto, Nobuyoshi Esaki and Tatsuo Kurihara

P135. Improvement of thermostability of a bacteriophage and a bacterium by heat-shock proteins Yun Jae Kim, Hyun Sook Lee, Jung-Hyun Lee and Sung Gyun Kang

P136. Characterization of the glutamine synthetase GlnA2 ofHalobacillus halophilus Anna Shyan, Melanie Thompson, Daniela Hartmann, Michaela Tausendschön, Inga Hänelt and Volker Müller

P137. Thermal stability of a [2Fe-2S] ferredoxin from Cyanidioschyzon merolae can be modified by a single amino acid substitution Yuko Ueno, Ayumi Sando, Hiroaki Tokiwa, Yukio Morimoto, Yasuyuki Kato-Yamada and Takeo Imai

P138. Cold-active and alkali-stable proteases from bacteria isolated from the polyextreme ikaite columns in Greenland Jeanette Eva Lylloff, Mikkel Glaring and Peter Stougaard

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P139. Purification of ribosome from extremely halophilic archaeonHaloarcula japonica and RFHR 2D analyses Kaoru Nakasone and Fuminori Aono

P140. Extremophilic enzymatic response for protection against UV-radiation damage Jenny M. Blamey, Bernardita Chirino, María Teresa Monsalves and Freddy Boehmwald

P141. A novel compensatory splicing mechanism for the Thermococcus kodakarensis Tko CDC21-1 intein without the highly conserved histidine Kazuo Tori, Manoj Cheriyan, Chandra Sekhar Pedamallu, Marleny A. Contreras and Francine B. Perler

SESSION 5. Genetics and gene expression

P142. Differential utilization of multiple Orc/Cdc6-determined replication origins govern polyploidy in Haloarcula hispanica Zhenfang Wu, Jingfang Liu, Hailong Liu, Xiaoqing Liu and Hua Xiang

P143. Transcriptional and functional analyses of Cas/CRISPR system in Haloferax mediterranei Ming Li, Zhenfang Wu, Hailong Liu, Jing Han and Hua Xiang

P144. Identification and characterization of the function and regulation of a sugar phosphotransferase system gene cluster in Haloferax mediterranei Lei Cai, Shuangfeng Cai, Xiaoqing Liu, Dahe Zhao, Jian Zhou and Hua Xiang

P145. Identification and characterization of the cognate anti-sigma factor and specific promoter elements of a Thermoanaerobacter tengcongensis ECF sigma factor Jingfang Liu, Jie Li, Zhenfang Wu, Huadong Pei, Jian Zhou and Hua Xiang

P146. Novel targets and cofactors for LysM from Sulfolobus solfataricus, a transcriptional regulator of the Lrp-family Ningning Song, Trong Nguyen Duc, Liesbeth van Oeffelen, Serge Muyldermans, Eveline Peeters and Daniel Charlier

P147. Involvement of universally conserved genes, ygjD and yeaZ orthologs, in DNA repair of Deinococcus radiodurans Takefumi Onodera, Katsuya Satoh, Toshihiro Ohta and Issay Narumi

P148. Functional analysis of multiple general transcription factors in S. acidocaldarius Bernadette Rauch and Bettina Siebers

P149. Involvement of two putative cyclopropanic fatty acid synthases in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens Francine Piubeli, Aggeliki Katsifa, Montserrat Argandoña, Joaquín J. Nieto, Manuel Salvador, Rosa García-Valero, Anna I. Koukkou and Carmen Vargas

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P150. Molecular characterization of a hybrid histidine-kinase involved in osmoadaptation in the halophilic bacterium Chromohalobacter salexigens Carmen Vargas, Rosa García-Valero, Montserrat Argandoña, Manuel Salvador, Francine Piubeli and Joaquín J. Nieto

P151. Molecular characterization of the uptake of ectoines inChromohalobacter salexigens Joaquín J. Nieto, Javier Rodríguez-Moya, Montserrat Argandoña, Fernando Iglesias-Guerra and Carmen Vargas

P152. New host-vector system for Deinococcus geothermalis Issay Narumi, Takefumi Onodera and Katsuya Satoh

P153. Role of the global regulator rpoS in the long- and short- term osmo- and thermoadaptation, in Chromohalobacter salexigens Manuel Salvador, Jose M. Pastor, Montserrat Argandoña, Vicente Bernal, Francine Pibueli, Rosa García-Valero, Manuel Cánovas, Joaquín J. Nieto and Carmen Vargas

P154. The array of transcriptional factors sequences in the genome ofD. hansenii reveals how this yeast orchestra a complex response to salt stress Marissa Calderón-Torres, Miguel Murguía-Romero, Daniela E. Castro and Antonio Peña

P155. Isolation and properties of acidophilic bacteria and the cryptic plasmid from acidic environments Tsubasa Sato, Kazuo Kamimura and Tadayoshi Kanao

P156. In vivo evolution research with chimeric Rpb5/RpoH subunits of the RNA polymerase in Pyrococcus furiosus Ingrid Waege, Christoph Reich, Bettina Sommer, David Pöllmann, Winfried Hausner and Michael Thomm

P157. Quorum sensing in Halomonadaceae Melanie Schwab, Ali Tahrioui, Emilia Quesada and Inmaculada Llamas

P158. Interference in the quorum-sensing system of pathogenic vibrios for fish and molluscs M. Torres, S. Prado, J. Dubert, A. Tahrioui, M. Romero, E. Quesada and I. Llamas

P159. The archaeal Holliday junction resolvase Hje has a regulatory role and is involved in DNA repair Yansheng Li, Qihong Huang, Chaoning Zeng, Yanze Li, Jinfeng Ni, Qunxin She and Yulong Shen

P160. An Hjc-interacting nuclease-ATPase inSulfolobus islandicus is essential for cell viability Binyuan Zhai, Yanze Li, Mingzhu Chu, Jinfeng Ni and Yulong Shen

P161. Clone library as tool for detection of multiple copies of 16S rRNA in Halobacteriaceae archaeon Leonardo H. Pinto, Gigliola R. B. Sallôto, Joyce Lemos, Ricardo P. Vieira, Orlando B. Martins and Maysa M. Clementino

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P162. Genetic engineering of Pyrococcus furiosus to use chitin as a carbon source Martina Kreuzer, Michael Thomm and Winfried Hausner

P163. N-glycosylation inHaloferax volcanii involves two distinct biosynthetic pathways Lina Kaminski, Ziqiang Guan, Sophie Yurist-Doutsch and Jerry Eichler

SESSION 6. Genomics and Metagenomics

P164. High through put mapping of transposon insertions for the identification of essential genes of the methanogenic archaeon Methanococcus maripaludis Felipe Sarmiento B., Jan Mrazek and William B. Whitman

P165. Towards an improved S. acidocaldarius expression system useful for functional metagenome analysis J. Kort, A. Wagner, S.V. Albers and B. Siebers

P166. New abundant microbial lineages in hypersaline systems Ana B. Fernandez, Rohit Ghai, Ana Belen Martin-Cuadrado, Cristina Sanchez-Porro, Francisco Rodriguez-Valera and Antonio Ventosa

P167. Genome analysis of Halanaerobium saccharolyticum DSM 6643T, a halophilic hydrogen producing bacterium Anniina Kivistö, Antti Larjo, Ville Santala, Christophe Roos and Matti Karp

P168. Unveiling microbial life and functional diversity in Mediterranean deep-sea hypersaline lakes María Alcaide, Mercedes V. Del Pozo, Álvaro Lafraya, Rafael Bargiela, Jesús Tornés, Tatyana N. Chernikova, Violetta LaCono, Olga V. Golyshina, Michail M. Yakimov, Peter N. Golyshin and Manuel Ferrer

P169. Functional diversity of microbial (meta-) genomes in the Iberian Peninsula Mónica Martínez, María Alcaide, Mercedes V. Del Pozo, Rafael Bargiela, Iván Lores, Celia Méndez, Jesús Sánchez and Manuel Ferrer

P170. Genomic insights into the life of the unusual extremophiles of the deep-sea brines of the Red Sea André Antunes, Intikhab Alam, Vladimir B. Bajic and Ulrich Stingl

P171. Isolation of sodium non-requirement alkaliphiles and its physiology predicted from comparative genome analysis with sodium dependent ones Makoto Fujisawa, Kazuaki Odakura, Manato Watanabe, Takuya Kamiyama and Kazuya Noda

P172. Integrating high-throughput technologies for the discovery of novel thermostable lignocellulases Colin Ohlhoff, Rob Huddy, Mariette Smart, Bronwyn Kirby, Inonge Mulako, Ana Casanueva, Rolene Bauer, Marla Tuffin and Don Cowan

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P173. Characterization of thermostable GH 9 cellulases from a metagenomic source Stephen Mackay, Robert Huddy, Marla Tuffin, Donald Cowan and Rolene Bauer

P174. Bioleaching heaps provide special opportunities for studying the adaption, ecophysiology and evolution of extreme acidophiles Raquel Quatrini and David S. Holmes

P175. Archaeal diversity in the geothermal field Los Azufres revealed by metagenomics Luis E. Servín Garcidueñas and Esperanza Martínez Romero

P176. Thermus thermophilus phage P23-77: archetype of a novel branch of an ancient viral lineage Alice Pawlowski, Ilona Rissanen, Jonathan M. Grimes, Karl Harlos, David I. Stuart and Jaana K.H. Bamford

P177. A constraint-based metabolic model designed to optimize ectoines production in Chromohalobacter salexigens Montserrat Argandoña, Francine Piubeli, Javier Rodríguez-Moya, Jose M. Pastor, Manuel Salvador, Vicente Bernal, Angel Sevilla, Manuel Cánovas, Joaquín J. Nieto and Carmen Vargas

P178. Metagenomic screening of extremophilic sediments for rhamnolipid production Wesley Trevor Williams, Johannes Kügler, Marla Tuffin, Christoph Syldatk and Don Cowan

P179. Functional metagenomics of thermophilic phage replisomes David Mead, Sally Floyd, Audrey Klingele, Brian Hedlund, Jeremy Dodsworth, Ron Godiska, Michael Lodes and Tom Schoenfeld

SESSION 7. Applications

P180. Natrialba sp. strain E21, an extremely halophilic bacterium isolated from a solar saltern in Ain Salah (Algeria) able to produce biosurfactant by free and entrapped cells Salima Kebbouche-Gana, Souad khemili, Mohamed Lamine Gana and Imene Ferioune

P181. Antagonistic activity of an extremely halophilic bacterium Bacillus sp. nov. B21 obtained from an Algerian oil field against sulfate-reducing bacteria consortium Mohamed Lamine Gana and Salima Kebbouche-Gana

P182. A reconstituted Mn(II)-peptide complex of Deinococcus radiodurans preserves immunogenicity of lethally irradiated vaccines against viruses and Staphylococcus aureus Elena Gaidamakova, Ian Myles, Dennis McDaniel, Cedar Fowler, Patricia Valdez, Manoshi Gayen,Paridhi Gupta, Anuj Sharma, Pamela Glass, Radha Maheshwari, Sandip Datta and Michael Daly

P183. Effect of the halophilic bacteria on wheat growth under water stress in saline soils Ahmad Ali Pourbabaee, Maryam Talebi, Mehdi Shorafa and Seyed Alireza Salami

P184. Enzymatic degradation of PrPSc by a protease secreted from Aeropyrum pernix K1 Marko Šnajder, Tanja Vilfan, Maja Černilec, Vladka Čurin Šerbec and Nataša Poklar Ulrih

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P185. Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4 Sotaro Sano and Shinsuke Fujiwara

P186. The potential biotechnological applications of the exopolysaccharides produced by moderately halophilic bacteria Hakima Amjres, Victoria Béjar, Inmaculada Llamas and Emilia Quesada

P187. Fast technique for finding naphthalene-degrading haloalkaliphilic actinomycetes Ninfa Ramírez-Durán, Reyna del Carmen Lara Severino, Hugo Ramírez Saad and Horacio Sandoval Trujillo

P188. Evaluation of the applicability of extremophiles for generation of EtOH and other valueable products from waste sulphite pulping liquor M. Weissgram, H. K. Weber and C. Herwig

P189. Health from extreme environments: searching for antimicrobial and antitumor activities in acidotolerant microalgae María Vázquez, Francisco Navarro, Alberto Toimil, Sandra Mora, Miguel Ángel Castaño, Adriana Márquez, Inés Garbayo, Eduardo Forján and Carlos Vílchez

P190. Coccomyxa onubensis, a novel acidotolerant microalga: identification and biotechnological value Isabel Vaquero, Mayca Márquez, Stefan Girlich, Juan Luis Fuentes, Benito Mogedas, Manuel González del Valle, Inés Garbayo, Eduardo Forján, Volker Huss and Carlos Vílchez

P191. Enhancement of polyunsaturated fatty acids and carotenoid production in the extremophile microalga Coccomyxa onubensis by UVA radiation Mª Carmen Ruiz, Živan Gojković, Nora Janzer, Mª José Dominguez, Carlos Vilchez, José M. Vega and Inés Garbayo

P192. Thermostable fluorescent protein palette for the study of thermophilic metabolism and its regulation Yamal Al-Ramahi, José Berenguer and Aurelio Hidalgo

P193. Usage of halotolerant bacteria toward bioconversion containing hydrophilic ionic liquid Atsushi Kurata, Humiya Seno, Chiaki Matsuhara, Takuma Tatsunami, Yasuyuki Ikeda and Noriaki Kishimoto

P194. Analysis of the immunological effect of Halorubrum sodomense Han-Seung Lee, Seulki Park, Ji Eun Oh, Danbie Jang, Byoung-Chan Kim, Dong-Woo Lee, Heung-Kyu Lee

P195. Thermodynamics and structure & function relationship of α-amylases from thermophilic bacteria from Tulsi Shyam, Gujarat, India Bhavtosh A. Kikani and Satya P. Singh

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abstracts abstracts

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60 abstracts book EXTREMOphiles_2012

keynotes lectures

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62 abstracts book EXTREMOphiles_2012OL1 Using genomes to track the evolution of Life on Earth James A. Lake

MCD Biology and Human Genetics, University of California Los Angeles (UCLA), USA E-mail: [email protected]

Today Evolutionary Genomics is in a state of crisis because we mistakenly assumed that once complete genome became available–the tree of life on Earth, including extemophile origins, could be easily reconstructed in considerable detail. Instead, all of us in the field agree that we cannot easily determine a single tree. Different genes have different histories. However, everyone seems to have different reasons for why they think that this happens. Here, I’ll make the case that Darwinian tree-like evolution, and the «survival of the fittest» metaphor give an incomplete view of evolution and that we need to focus more upon both trees like evolution and cooperation between organisms (endosymbioses, symbioses, and other types of gene sharing) to accurately reconstruct evolutionary histories. Trees are easy to calculate from genomic data, but we must combine «survival of the fittest» and «cooperation», if we are to reconstruct the evolution of life on Earth. Methods to do this are vastly more complex and are just being developed. But they are much more revealing about the history of life. I’ll describe some of the remarkable findings that are now being obtained using these new methods.

References · Cox, CJ, Foster, PJ, Hirt, RP, Harris, SR, Embley, TM. (2008). The archaebacterial origin of eukaryotes. Proc. Nat. Acad. Sci. USA 105: 20356-20361. · Lake, JA. (2009). Evidence for an early prokaryotic endosymbiosis. Nature 460: 967-971.

abstracts book 63 CL1EXTREMOphiles_2012 Basic and applied studies of life near 100°C Michael W. W. Adams

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA E-mail: [email protected]

Microorganisms that can grow at or above 95°C were discovered three decades ago (Stetter, 1982). Most of the species currently known are anaerobic heterotrophs that utilize peptides or carbohydrates as carbon sources and depend to a greater or lesser extent upon elemental sulfur (S0) for growth. Other anaerobic species include methanogens, sulfate-reducers and nitrate-reducers, and some microaerophilic and aerobic species are also known. With one notable exception (Aquifex), all genera are classified within the Archaea domain. Many of these organisms, including species of Pyrobaculum, Pyrococcus, Thermococcus, Archaeoglobus and Methanopyrus, have been extensively investigated from physiological, metabolic, biochemical and molecular perspectives and complete genome sequences are available for many of them. These studies have frequently revealed many novel pathways and modes of energy conservation, and proteins from these organisms have served as model systems for X-ray and NMR analyses through structural genomics projects. Microbes that thrive near 100°C have significant biotechnological potential. In particular, they afford new approaches to biomass conversion and the production of various biofuels, including hydrogen gas and alcohols. This potential is likely to be realized in the near future with the development of genetic tools for some of these organisms. These include the pioneering efforts with the heterotrophic S0-reducer, Thermococcus kodakarensis

(Topt 85°C: Sato et al., 2003). Even more recently, molecular tools have been developed for Pyrococcus furiosus

(Topt 100°C), allowing mutational analyses and metabolic engineering (Bridgeret al., 2011; Basen et al., 2012). Potential biotechnological developments with these organisms will be discussed.

References · K. O. Stetter (1982). Ultrathin mycelia-forming organisms from submarine volcanic areas having an optimum growth temperature of 105°C. Nature 300: 258-260. · T. Sato, T. Fukui, H. Atomi and T. Imanaka (2003). Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Bacteriol 185: 210-220. · S. L. Bridger, S. M. Clarkson, K. Stirrett, M. DeBarry, G. L. Lipscomb, G. J. Schut, J. Westpheling, R. A. Scott and M. W. W. Adams (2011). Deletion strains reveal metabolic roles for key elemental sulfur responsive proteins in Pyrococcus furiosus. J. Bacteriol. 193: 6498-6504. · M. Basen, J. Sun and M. W. W. Adams (2012). Engineering a hyperthermophilic archaeon for temperature- dependent product formation. mBio 3: e00053-12.

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main symposia

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66 abstracts book EXTREMOphiles_2012S1.1 Life in extreme environments – what does it tell us about the prospects for life elsewhere? Charles Cockell

UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, James Clerk Maxwell Building, Edinburgh, EH9 3JZ, UK E-mail: [email protected]

The study of life in extreme environments informs us about aspects of microbial physiology that have relevance to the search for life beyond the Earth. By studying microorganisms at physical extremes such as pressure, temperature, salinity, etc we begin to map out the ‘biospace’ of life on the Earth. Despite remarkable progress in the field there still remains very little knowledge about the adaptations of microorganisms to multiple extremes (polyextremophiles), which limits our capacity to assess the habitability of complex extraterrestrial environments for which there are limited data, let alone terrestrial extreme environments, which are rarely characterized by a single extreme. A further question important question is whether the limits to life on the Earth represent a universal boundary set by fundamental biophysical constraints or whether life’s limits are an idiosyncrasy of terrestrial life, reflecting bicohemical limitations locked into terrestrial life in its early evolution. If the tape of evolution was rerun, would the limits to life be different? It seems unlikely. Astrobiologists often focus on physical extremes to life, but energetic requirements might limit the potential for life elsewhere. On Mars, for instance, conditions for chemolithotrophs are likely to be electron acceptor limited and for anaerobic respiring organisms, electron donor limited. The search for extraterrestrial redox couples is an essential task in examining extraterrestrial environments. It is in these investigations that planetary sciences and microbiology converge. Although extraterrestrial environments might harbour habitable environments, we still do not know what factors are required for an origin of life. There is a possibility that the Universe contains many uninhabited or vacant habitats – environments where conditions are suitable for life, but there no life has emerged to take advantage of them. In our own Solar System, environments on Mars and the water bodies of moons such as the Saturnian satellite, Enceladus, might be potential examples of uninhabited habitats.

References · Cockell CS. 2011. Vacant habitats in the Universe. Trends Ecol. Evol. 26, 73-80. · Nixon S, Cockell CS, Tranter M. 2012. Limitations to a biological iron cycle on Mars. Planet. Space Sci. (in press).

abstracts book 67 S1.2EXTREMOphiles_2012 Subsurface geomicrobiology of the Iberian Pyrite Belt R. Amilsa,b, D. Fernández-Remolara, V. Parroa, J.A. Manfredia, K. Timmisc, M. Oggerina, M. Sánchez- Romána, F.J. Lópeza, J.P. Fernándeza, E. Omoregiea, D. Gómez-Ortizd, C. Brionesa, F. Gómeza, M. Garcíaa, N. Rodrígueza, J.L. Sanze and the IPBSL Team

aCentro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Spain bCentro de Biología Molecular Severo Ochoa (CSIC-INTA), Cantoblanco, Spain cTechnical University of Braunschweig, Germany dUniversidad Rey Juan Carlos, Madrid, Spain eDepartamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Spain E-mail: [email protected]

The recent geomicrobiological characterization of Río Tinto, Iberian Pyrite Belt (IPB), has proven the importance of the iron cycle, not only in generating the extreme conditions of the habitat (low pH, high concentration of toxic heavy metals) but also in maintaining the high level of microbial diversity, both prokaryotic and eukaryotic, detected in the water column and the sediments. The extreme conditions of the Tinto basin are not the product of industrial contamination but the consequence of the presence of an underground bioreactor that obtains its energy from the massive sulfide minerals of the IPB. To test this hypothesis, a drilling project was carried out to intersect ground waters that interact with the mineral ore in order to provide evidence of subsurface microbial activities and the potential resources to support them. The Iberian Pyrite Belt Subsurface Life (IPBSL) is a drilling project specifically designed to characterize the subsurface ecosystems operating in the IPB. A dedicated geophysical characterization of the area selected two drilling sites due to the possible existence of water with high ionic content. Two wells have been drilled in the selected area (Peña de Hierro), BH11 and BH10, with depths of 340 and 630 meters respectively, with recovery of cores and generation of samples in anaerobic and sterile conditions. Preliminary results showed an important alteration of mineral structures associated with the presence of water, with production of expected products from the bacterial oxidation of pyrite (sulfates and soluble iron). Ion chromatography of water soluble compounds from uncontaminated samples showed the existence of putative electron donors (ferrous iron, nitrite, hydrogen and methane, in addition of the metal sulfides), electron acceptors (sulfate, nitrate, ferric iron) as well as variable concentration of metabolic organic acids (mainly acetate, formate, propionate and oxalate), which are strong signals of the presence of an active subsurface ecosystem associated to the high sulfidic mineral content of the IPB. The system is driven by oxidants that appear to be provided by the rock matrix, only groundwater is needed to launch microbial metabolism. The geological, geomicrobiological and molecular biology analysis which are under way, should allow the characterization of this ecosystem of astrobiological interest which is not dependent on radiation.

68 abstracts book EXTREMOphiles_2012S1.3 Atacama Desert–a Mars analog inhabited by polyextremophiles Fred A. Rainey

Department of Biological Sciences, University of Alaska Anchorage, Anchorage, AK 99508, USA E-mail: [email protected]

A number of reports have characterized the Atacama region of Chile as a Mars analog that contains highly desiccated Mars-like soils with low concentrations of organic material, extremely low numbers of culturable microorganisms, and high concentrations of nitrate and perchlorate. The microorganisms that have been isolated from or detected in these soils can be considered polyextremophiles based on their ability to survive under a combination of extreme conditions. Bacterial isolation studies on a large number of surface and subsurface soil samples from the hyper-arid region of the Atacama have yielded a collection of bacterial strains that are somewhat limited in diversity with the majority of them belonging to the Actinobacteria lineage. Many Atacama soils yield only one or two colonies from dilution plating experiments. We tested the survival of Atacama isolates as well as control organisms in desiccated conditions, their ability to tolerate and grow in the presence of high concentrations of perchlorates and their resistance to UV and gamma radiation. The study demonstrates the ability of bacterial isolates obtained from the Mars-like soils of the Atacama hyper-arid region to survive under polyextreme conditions and grow in the presence of high concentrations of perchlorate. These strains represent a resource for further studies on the mechanism of tolerance to extreme conditions and to high levels of perchlorates by environmental isolates of certain Actinobacteria genera. The study could have important implications for possible microbial activity on transient cold aqueous environments in Mars polar regions.

abstracts book 69 S1.4EXTREMOphiles_2012 Evolutionary relationships between membrane vesicles, plasmids and viruses Marie Gaudina, Evelyne Margueta, Mart Krupovica,b, Jacques Obertoa and Patrick Forterrea,b

aInstitut de Génétique et de Microbiologie-CNRS UMR 8621-bât.409 bUniversité Paris-Sud, France, Institut Pasteur, 25 rue du Docteur Roux, 75015, Paris, France E-mail: [email protected]

Production of membrane vesicles (MVs) is ubiquitous in Bacteria, Eukarya (exosomes, ectosomes) and Archaea (Soler et al., 2008; Ellen et al., 2009), suggesting that MVs production is an ancient conserved biological feature. We are studying MVs in Thermococcus species [Soler et al., 2008; Gaudin et al., 2012). Electron microscopy and biochemical analyses have shown that these archaeal MVs are produced by budding from the membrane, as in the case of eukaryotic ectosomes. These MVs harbor DNA and protect DNA against thermodegradation (Soler et al., 2008). T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harboring this plasmid. Notably, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature (Gaudinet al., 2012). MVs produced by Thermococcus nautilus 30-1 only harbor two of the three plasmids present in that strain, pTN1 and pTN3. These two plasmids share with pLC70 genes encoding a putative mobility protein that could be used to package plasmids inside MVs. Sequencing of pTN3 revealed that this plasmid encodes for a capsid protein and a packaging ATPase typical of the PRD1-adenovirus lineage. This shows that MVs can be used to transfer viral genomes. I will discuss this intriguing evolutionary connection between MVs and viruses, also observed in eucaryotes. The ubiquity of MVs suggests that LUCA and possibly more ancestral cells already produced MVs. One can speculate that some viral lineages originated when ribocells producing MVs were transformed into virocells producing virions.

References · Soler et al. 2008. Res. Microbiol. 159: 390-399. · Ellen et al.2009. Extremophiles 13: 67-79. · Gaudin et al. 2012. Environ Microbiol. Report, in press.

70 abstracts book EXTREMOphiles_2012S2.1 Genetic tools and their optimization for the crenarchaeote Sulfolobus acidocaldarius Michaela Wagner, Alexander Wagner and Sonja-Verena Albers

Molecular Biology of Archaea, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany E-mail: [email protected]

Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon which grows optimally at 76°C and pH 3. We constructed a pyrE deletion mutant ofS. acidocaldarius wild type strain DSM 639 missing 322 bp called MW001. Using this strain we were able to obtain using single as well as double crossover events deletion mutants, tag genes genomically and ectopically integrate foreign DNA into MW001. These methods enable us to construct single, double, and triple deletions strains that can still be complemented with the pRN1 based expression vector. Moreover, we have optimized the expression vector by screening different mutations in the maltose inducible maltose binding promoter psac1161 that is employed in the expression plasmid pCMal (Berkner et al., 2010). Furthermore, we have identified the maltose regulatory protein MRP that is an activator of the psac1161 promoter. Coexpression of MRP from the expression vector with the gene of interest increased the expression levels 5 fold. Taken together we have developed a versatile and robust genetic tool box for the crenarchaeoteS. acidocaldarius that will promote the study of unknown gene functions in this organism and makes it a suitable host for synthetic biology approaches (Wagner et al., 2012).

References · Wagner, M., van Wolferen, M., Wagner, A., Lassak, K., Meyer, B., Reimann, J., and S.V. Albers (2012) Versatile genetic tool box for the crenarchaeoteSulfolobus acidocaldarius. Front. Microbiol. 3: 214. doi: 10.3389/ fmicb.2012.00214. · Berkner, S., Wlodkowski, A., Albers, S.V. and G. Lipps (2010) Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius. Extremophiles 14: 249-259.

abstracts book 71 S2.2EXTREMOphiles_2012 The genetic toolbox for Thermus thermophilus José Berenguer, Laura Alvarez, Carlos Bricio, Carolina E. César and Aurelio Hidalgo

Centre of Molecular Biology Severo Ochoa (UAM-CSIC), Faculty of Sciences, Autonomous University of Madrid, 28049 Madrid, Spain E-mail: [email protected]

A great diversity of Thermus spp. strains exists, some of which having a very active natural competence system. Among them the strains HB27 and HB8 of T. thermophilus have been widely used as laboratory models because of the easy by which they can accept external DNA and grow at great rates under laboratory conditions (Cava et al., 2009). These properties have allowed the development of a complete genetic toolbox functional in the range 60 to 70ºC that includes: (i) selectable genes, (ii) cloning and expression plasmids, (iii) reporter enzymes, and (iv) fluorescent localization markers. Selectable genes that complement auxotrophies have been used by different groups for the co-selection of accompanying genes, and uridine biosynthesis genes are among the most useful tools for insertion-deletion strategies because of the possibility of negative selection in presence of substrate homologue 5´fluoro-orotic acid. Antibiotic thermostable resistances to kanamycin, bleomycin and hygromycin B are also available and have been used for directed knockout of genes and plasmid development. Homologous mutant rpsL genes providing dependence to streptomycin has been also developed for insertion-deletion methods. Plasmids for the cloning of genes and for the construction of genetic libraries have been constructed, as also derivatives in which the cloned genes can be expressed from regulated promoters, allowing the controlled expression of enzymes that otherwise are not expressed in active forms in more conventional systems. Folding vectors also exists that allow the selection of thermostable variants of enzymes and proteins from mesophiles. Promoter probe and protein localization vectors are available that are based on the use of thermostable reporter enzymes such as α or β glycoxylases or in thermostable variants of fluorescent proteins. An overview of these tools and examples of the use of the latest developments on this field will be presented.

References · Cava et al. (2009). Thermus thermophilus as biological model. Extremophiles 13: 213-231.

72 abstracts book EXTREMOphiles_2012S2.3 One stator that couples to two different ions: flagellar stator and motility of alkaliphilic Bacillus species Masahiro Itoa,b, Naoya Teraharac and Motohiko Sanoa

aGraduate School of Life Sciences, Toyo University, Oura-gun, Gunma 374-0193, Japan bBio-nano Electronics Research Centre, Toyo University, Kawagoe, Saitama, 350-8585, Japan cGraduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]

The bacterial flagellum acts as the propeller for cell locomotion in a variety of environments. The flagellar motor consisting of the rotor and the stator rotates the flagellar filament. All the bacterial flagellar motors characterized so far are energized by either transmembrane electrochemical gradients of protons or sodium ions that are coupled to motility via membrane embedded stator complexes. Some bacteria possess stator complexes of only one ion-coupling type, either protons or sodium, while others have two flagellar systems that have different ion-coupling properties. Use of sodium ions is associated with elevated pH and a high salt environment. Among the reported stators, MotAB-type stators use protons while MotPS- and PomAB-type stators use Na+ as coupling ions. Previously, our group reported that the flagellar motor of Bacillus subtilis is a hybrid motor consisting of both MotAB and MotPS (Ito et al., 2005), and that of Bacillus clausii is a dual-specificity motor consisting of MotAB that has selectivity for both H+ and Na+ (Terahara et al., 2008). It is thought that the flagellar stator ofBacillus species evolved to adjust to the environment. Here, we systematically amplified the uncharacterized flagellar stator genes from each genomic DNA of nineteen kinds of alkaliphilic Bacillus species by using PCR with primers of conserved sequences among motAB and motPS genes of Bacillus species, and then sequenced each PCR product. We also tested swimming behaviour of each bacterium under several conditions. These results suggested that the stators of alkaliphilicBacillus species have been diversely evolved. Among them, we identified a novel type flagellar motor which stimulates swimming speed under elevated+ K or Rb+ concentrations. The stator protein of this bacterium is identified as a MotPS-type. In this organism,+ Na , K+ and Rb+ can be used as coupling ions for flagellar rotation. This is the first report that describes a flagellar motor that can use both K+ and Rb+ as coupling ions.

References · M. Ito, N. Terahara, S. Fujinami and T.A. Krulwich (2005). Properties of motility in Bacillus subtilispowered by the H+-coupled MotAB flagellar stator, Na+-coupled MotPS or hybrid stators MotAS or MotPB. J Mol Biol 352: 396-408. · N. Terahara, T. A. Krulwich and M. Ito (2008) Mutations alter the sodium versus proton use of aBacillus clausii flagellar motor and confer dual ion use on Bacillus subtilis motors. Proc Natl Acad Sci USA 105: 14359-14364.

abstracts book 73 S2.4EXTREMOphiles_2012 Virus-host relationships in hyperthermophiles David Prangishvili

Institut Pasteur, Department of Microbiology, Molecular Biology of the Gene in Extremophiles Unit, 25 rue du Doctour Roux, 75015 Paris, France E-mail: [email protected]

One of most remarkable, albeit not fully recognized features of the biodiversity in extreme geothermal environments is the exceptional nature of DNA viruses infecting archaeal hosts thriving at temperatures above 80 °C. About three dozen of such viruses isolated and characterized to date form a singular group in the viral world, not related to any other viruses (Pina et al., 2011). They reveal diverse morphologies, never observed among DNA viruses of Bacteria or Eukarya, and carry genomes with the considerable majority of genes without detectable homologs in other viruses or cellular life forms. Moreover, recent studies reveal unique features of interaction of these viruses with host cells, which I will illustrate on the example of replication, transcription and egress mechanisms of filamentous viruses from the familiesRudiviridae and Lipothrixviridae.

Reference Pina M, Bize A, Forterre P, Prangishvili D. 2011. The Archeoviruses. FEMS Microbiol. Rev. 35: 1035-1054.

74 abstracts book EXTREMOphiles_2012S3.1 Understanding complexity and processes in Antarctic niche habitats Don Cowana,b, Thulani Makhalanyaneb and Marla Tuffinb

aDepartment of Genetics, University of Pretoria, Pretoria 0002, South Africa bInstitute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Cape Town 7353, South Africa E-mail: [email protected]

Antarctic Dry Valley desert soils are characterised by a range of environmental extremes, dominated by very low temperatures and extreme desiccation. Contrary to expectations, metagenomic surveys have revealed wide bacterial phylotypic diversity in surface soils, sub-surface horizons and in specialised crypto-lithic habitats. Hypolithons, photosynthetic communities found under translucent rocks in desert pavements, are a significant source of biomass and biocomplexity in such hyperarid environments (Cary et al., 2010). These isolated communities are clearly separable into three morphotypes (Cowan et al., 2010), each of which show clearly separate bacterial phylotypic ‘fingerprints’ (Khan et al., 2011). However, deep sequencing of 16S rRNA gene amplicon sets suggests that while the different hypolithon classes recruit specific dominant signature species from the background ‘soil’ community, most phylotypes are ubiquitous. The contributions of Antarctic soil microorganisms to biogeochemical cycling remain poorly understood. Terminal Restriction Fragment Length Polymorphism (T-RFLP) and barcoded amplicon pyrosequencing were used to assess the functional guilds implicated in C and N cycling organisms in both hypolithic and open soil samples. cbbL gene primers, targeting both green- and red-like ribulose 1,5-bisphosphate carboxylase (RubisCO) genes, were chosen as a functional marker for carbon fixation. We found a wide range cbbLof -positive phylotypes (i.e., with the genetic capacity for photosynthesis. T-RFLP profiles showed significant differences between red-like and green-like cbbL-distributions between different soil samples. Genes implicated in each of the three principal phases of nitrogen cycling, nitrogen fixation (nifH), de- nitrification (nirK, nirS, narG) and nitrification (amoA), were also examined. Multivariate analysis (PERMANOVA) of T-RFs showed that hypolithic and soil communities were structurally distinct. Sequence analysis suggested that phylotypes assigned to the Actinobacteria, cyanobacteria and á- and â-Proteobacteria were all implicated in nitrogen cycling. The distribution of genes for the various N-cycling and the absence of detectable genes in key denitrification processes together suggest stringent nitrogen conservation in these oligotrophic Antarctic soil habitats.

References: · Cary, SC, McDonald, I, Barrett, JE and Cowan, DA. (2010) On the rocks: Microbial ecology of Antarctic cold desert soils. Nature Rev. Microbiol. 8: 129-138. · Cowan, DA, Khan, N, Pointing, S, Cary, SC. (2010) Diverse hypolithic refuge communities in Antarctic Dry Valleys, Antarct. Sci. 22: 714-720. · Khan, N, Tuffin, IM, Stafford, W, Cary, SC, Lacap, DC, Pointing, SB, Cowan, DA (2011) Hypolithic microbial community colonization of quartz rocks from Miers Valley, McMurdo Dry Valleys, Antarctica. Polar Biol. 34: 1657-1668.

abstracts book 75 S3.2EXTREMOphiles_2012 Assembly-driven metagenomics of a hypersaline microbial ecosystem Sheila Podella, Juan A. Ugaldea, Priya Narasingaraoa, Jillian F. Banfieldb, Karla B. Heidelbergc and Eric E. Allena,d

aMarine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA bDepartment of Earth and Planetary Sciences, University of California, Berkeley, Berkeley, CA USA cDepartment of Biological Sciences, University of Southern California, Los Angeles, CA USA dDivision of Biological Sciences, University of California, San Diego, La Jolla, CA USA E-mail: [email protected]

Microbial populations inhabiting a natural hypersaline lake ecosystem, Lake Tyrrell, Victoria, Australia, have been characterized using deep metagenomic sampling, iterative de novo sequence assembly, and multidimensional phylogenetic binning. Consensus genomes were reconstructed for twelve environmental microbial populations, eleven archaeal and one bacterial, comprising between 0.6-14.1% of the planktonic community. Eight of the twelve genomes recovered represent microbial populations not characterized previously in laboratory culture. The extent of genome assembly achieved has allowed extensive lineage-specific compartmentalization of predicted metagenomic functional capabilities and cellular properties associated with both dominant and less abundant members of the community. Among the sampled populations, two representative genomes belonging to a new major lineage of Archaea, Class Nanohaloarchaea, were obtained (Narasingarao et al., 2012). Phylogenomic analysis and metabolic characteristics of the Nanohaloarchaea support their distinct taxonomic placement relative to previously described halophilic species of Class Halobacteria. Reanalysis of genetic datasets from globally distributed hypersaline habitats reveals the Nanohaloarchaea are in high abundance and broadly distributed worldwide. The generation of habitat-specific, environmental reference genomes based on metagenomic sequence assembly has provided rich opportunities for discovery of both the overall genetic composition and community architecture of multiple microbial groups constituting a native microbial ecosystem. The degree of connectivity between microbial populations based on metabolic network analysis reveals exquisite functional redundancy in hypersaline microbial communities. This constrained metabolic repertoire may reflect limitations of adaptation to extreme salinity and is consistent with the hypothesis that temporal variability in community membership may be dominated by top-down forcing dynamics (e.g. grazing and viral predation) rather than nutrient availability and assimilation.

References · Narasingaro P et al. (2012). De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities.ISME J 6:81-93.

76 abstracts book EXTREMOphiles_2012S3.3 Genomic diversity of a Haloquadratum walsbyi population in a single saltern-pond a a b a Francisco Rodriguez-Valera , Ana-Belen Martin-Cuadrado , Lejla Pasic and Rohit Ghai

aDepartment of Microbiology, Universidad Miguel Hernandez, 03550 Alicante, Spain bDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia E-mail: [email protected]

Haloquadratum walsbyi is a square haloarchaeon that dominates neutral saturated brines around the world. For many years this microbe resisted isolation till finally in 2004 two isolates, HBSQ001 (from crystallizer CR30 in Spain) and C23 (from Australia) were obtained in pure culture. The genome of HBSQ001 was published in 2006 and that of C23 last year. We have sequenced metagenomes from the same location taken some years later. We have accumulated Gbps of sequence from environmental clones from this microbe throughout the years. The analysis of so much sequence from the same microbe both from pure cultures and (even more important) from its natural habitat has shed light over one of the toughest conundrums faced by Microbiology-the genomic diversity of prokaryotic species. The data that we have found indicates that several clonal lineages coexist in the crystallizer, probably within the range of 100 but certainly not less than 10. These lineages share high similarity over a large part of their genomes, the core, but differ enormously in some gene complements, particularly the genes involved in the synthesis of the cell wall. First of all we have been able to identify two types of cell walls in this very restricted taxon (similarity of the core>98% ANI). The Australian isolate and some Alicante clones have a simplified version with only two major genes one of them coding for the major cell surface glycoprotein (MCSG) of the S-layer. The other contains homologous to these two genes but is more complex and appears to have doubled in size. From a metagenomic fosmid library from the same saltern-pond from where the first isolate was obtained we have sequenced 6 fosmids that cover the genomic island where is the MCSG is found. Some of these lineages have been lost since some of their genes cannot be recovered from the environment regardless of the coverage. The variability found of cell surface glycoproteins in each of the fosmids probably reflects the pressure to which this kind of proteins are as they supposed one the recognized targets of phages (Rodriguez-Valera et al., 2009), ten times more abundant in this habitat than in any other described so far.

References · F. Rodriguez-Valera, A-B Martin-Cuadrado, B. Rodriguez-Brito, L. Pašic, T. F. Thingstad, F. Rohwer, A. Mira. Explaining microbial population genomics through phage predation Nat Rev Microbiol 7: 828-835 (2009).

abstracts book 77 S3.4EXTREMOphiles_2012 Biased gene flow in haloarchaea: can it account for the appearance and maintenance of phylogenetic clusters? R. Thane Papke

Department of Molecular and Cell Biology, University of Connecticut, Storrs, USA E-mail: [email protected]

How prokaryote diversity is distributed among individuals, how it got there, and how evolutionary forces produce phylogenetic clusters, are interconnected questions, and their answers and interpretations represent contentious issues in prokaryote evolution and taxonomy. It has been thought that prokaryotes in the environment are largely clonal and subject to diversity purging genome-wide selective sweeps upon acquisition of adaptive traits. This hypothesis can account for the maintenance and diversification of species, genera, etc. However, the assumption of population clonality in nature has not always held up to scrutiny. Observation of natural prokaryotic populations has shown genetically they can appear more like randomly mating sexually reproducing species, despite their asexual reproductive mechanism. In these populations, homogenization of species traits is therefore probably the result of frequent intraspecies recombination rather than selective sweeps. Additionally, because barriers to genetic transfer can be very leaky, interspecies exchange acts as a diversifying force that obscures and baffles our understanding of Darwinian descent from a common ancestor as a model for comprehending how prokaryotic species, genera, etc., formed and are related: e.g., alleles/ genes are ‘free agents’ unconfined to a single lineage. How then is it possible for clusters to be generated by a single mechanism displaying opposing forces? Through the utilization of multilocus sequence analysis (MLSA) and whole genome comparisons, we were able to analyze and investigate thoroughly a class of Archaea, Halobacteria (also called haloarchaea) as a model group of organisms sharing a common requirement for extreme concentrations of NaCl. These analyses ultimately provide insight towards comprehending how gene exchange can generate clusters and give the appearance of species, genera, etc. Evidence will be presented in support for the hypothesis that biased gene exchange can mimic the effect of common ancestry in the creation and maintenance of phylogenetic clusters.

78 abstracts book EXTREMOphiles_2012S4.1 Unravelling protein adaptation to high salt concentration using resurrected ancestral-halophilic archaeal enzymes Romain Talona, Eric Girarda, Bruno Franzettia, Samuel Blanquardb, Mathieu Groussinc, Manolo Gouyc, Roxane Lestinid, Hannu Myllykalliod and Dominique Maderna

aExtremophilic and Large Molecular Assemblies Team, Institut of Structural Biology (IBS) 41 Rue Jules Horowitz, 38042 Grenoble Cedex, France bTeam Bonsai, INRIA Lille Nord Europe, UMR 8022, 40 avenue Halley, 59650 Villeneuve d’Ascq, France cLBBE «Biométrie et Biologie Évolutive», UMR CNRS 5558, UCB Lyon 1-Bât. Grégor Mendel, 43 bd du 11 novembre 1918, 69622 Villeurbanne , France dLaboratoire d’Optique et Biosciences (LOB), Ecole Polytechnique, CNRS UMR7645- INSERM U696, Route de Saclay, 91 128 Palaiseau Cedex, France E-mail: [email protected]

At any time in the cytoplasm, a given protein exists as a population of conformers, each(i) differing slightly from others by its structure, (ii) being characterized by a given free energy, (iii) and being statistically represented in a specific proportion. Environmental parameters that modify the thermodynamic equilibrium of this population will therefore impact protein properties. Shape, energy and proportion of conformers are primarily determined at the level of the amino-acid sequence, upon which acts the mutation process. Each mutation accumulated during the adaptive pathway of protein facing extreme physico chemical conditions will impacts the protein energy landscape, the rates of inter-conversion between conformers, and the population structure. Determining the evolvability potential of each amino acid replacement, or in other words, determining the magnitude of its impact on the protein conformational landscape, is therefore a key point to clearly understand the adaptive history of proteins. In the presentation, I will present biochemical and crystallographic data of several malate dehydrogenase (MalDH) from extremely halophilic enzymes together with their non halophilic counterpart. These enzymes have a high level of sequence identity, but intriguingly, they display very different properties in term of stability and activity depending on the salt concentration. To understand with accuracy the history of substitutions that has driven their change in properties, we have reconstructed the evolutionary pathway of these enzymes in the Archaea Domain. I will show preliminary results, including the resurrection of the halophilic malate dehydrogenase ancestor using public available sequence data from halophilic and halotolerant archaeal species and homogeneous methods to infer ancestral sequences. I will discuss the requirement of more robust algorithms and the use of phylogenomic data that will fully account for the artefacts produced by the frequent lateral gene transfer during reconstruction of ancestral sequences when large taxonomic data set are analysed. According to our findings, the various trades-offs between biophysical biochemical and dynamical parameters that took place during halophilic adaptation of protein will be discussed.

abstracts book 79 S4.2EXTREMOphiles_2012 From non-homogeneous evolutionary models to molecular thermometers Manolo Gouy, Mathieu Groussin and Bastien Boussau

Laboratoire de Biométrie et Biologie Evolutive, Université Lyon 1, 69622 Villeurbanne, France E-mail: [email protected]

It was recently recognized that the effects of environmental temperature on ancestral organisms left genetic footprints that could be uncovered in extant genomes. These effects allow to define «molecular thermometers» that relate ancestral environmental temperatures to the composition of ancestral molecules in nucleotides and amino acids. The application of molecular thermometers is therefore determined by the accuracy of the reconstruction of ancestral molecular compositions. Recent progress in the definition of probabilistic models of the evolutionary process have improved the biological realism of these models by accounting for the variation of patterns of molecular evolution among lineages. These new non-homogeneous methods allow to reconstruct ancestral molecular compositions more accurately than traditional homogeneous methods. Analyses of genomic data using these tools allow to attempt to reconstruct the evolutionary history of the adaptation to environmental temperatures at the scale of the tree of life.

References · M. Groussin and M. Gouy (2011) Adaptation to environmental temperature is a major determinant of molecular evolutionary rates in archaea.Mol. Biol. Evol. 28: 2661–2674. · B. Boussau, S. Blanquart, A. Necsulea, N. Lartillot and M. Gouy (2008) Parallel adaptations to high temperatures in the archean eon. Nature 456: 942-945.

80 abstracts book EXTREMOphiles_2012S4.3 Function and evolution of proteins encoded by interrupted genes in Archaea Beatrice Cobucci-Ponzano, Mosè Rossi and Marco Moracci

Institute of Protein Biochemistry – CNR, Via P. Castellino 111, 80131 Naples, Italy E-mail: [email protected]

In translational recoding, specific genes are expressed by following rules different from those of the standard translational decoding leading to polypeptides that do not match the primary sequence deduced from the corresponding genes. This process, including programmed -/+1 frameshifting, ribosome hopping, and codon reassignment, has been identified in all living domains (Dinman, 2012). In Archaea, since recently, the only known recoding events were termination codon readthrough leading to the decoding of the 21st and 22nd amino acids selenocysteine and pyrrolysine, respectively (Cobucci-Ponzanoet al. 2005), however, we also demonstrated that the gene fucA1, encoding for a alpha-L-fucosidase from the archaeon Sulfolobus solfataricus, was expressed by programmed -1 frameshifting. In fact, its ORFs SSO11867 and SSO3060 led to synthesis, both in vitro and in vivo, of a fully functional polypeptide of 495 amino acids (Cobucci-Ponzanoet al. 2003), In addition, more recently, we demonstrated by functional highthroughput proteomic approaches, that disrupted genes leading to functional products can be frequently find in archaeal genomes and possible novel examples of recoding events have been proposed (Cobucci-Ponzano et al. 2010), These results will be described here and discussed with particular focus on the functional meaning of interrupted genes and their mechanisms of expression. The relevance of disrupted genes and translational recoding in the evolution of Archaea and the origin of the genetic code will be also presented. This work was supported by the project MoMa n. 1/014/06/0 of the Agenzia Spaziale Italiana.

References · Dinman JD. (2012) Control of gene expression by translational recoding. Adv Protein Chem Struct Biol. 86:129-49. · Cobucci-Ponzano B. et al. (2005) Recoding in archaea. Mol Microbiol. 2: 339-348. · Cobucci-Ponzano B. et al. (2003) Identification of an archaeal alpha-L-fucosidase encoded by an interrupted gene. Production of a functional enzyme by mutations mimicking programmed -1 frameshifting. J Biol Chem. 278: 14622-14631. · Cobucci-Ponzano, B. et al. (2010) Functional characterization and high-throughput proteomic analysis of interrupted genes in the archaeon Sulfolobus solfataricus. J Proteome Res. 9: 2496-2507.

abstracts book 81 S4.4EXTREMOphiles_2012 Hyperstability in resurrected Precambrian proteins Jose M. Sanchez-Ruiz

Department of Physical Chemistry, Faculty of Sciences, University of Granada, 18071 Granada, Spain E-mail: [email protected]

About 50 years ago, Linus Pauling and Emile Zuckerkandl (Pauling, 1963) pointed out the possibility of reconstructing the sequences of ancestral proteins from the comparative analysis of related sequences in living organisms. The field of Paleobiochemistry they imagined came to life in the mid-nineties thanks to the increasing number of known sequences, the development of reliable statistical procedures for sequence reconstruction and advances in genetic engineering that allow ancestral proteins to be «resurrected» and studiedin vitro. Ancestral resurrection studies have led to a wealth of information about the physical environment surrounding extinct organisms and how their proteins (and the organisms themselves) adapted to changes in that environment. Furthermore, recent work indicates that that resurrected proteins may show much enhanced stability-related and function-related properties (Gaucheret al., 2008; Perez-Jimenez et al., 2011). The biotechnological relevance of this result can be easily realized by considering that protein engineering usually involves modifications in amino acid sequence that bring about changes in protein properties. In mathematical terms, engineering of a given protein can be viewed as the movement of a point representing the molecule in a space of properties versus sequence. Single mutations or analysis of variant libraries achieve small changes in sequence and, consequently, lead typically to modest modulations in properties (the point moves a short distance in properties/sequence space). On the other hand, ancestral protein resurrection (in particular when resurrecting proteins from the Precambrian: about 4500-500 million years ago) involves large changes in sequence and may bring about huge modulations in protein properties (the point moves a large distance in properties/sequence space). Ancestral resurrection (in particular, Precambrian ancestral resurrection) may therefore become one of the main sources of the next generation of proteins for biotechnological and biomedical applications.

References · Pauling, Zuckerkand (1963) Acta Chem Scan 17:S9-S16. · Gaucher, Govindarajan, Ganesh (2008) Nature 451:704-707. · Perez-Jimenez, Ingles-Prieto, Zhao, Sanchez-Romero, Alegre-Cebollada, Kosuri, Garcia-Manyes, Kappock, Tanokura, Holmgren, Sanchez-Ruiz, Gaucher, Fernandez (2011) Nat Struct Mol Biol 18: 592-596.

82 abstracts book EXTREMOphiles_2012S5.1 Microbial community development in and beneath deep-sea hydrothermal vents in the Earth, and probably in the Enceladus Ken Takai

Subsurface Geobiology Advanced Research (SUGAR) project, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Japan E-mail: [email protected]

Over the past 35 years, researchers have explored seafloor and subseafloor deep-sea hydrothermal vent environments around the globe and studied a number of microorganisms, their unique metabolic and physiological properties, and their vast phylogenetic diversity. Bioinformatics and interdisciplinary geochemistry- microbiology approaches are providing new information on the diversity and community composition of archaea living in deep-sea vents. In particular, recent investigations have revealed that the community structure and productivity of chemolithotrophic microbial communities in the deep-sea hydrothermal environments are controlled primarily by variations in the geochemical composition of hydrothermal fluids. This was originally predicted by a thermodynamic calculation of energy yield potential of various chemolithotrophic metabolisms in a simulated hydrothermal mixing zone (McCollom and Shock, 1997) and has been called as McCollom and Shock’s prediction. The prediction has been finally justified by the relatively quantitative geomicrobiological characterizations in various deep-sea hydrothermal vent environments all over the world. Thus, there should be a possible principle that the thermodynamic estimation of chemolithotrophic energy yield potentials could predict the realistic chemolithotrophic living community in any of the deep-sea hydrothermal vent environments. In 2005, a spacecraft Cassini discovered a water vapour jet plume from the sole pole area of the Saturnian moon Enceladus (Hansen et al., 2008). The chemical composition analyses of Cassini’s mass spectrometer strongly suggested that the Enceladus could host certain extent of extraterrestrial ocean beneath the surface ice sheet and possible ocean-rock hydrothermal systems. If McCollom and Shock’s prediction is a rule for the microbial communities in the oceanic hydrothermal systems of the Earth, it will be applied to the potential living communities in the extraterrestrial oceanic hydrothermal systems of the Enceladus. In this presentation, I would like also to introduce a Japanese Astrobiology Project: Capture of Extraterrestrial Life from Enceladus plumes by Hayabusa-flyby system.

References · T. M. McCollom and E. L. Shock (1997). Geochemical constraints on chemolithoautotrophic metabolism by microorganisms in seafloor hydrothermal systems.Geochim Cosmochim Acta 61: 4375-4391. · C.J. Hansen, L.W. Esposito, A.I. F. Stewart, B. Meinke, B. Wallis, J.E. Colwell, A.R. Hendrix, K. Larsen, W. Pryor & F. Tian (2008). Water vapour jets inside the plume of gas leaving Enceladus. Nature 456: 477-479.

abstracts book 83 S5.2EXTREMOphiles_2012 Extremophiles in Antarctica: insight into adaptation, evolution and ecosystem function of cold aquatic systems using metagenomics and metaproteomics Ricardo Cavicchioli, Federico M. Lauro, Matthew Z. DeMaere, Timothy J. Williams, Mark V. Brown, Sheree Yau and David Wilkins

School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia E-mail: [email protected]

Antarctica is arguably the world’s most important continent for influencing the Earth’s climate and global ocean ecosystem but the functions and dynamics of its microbiota are still poorly understood. Antarctica has also been shown to be extremely sensitive to the impact of human induced global warming. This is sobering given that Antarctica contains 90% of the ice on earth, and the Southern Ocean accounts for ~30% of the global ocean uptake of carbon dioxide. By exerting a major influence over the global ocean, Antarctic and Southern Ocean water masses affect the climate of all continents. The geographic distribution and diversity of types of lake systems across Antarctica is also considerable – the majority of which have not been studied.

In this presentation I will overview our published and unpublished findings that use metagenomics and metaproteomics to examine Antarctic lake and ocean systems: these include marine-derived Ace (meromictic), Organic (meromictic, hypersaline) and Deep (extremely hypersaline) lakes in the Vestfold Hills, glacier lagoons and pools on Heard Island, and ocean sites spanning long latitudinal transects from Australia to Antarctica. Our studies describe unanticipated and important roles for specific microbial components of the microbial loop, and highlight the apparent «peculiarities» of polar ecosystems – knowledge we have only been able to gain by using modern «omic» approaches to generate a new level of information about microbial communities, and linking that information to associated chemical, physical, geological and biological data to obtain an integrated view of the aquatic ecosystems. Our studies provide baselines for monitoring how environmental perturbations affect microbial dynamics over time.

References · Lauro FM, DeMaere MZ, Yau S et al. (2011) An integrative study of a meromictic lake ecosystem in Antarctica. ISME J 5: 879–895. · Ng C, DeMaere MZ, Williams TJ, et al. (2010) Metaproteogenomic analysis of a dominant green sulfur bacterium from Ace Lake, Antarctica.ISME J 4: 1002–1019. · Yau S, Lauro FM, DeMaere MZ et al. (2011) Virophage control of Antarctic algal host–virus dynamics.Proc Natl Acad Sci USA 108: 6163–6168. · Williams TJ, Long E, Evans F et al. (2012) A metaproteomic assessment of winter and summer bacterioplankton from Antarctic Peninsula coastal surface waters. ISME J doi: 10.1038/ismej.2012.28

84 abstracts book EXTREMOphiles_2012S5.3 The benefits of James Cameron’s Deepsea Challenge Expedition to Hadal Microbiology Douglas H. Bartlett

Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202, USA E-mail: [email protected]

During the winter and spring of 2012 the Deepsea Challenge Expedition conducted submersible and lander operations in the New Britain Trench (down to ~8.2 km depth) and the Mariana Trench (down to ~10.9 km depth). This expedition marked the first manned mission to the Challenger Deep in more than half a century. In addition to the extensive undersea filming and photography that was undertaken, sediment, seawater, and scavenging amphipod samples were also recovered. Many samples were processed for the cultivation of piezophiles (high pressure adapted microbes) and for culture-independent phylogenetic and genomic characterization.

During this presentation I will present an overview of the two legs of this cruise and describe the significance of the samples collected in the context of ongoing efforts to better understand microbial life in hadal settings. Prior and ongoing analyses of samples recovered from the deepest portion of the Atlantic Ocean (the Puerto Rico Trench [PRT] at ~ 8.5 km depth) will also be presented.

The deep-sea PRT Trench microbes have more in common at the species level with other deep-sea microbial communities in the Pacific Ocean and the Mediterranean Sea than with the microbial populations above them in shallow waters. They also harbor larger genomes with more COGs assigned to signal transduction, transcription, replication, recombination and repair and inorganic ion transport. The overrepresented transporters in the PRT metagenome include di- and tri-carboxylate transporters that correspond to the prevailing catabolic processes such as butanoate, glyoxylate and dicarboxylate metabolism. A surprisingly high abundance of sulfatases for the degradation of sulfated polysaccharides were also present in the PRT. But, perhaps the most dramatic adaptational feature of the PRT microbes is heavy metal resistance, as reflected in the high numbers of metal efflux systems present. Single-cell genomics approaches have proven particularly useful for placing PRT metagenomic data into context.

References · E. A. Eloe, D. W. Fadrosh, M. Novotny, L. Zeigler Allen, M. Kim, M. J. Lombardo, J. Yee-Greenbaum, S. Yooseph, E. E. Allen, R. Lasken, S. J. Williamson and D. H. Bartlett . (2011). Going deeper: metagenome of a hadopelagic microbial community. PLoS One 6:e20388. · E. A. Eloe, C. N. Shulse, D. W. Fadrosh, S. J. Williamson, E. E. Allen and D. H. Bartlett (2011). Compositional differences in particle-associated and free-living microbial assemblages from an extreme deep-ocean environment. Env. Microbiol. Rpts 3: 449-458 · E. A. Eloe, F. Malfatti, J. Gutierrez, K. Hardy, W. E. Schmidt, K. Pogliano, J. Pogliano, F. Azam, and D. H. Bartlett (2011). Isolation and characterization of a psychropiezophilic alphaproteobacterium. Appl Environ Microbiol. 77: 8145-8153.

abstracts book 85 S5.4EXTREMOphiles_2012 Radiation and desiccation resistance of hyperthermophilic microorganisms Petra Rettberga, Kristina Bebloa, Reinhard Rachelb, Reinhard Wirthb, Harald Huberb, Günther Reitza

aDepartment of Radiation Biology, Institute of Aerospace Medicine, German Aerospace Center (DLR), Germany bInstitute for Microbiology and Archaea Center, Faculty of Biology and Preclinical Medicine Natural Sciences III, University of Regensburg, Regensburg, Germany E-mail: [email protected]

In our study, we examined the desiccation tolerance of several strains of thermophilic and hyperthermophilic Archaea and thermophilic phylogenetically deep-branching Bacteria. Seventeen organisms showed a significant high ability to withstand desiccation. The desiccation tolerance turned out to be species-specific and was influenced by several parameters like storage temperature, pH, substrate or presence of oxygen. The distribution of desiccation tolerance in the phylogenetic tree of life is not domain specific. There are significant differences in desiccation tolerance among organisms from the same order and even from closely related strains of the same genus. Our results show that tolerance of vegetative cells to desiccation is a common phenomenon of thermophilic and hyperthermophilic microorganisms although they originated from quite different non- arid habitats like boiling acidic springs or black smoker chimneys. Based on the analysis of the radiation resistant mesophilic bacterium Deinococcus radiodurans, a correlation between desiccation tolerance and radiation resistance was supposed. So far, no systematic investigations with (hyper-) thermophilic Archaea and phylogenetically deep-branching thermophilic Bacteria have been performed. Therefore, we investigated the ability of several of these species to survive high fluences of monochromatic UV-C (254 nm) and high doses of ionizing radiation, respectively. Nine out of fourteen tested microorganisms showed a surprisingly high tolerance against ionizing radiation and two species (Aquifex pyrophilus, Ignicoccus hospitalis) were even able to survive 20 kGy. In contrast, there was nearly no difference in survival of the tested strains after exposure to UV-C under anoxic conditions of all tested organisms. By exposure to UV-C, photoproducts were formed in the DNA of irradiated Archaea and Bacteria. The distribution of the main photoproducts was species specific but the amount of the photoproducts was only partly dependent on the applied fluence. Overall, our results show, that tolerance to radiation seems to be a common phenomenon among thermophilic and hyperthermophilic microorganisms, but there is no general correlation between desiccation tolerance and radiation resistance.

References · Beblo K, Rabbow E, Rachel R, Huber H, Rettberg P (2009). Tolerance of thermophilic and hyperthermophilic microorganisms to desiccation. Extremophiles 13: 521-531. · Beblo K, Douki T, Schmalz G, Rachel R, Wirth R, Huber H, Reitz G, Rettberg P (2011). Survival of thermophilic and hyperthermophilic microorganisms after exposure to UV-C, ionizing radiation and desiccation.Arch Microbiol 193: 797-809.

86 abstracts book EXTREMOphiles_2012S6.1 One-carbon Metabolism in a Thermococcus strain and potential for hydrogen production Sung Gyun Kang, Hyun Sook Lee, Yun Jae Kim and Jung-Hyun Lee

Korea Ocean Research & Development Institute, Ansan, P.O. Box 29, Seoul 425-600, Korea E-mail: [email protected]

Thermococcus onnurineus NA1 was isolated from a deep-sea hydrothermal vent region to understand the adaptation mechanism of microorganisms in the highly variable hydrothermal vent environment. Based on its genome sequencing (Lee et al., 2008), genes were identified coding for a CO dehydrogenase (Codh), three formate dehydrogenases (Fdh) and proteins that function in the hydrolysis of starch, and multiple hydrogenases. The analysis of the genome suggested that T. onnurineus NA1 has the metabolic potential to grow with CO and/or formate as well as starch or proteinous sources as a substrate (Lim et al., 2010), followed by empirical demonstration (Kim et al. 2010).T. onnurineus NA1 is capable of producing hydrogen under anaerobic growth conditions supplemented with either CO, formate, or starch. Recently, the polyphasic approach by employing transcriptomic, proteomic and metabolomic tools allowed us to understand how the strain regulates the metabolism upon the presence of each substrate. The kinetic analysis of hydrogen production from CO, formate, or starch would be also discussed in comparison with those of other hydrogen-producing microorganisms.

References · H. S. Lee et al (2008). The complete genome sequence of Thermococcus onnurineus NA1 reveals a mixed heterotrophic and carboxydotrophic metabolism. J Bacteriol 190:7491-7499. · J. K. Lim et al (2010). Identification of a novel class of membrane-bound [NiFe]-hydrogenases in Thermococcus onnurineus NA1 by in-silico analysis. Appl Env Microbiol 76: 6286-6289. · Y. J. Kim (2010). Formate-driven growth coupled with hydrogen production. Nature 467: 352-355.

abstracts book 87 S6.2EXTREMOphiles_2012 Biosynthesis, evolution and role of unique compatible solutes in heat stress adaptation of marine hyperthermophiles Nuno Borges, Marta V. Rodrigues, Luís G. Gonçalves, Ana M. Esteves and Helena Santos

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Email: [email protected]

Many marine hyper/thermophiles accumulate compatible solutes not only in response to osmotic stress but also in response to heat stress. Most of these solutes have not been found or are rarely encountered in mesophiles (Santos et al., 2012). The best examples are di-myo-inositol-phosphate (DIP) and its derivatives (mannosyl-DIP and glycero-phospho-inositol). In general, the level of DIP increases remarkably at supra-optimal temperature, leading to the assumption that this solute plays a role in adaptation of hyper/thermophiles to heat stress. However, a definite proof of the physiological function of DIP is lacking. The biosynthesis of DIP involves the consecutive actions of inositol-1-phosphate cytidylyltransferase (IPCT) and di-myo-inositol-phosphate phosphate synthase (DIPPS). The synthesis of mannosyl-DIP was also established and comprises a single step reaction catalyzed by mannosyl-DIP synthase (Santoset al., 2011). Using a genomic approach, putative genes for these enzymes were identified and their assignments confirmed by functional expression in E. coli. Functional and structural properties of these enzymes will be presented. To obtain insight into the origin and evolution of DIP synthesis, a phylogenetic analysis of the DIP biosynthetic enzymes was performed (Gonçalves et al., 2012). In comparison with the 16S rRNA-based phylogenetic tree, significant topology differences were observed in the IPCT/DIPPS-based tree, suggesting the occurrence of multiple horizontal gene transfer events. To determine the physiological role of DIP, a Thermococcus kodakarensis mutant was constructed by deleting the gene encoding the key-enzyme implicated in DIP synthesis. Surprisingly, the DIP-deficient mutant and the parental strain exhibited the same growth rate under heat stress (Santos et al., 2011). Analysis of the solute pools revealed that the DIP-deficient strain substituted the missing DIP by aspartate, another negatively charged solute. Therefore, we conclude that DIP is part of the global strategy used by T. kodakarensis to cope with heat stress.

References · Santos et al. (2012) Organic compatible solutes of prokaryotes that thrive in hot environments: the importance of ionic compounds for thermostabilization. InExtremophiles Handbook, K. Horikoshi, G. Antranikian, A.T. Bull, F.T. Robb, K.O. Stetter (Eds), Springer, Tokyo, pp. 497-520. · Gonçalves et al. (2012) Evolution of the biosynthesis of di-myo-inositol phosphate, a marker of adaptation to hot marine environments. Environ Microbiol. 14: 691-701.

88 abstracts book EXTREMOphiles_2012S6.3 Microbial life under extreme energy limitation: how to make a living by producing acetate from carbon dioxide and hydrogen Volker Müller

Department of Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, University of Frankfurt, 60438 Frankfurt, Germany E-mail: [email protected]

Synthesis of acetate from carbon dioxide and molecular hydrogen via the Wood-Ljungdahl pathway is thermodynamically at the edge of life since it allows or the synthesis of only a fraction of an ATP. Recently, new insights have been obtained how this pathway is coupled to energy conservation.Acetobacterium woodii uses an ancient version of the pathway without cytochromes and quinones and couples the pathway to the generation of a transmembrane electrochemical sodium ion gradient that in turn drives ATP synthesis via a unique Na+

F1FO ATP synthase (Schmidt et al., 2009; Poehlein et al., 2012). None of the enzymes of the carbon flow are membrane-bound, but one enzyme of the electron transfer pathway, the ferredoxin:NAD+ oxidoreductase (Fno). Electron flow from reduced ferredoxin to NAD+ is coupled to Na+ export across the cytoplasmic membrane (Biegel & Müller 2010). The Fno is encoded by the rnf genes that code for a membrane-bound electron transfer complex comprising six subunits with FeS cluster and flavins as electron carriers (Biegelet al., 2011). Inspection of the genome sequence revealed that Rnf it is the only ion pump connect to the pathway. The Rnf complex is «fueled» with reduced ferredoxin and A. woodii has evolved a metabolism designed to overcome the endergonic ferredoxin with hydrogen as reductant reduction by soluble, electron bifurcating enzymes such as a hydrogenase (Schuchmann et al., submitted).

References · Schmidt S, Biegel E, Müller V (2009). The ins and outs of Na+ bioenergetics in Acetobacterium woodii. Biochim Biophys Acta 1787: 691-696. · Poehlein A, Schmidt S, Kaster AK, Goenrich M, Vollmers J, Thürmer A, Bertsch J, Schuchmann K, Voigt B, Hecker M, Daniel R, Thauer RK, Gottschalk G, Müller V (2012). An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis. PloS One 7: e33439. · Biegel E & Müller V (2010). Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase. Proc Natl Acad Sci USA 107: 18138-18142. · Biegel E, Schmidt S, González JM, & Müller V (2011). Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes. Cell Mol Life Sci 68: 613-634. · Schuchmann K & Müller V, submitted.

abstracts book 89 S6.4EXTREMOphiles_2012 Novel insights into sugar metabolism of extremophilic archaea Peter Schönheit

Institut für Allgemeine Mikrobiologie, University of Kiel, 24118 Kiel, Germany E-mail: [email protected]

Comparative analysis of sugar catabolism in archaea revealed several unusual pathways and enzymes not found in bacteria and eukarya. Here, recent data of sugar catabolism in hyperthermophilic and halophilic archaea are reported. 1. Most hyperthermophilic archaea degrade glucose via modified Embden-Meyerhof (EM) pathways, which differ from the classical EM pathway by a reduced potential of allosteric regulation. E.g. pyruvate kinases (PKs) do not respond to any known allosteric effectors of PKs from bacteria and eukarya, such as fructose-1,6- bisphosphate or AMP (Johnsen et al., 2003). We solved the crystal structure of PK from Pyrobaculum aerophilum and discovered a novel allosteric activator (Solomons et al., 2012). 2. The pathway of fructose degradation was analysed in the haloarchaeon Haloferax volcanii. It is shown that fructose uptake is mediated by a bacterial like PEP dependent phosphotransferase system (PTS), generating fructose-1-phosphate (F1P). Further degradation to triose phosphates involves F1P kinase and a bacterial like class II fructose-1,6-bisphosphate aldolase. This is the first report of the functional involvement of a bacterial type PTS system in sugar metabolism of archaea (Pickl et al. 2012). 3. The catabolism of D-xylose (Johnsen et al., 2009) and L-arabinose was studied in H. volcanii. Both pentoses were found to be oxidatively degraded toα- ketoglutarate as an intermediate. The initial oxidation is catalyzed by distinct D-xylose specific and L-ara- binose specific dehydrogenases, whereas subsequent conversion of D-xylonate and L-arabinoate to α-ketoglutarate are mediated by the same set of enzymes. These pentose degradation pathways in H. volcanii will be compared to the D-xylose and L-arabinose degradation pathways in Sulfolobus species (Nunn et al., 2010)

References · J. Solomons, U. Johnsen, P. Schönheit and C. Davies (2012). In preparation. · U. Johnsen, T. Hansen and P. Schönheit (2003). Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus, Aeropyrum pernix, Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima. JBC 278: 25417-25427. · A. Pickl, U. Johnsen and P. Schönheit (2012). Fructose degradation in the haloarchaeonHaloferax volcanii involves a bacterial type phosphoenolpyruvate-dependent phosphotransferase system, fructose-1-phosphate kinase, and class II fructose-1,6-bisphosphate aldolase. J Bacteriol 194: 3088-3097. · U. Johnsen, M. Dambeck, H. Zeiss, T. Fuhrer, J. Soppa, U. Sauer and P. Schönheit (2009). D-Xylose degradation pathway in the halophilic archaeon Haloferax volcanii. JBC 284: 27290-27303. · C. Nunn, C.E., Johnsen, U., Schönheit, P., Fuhrer, T., Sauer, U., Hough, D.W., Danson, M.J. (2010) Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus. JBC 285: 33701-33709.

90 abstracts book EXTREMOphiles_2012

oral parallel sessions

abstracts book 91 OL1EXTREMOphiles_2012

92 abstracts book EXTREMOphiles_2012O1 Martian Habitability Index Studies for Mars Science Laboratory (MSL) in four extreme environments (Earth analogues) and laboratory atmospheres simulation facilities Felipe Gómeza, J. A. Rodríguez-Manfredib, J. Gómez-Elvirab and REMS team

aDepartment of Planetary Geology and Habitability, Centro de Astrobiología (INTA-CSIC), Torrejón de Ardoz, Madrid 28850, Spain bDepartment of Robotics and Space Instrumentation, Centro de Astrobiología (INTA-CSIC), Torrejón de Ardoz, Madrid 28850, Spain E-mail: [email protected]

We are using extreme environments as test facilities from an Astrobiological perspective, in order to develop an Universal Habitability Index for space astrobiological mission application. We are developing a Universal Habitability Index (Madahvi, 1998) for life prospection studies in Mars surface during Mars Science Laboratory mission using data from the environmental station of MSL in four field case studies: Alaskan permafrost, Chott El Jerid salty arid region, Rio Tinto in Spain and Atacama Desert. Also some results of survivability of bacteria to Mars surface conditions in simulation facilities. First case: studying the permafrost in the Imuruk lake volcanic field area (Alaska): In order to map the permafrost underground, electric tomography sounding was performed. Resulting tomographic data indicate that the permafrost of the studied area is at a mean depth of 0.50 meter from the surface, sometimes even shallower. Drilling points were selected depending on the permafrost depth known from the tomographic data analysis. First results report enrichment in several inoculated media including some specific for heterotrophic aerobic bacteria, anaerobic chemolithotrophic and methanogen bacteria. Second case: studying Atacama Desert: Habitability studies in the Atacama Desert were developed by the selection of a 36 square meters area where an atmospheric station was installed. The following parameters were measured: UV radiation, atmosphere temperature, ground temperature at three different depths, wind direction. Samples at the pre-selected depths were taken in order to develop microbiological studies in the laboratory. Third case: Chott El Jerid (Tunisia): The salty arid region located in the south of Tunisia near Algerian border was used as third extreme region where to validate the data obtained in previous campaigns. 36 square meter area was used for the installation of an atmospheric station for environmental parameters measurement in similar conditions as in Atacama Desert. Microbiological studies were conducted later on in laboratory. A ground temperature sensor similar to REMS Ground Temperature Sensor (GTS) was tested in this field campaign. Data will be shown. Fourth case: Rio Tinto (Spain): Micro niches in layered Jarosite deposits in Rio Tinto have been identified and studied. Endolithic niches inside salt deposits used by phototrophs for taking advantage of sheltering particular light wavelengths. These acidic salts deposits located in Río Tinto shelter life forms which are difficult to localize by eye from adverse radiation conditions. Five case: We will present some results of chemolithotrophic bacteria survivability exposed to Mars simulated conditions. References Madahvi (1998) Steps to a General Theory of Habitability. Human Ecology Review 5: 23.

abstracts book 93 O2EXTREMOphiles_2012 Probing the limits of extremophilic life in extraterrestrial environment simulated experiments Claudia Lagea, Gabriel Dalmasoa, Lia Teixeiraa, Amanda Bendiaa, Ivan Paulino-Limab, Douglas Galantec, Eduardo Janot-Pachecoc, Ximena Abrevayad, Armando Azúa-Bustose, Vivian Pelozzarif and Alexandre Rosadog

aLaboratório de Radiações em Biologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil bNASA-Ames Research Center, USA cInstituto de Astronomia, Geofísica e Ciências Atmosféricas, Universidade de São Paulo, Brazil dInstituto de Astronomía y Física del Espacio, Universidad de Buenos Aires - CONICET, Argentina ePontificia Universidad Catolica de Chile, Chile fInstituto Oceanográfico, Universidade de São Paulo, Brazil gInstituto de Microbiologia Prof Paulo Góes, Universidade Federal do Rio de Janeiro, Brazil Email: E-mail: [email protected]

Astrobiology is a brand new area of science that seeks to understand the origin and dynamics of life in the universe. Several hypotheses to explain life in the cosmic context have been developed throughout human history, but only now technology has allowed many of them to be tested. Laboratory experiments have been able to show how chemical elements essential to life, carbon, nitrogen, oxygen and hydrogen combine in biologically important compounds. Interestingly, these compounds are found universally. As these compounds were combined to the point of originating cells and complex organisms is still a challenge to be unveiled by science. However, our 4.5 billion years old solar system was born within a 10 billion years old universe. Thus, simple cells like microorganisms may have had time to form in planets older than ours or other suitable molecular places in the universe. One hypothesis to explain the origin of life on Earth is called panspermia, which predicts that microbial life could have been formed in the universe billions of years ago, traveling between planets, and inseminating units of life that could have become more complex in habitable planets like ours. A project designed to test viability of extremophile microorganisms exposed to simulated extraterrestrial environments is ongoing at the Carlos Chagas Filho Institute of Biophysics to test whether microbial life could withstand those inhospitable environments. Ultra resistant (known or novel ones) microorganisms collected from terrestrial extreme environments, extremophiles, have been exposed to intense radiation sources simulating solar radiation (synchrotron accelerators), capable of emitting in a few hours radiation equivalent of million years accumulated doses. The results obtained in these experiments reveal the interesting possibility of the existence of microbial life beyond Earth.

Table 1. Summary of results observed under extraterrestrial-simulated environments

94 abstracts book EXTREMOphiles_2012O3 Archipelago of endolithic microbial life in the hyper arid core of the Atacama Desert Jacek Wierzchosa, Alfonso F. Davilab, Jocelyne DiRuggieroc, Asunción de los Ríosa, Sergio Valeaa, a d a c c Beatriz Cámara , Octavio Artieda , Mariela Speranza , Courtney Robinson , Tiffany Souterre , Virginia Souza-Egipsye and Carmen Ascasoa

aMuseo Nacional de Ciencias Naturales, MNCN-CSIC, 28006 Madrid, Spain bNASA Ames Research Center, MS 245-6, Moffett Field, California 95136, USA cThe Johns Hopkins University, Baltimore, MD 21218-2685, USA dUniversidad de Extremadura, 10600 Plasencia, Spain eInstituto de Ciencias Agrarias - CSIC, 28006 Madrid, Spain E-mail: [email protected]

The hyper-arid core of the Atacama Desert is considered the driest and most life-limited place on Earth. As such, this desert is an ideal environment to study the survival and biological adaptation strategies of microorganisms to this extremely dry environment. The extreme aridity of the Atacama Desert allows us to address questions such as the potential for life in extraterrestrial environments such as the surface of Mars. Our results over the last six years of searching for microbial life in the hyper-arid core of the Atacama Desert shows the presence of various habitats capable of sustaining active microbial ecosystems. All of them belong to endolithic – inside the rocks – microbial habitats. We suggest that these endolithic habitats protect microbial communities from UV and excessive PAR radiation and supply increased moisture, thus providing environmental refuges – or islands of life - in the desert. This «archipelago» of microbial islands of life is composed by the following endolitically colonized substrates: a) halite rocks (Wierzchos et al., 2006; de los Ríos et al., 2010); b) gypsum crusts (Wierzchos et al., 2011), c) volcanic origin ignimbrite (Wierzchos et al., 2012) and d) rhyolite and calcite rocks (DiRuggiero et al., submitted).

References · A. de los Ríos et al. (2010). Comparative analysis of the microbial communities inhabiting halite evaporites of the Atacama Desert. Int Microbiol 2: 79-89. · J. Wierzchos, C. Ascaso and C.P. McKay (2006) Endolithic cyanobacteria in halite rocks from the hyper-arid core of the Atacama Desert. Astrobiology 6: 415-422. · J. Wierzchos et al. (2011). Microbial colonization of Ca-sulfate crusts in the hyperarid core of the Atacama Desert: implications for the search for life on Mars. Geobiology 9: 44-60. · J. Wierzchos et al. (2012). Ignimbrite as a substrate for endolithic life in the hyper-arid Atacama Desert: implications for the search for life on Mars. Icarus, in press.

abstracts book 95 O4EXTREMOphiles_2012 The energy sulfur metabolism of the hyperthermophilic bacterium Aquifex aeolicus Marianne Guiral, Clément Aussignargues, Laurence Prunetti, Pascale Infossi, Marianne Ilbert and Marie-Thérèse Giudici-Orticoni

IMM-CNRS-AMU, Marsella, France E-mail: [email protected]

Aquifex aeolicus, a highly thermophilic bacterium, grows at 85°C with molecular hydrogen as electron donor and molecular oxygen as electron acceptor. A sulfur compound (elemental sulfur, thiosulfate or hydrogen sulfide) is also absolutely required for growth. Stimulated by its exceptional physiological properties, we have set out to study the energy metabolism and the proteins involved in the bioenergetics pathways of this bacterium. We have associated tools of proteomics, biochemistry, and physico-chemistry to characterize two high molecular weight membrane-bound protein edifices representing two complete «respirasomes» involved in two distinct respiratory pathways. One superstructure, formed by the stable association of a hydrogenase and a sulfur reductase, is responsible for sulfur reduction using H2 (Guiral et al., 2005). The second one is a new supercomplex involved in oxygen respiration and contains all proteins and complexes required for the electron transfer from H2S to O2. It contains the Sulfide quinone reductase (Sqr), thebc complex and the cytochrome c oxidase (Prunettiet al., 2010). Thus two different energetic pathways (sulfur reduction and sulfur oxidation) are organized in this bacterium as supramolecular structures in the membrane. Moreover, additional cytoplasmic enzymes involved in sulfur compounds utilization were characterized from A. aeolicus: the sulfur oxygenase reductase (SOR), which catalyzes the simultaneous oxidation and reduction of elemental sulfur, in the presence of oxygen (Pelletieret al., 2008) and a sulfur transferase SbdP (Giuliani et al., 2007) which might function as a sulfur supplier, distributing elemental sulfur to some enzymes of energy sulfur metabolism, and thus optimizing respiration on elemental sulfur (Aussignargues et al., 2012). Taking the advantage of the complete genome sequence of A. aeolicus in addition to the experimental data, we propose a model for the energetic optimization of sulfur compounds in this bacterium, integrating known and hypothesized pathways.

References · Guiral M et al. (2005) J Biol Chem 280, 42004-42015. · Prunetti L et al. (2010) J Biol Chem 285, 41815-41826. · Pelletier N et al. (2008) Extremophiles 12, 205-215. · Giuliani MC et al. (2007) FEBS J. 274, 4572-4587. · Aussignargues C et al. (2012) J. Biol. Chem., in press.

96 abstracts book EXTREMOphiles_2012O5 Genetic and functional analysis of CdvB paralogs in Sulfolobus acidocaldarius Nuan Yang and Arnold J.M. Driessen

Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands E-mail: [email protected]

The exact mechanism of cell division in Archaea is unknown. While Euryarchaeota seem to rely on an FtsZ- based mechanism, the majority of Crenarchaeota and all Thaumarchaeota seem to utilize the Cell division system (Cdv) (Bernander and Ettema, 2010) that has also been implicated in the formation of small cell surface derived membrane vesicles (Ellen et al., 2009) The Cdv system consists of three genes, cdvA, cdvB and cdvC that are organized in an operon. CdvC is homologous to the AAA-type ATPase, Vps4, which involved in multivesicular body (MVB) biogenesis in eukaryotes, also termed the ESCRT-III machinery. CdvB is homologous to Vps24 and forms helical filaments. Much akin the ESCRT-III machinery, CdvA, CdvB and CdvC are interacting proteins. In addition to the cdvABC operon, Sulfolobus acidocaldarius contains three additional genes that encode CdvB paralogs that are differentially expressed under conditions of the cell cycle, viral infection and others. We have used a genetic approach to determine the function of these additional archaealcdv genes. Independent deletion mutants of the three cdvB-like genes have been isolated, and mutant strains were analyzed for growth, cell division and membrane vesicle formation using biochemical and microscopic techniques. Two of the mutants showed a severe growth defect, but differ in their flow cytometry characteristics and morphology. The results suggest that one of the CdvB-like proteins serves a role in chromosomal segregation, whereas the other mutant shows a cell division defect.

References · R. Bernande and T. J. Ettema (2010) FtsZ-less cell division in archaea and bacteria. Curr Opin Microbiol 13: 747-752. · A. F. Ellen. S. V. Albers, W. Huibers, A. Pitcher, C. F. Hobel, H. Schwarz, M. Folea, S. Schouten, E J. Boekema, B. Poolman and A. J M. Driessen (2009) Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components. Extremophiles 13: 67-79.

abstracts book 97 O6EXTREMOphiles_2012 Enzymes and regulation involved in archaeal coenzyme A biosynthesis Hiroya Tomitaa, Yuusuke Yokoojia, Takuya Ishibashia, Tadayuki Imanakab,c and Haruyuki Atomia,c

aDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, , 615-8510 Japan bDepartment of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, 525-8577 Japan cJST, CREST, Sanbancho, Chiyoda-ku, Tokyo, 102-0075 Japan E-mail: [email protected]

Coenzyme A (CoA) is a major acyl group carrier in all living systems forming high energy thioester bond with various carbonyl compounds. CoA and its derivatives are essential cofactors in diverse metabolic pathways such as glycolysis, the tricarboxylic acid cycle, ?-oxidation, and fatty acid and isoprenoid biosynthesis. Although bacteria and eukaryotes share a common pathway for CoA biosynthesis, we previously clarified that Thermococcus kodakarensis, a sulfur-reducing, hyperthermophilic archaeon isolated from Kodakara Island, Japan, utilize a different pathway. In the conversion of pantoate to 4’-phosphopantothenate, bacteria and eukaryotes utilize pantothenate synthetase (PS) and pantothenate kinase (PanK). In contrast, T. kodakarensis uses two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS) (Yokooji et al., 2009). In order to synthesize one molecule of CoA, one molecule each of L-valine, alanine, L-cysteine are consumed along with 5 ATPs and an NADPH. Therefore, it can be expected that a regulatory mechanism exists to prevent the excess synthesis of CoA. In bacteria and eukaryotes, it has been well known that the PanK activity is controlled by feedback inhibition by CoA and its thioesters (Rocket al., 2003)]. As the archaea do not utilize PanK, it is of interest as to how CoA biosynthesis is regulated in these organisms. Here we have carried out a detailed biochemical characterization of PoK and PPS, as well as ketopantoate reductase (KPR), the enzyme that provides the PoK substrate pantoate from 2-oxopantoate. Recombinant PPS and KPR were produced in Escherichia coli, and PoK was produced in T. kodakarensis. The individual proteins were purified and their enzymatic properties examined. Kinetic studies revealed that PoK exhibits substrate inhibition with pantoate and PPS exhibits substrate inhibition with 4-phosphopantoate and ATP. Site-directed mutagenesis of PoK identified several residues (Ser104, Glu134, and Asp143) essential for activity, and others involved in substrate binding. Other results on product/feedback inhibition will be presented and discussed in relation with the regulation of CoA biosynthesis.

References · Yokooji et al. (2009). J. Biol. Chem. 284: 28137-28145. · Rock et al. (2003). J. Bacteriol. 185: 3410-3415.

98 abstracts book EXTREMOphiles_2012O7 Prokaryotic diversity in hydrothermal springs of the Azores analysed by a network of 16S rRNA gene based methods Kerstin Sahma, Patrick Johna, Heiko Nackeb, Ralf Grotea, Rolf Danielb and Garabed Antranikiana

aInstitute of Technical Microbiology, Hamburg University of Technology, Germany bInstitute of Microbiology and Genetics, Georg August University Göttingen, Germany E-mail: [email protected]

Two hot springs situated in the Furnas Valley at Sao Miguel (Azores) were analysed with regard to composition of the prokaryotic community. Based on pyrosequencing of PCR-amplified variable regions of 16S rRNA-genes complemented by metagenomic analysis and fluorescencein-situ hybridisation (FISH) we obtained an in-depth picture of the phylogenetic composition of this complex ecosystems. Common features from hydrothermal springs across continents were detected as well as differences in the quantitative contribution of heterotrophic vs. autotrophic organisms. While in site AI (92°C, pH 8) the relative contribution of the two domains was almost even, the acidic spring AIV (51°C, pH3) was dominated by Bacteria. The combination of 16S rRNA gene sequence analysis and FISH revealed high abundance of heterotrophic bacterial genera in the hot, slightly alcaline spring AI. In addition chemolithoautotrophic Aquificales made up app. 28% of the community. The archaeal population was made up of Crenarchaeota, exclusively, dominated by sequences of Desulfurococcaceae, in particularSulfophobococcus , Desulfurococcus, and Thermosphaera. In addition Thermoproteaceae-sequences related to the genus Pyrobaculum made up almost 25%. Overall diversity in the acidic spring (AIV) was lower and the bacterial community was dominated by sequences related to the chemolithoautotrophic genus Acidithiobacillus, to the heterotrophic Acidicaldus, and to Anoxybacillus. The quantitatively small archaeal community was made up almost exclusively of the euryarchaeal genus Thermoplasma.

abstracts book 99 O8EXTREMOphiles_2012 Continuous enrichment cultures using diluted hydrothermal fluid as medium: New insights into active microbial diversity inhabiting active deep-sea vent chimney of Guaymas Basin Nolwenn Callaca,b,c, Olivier Rouxelc,d, Françoise Lesongeurb, Carole Deckere, Céline Liorzouc, Claire Bassoulletc, Karine Estèveb, Patricia Pignetb, Sandrine Cherond, Yves Fouquetd, Céline Rommevaux-Jestinf and Anne Godfroyb aUniversité Européenne de Bretagne, Brest, France bIfremer, Laboratoire de Microbiologie des Environnements Extrêmes, UMR6197, 29280 Plouzané, France cInstitut Universitaire Européen de la mer Domaines Océaniques, UMR6538, 29280 Plouzané, France dIfremer, Laboratoire de Géochimie et de Métallogénie, 29280 Plouzané, France eIfremer, Laboratoire des Environnements Profonds, 29280 Plouzané, France fIPGP, Laboratoire Géobiosphère Actuelle et Primitive, UMR7154, 75238 Paris, France E-mail: [email protected]

It is now established that in hydrothermal systems host diverse microbial communities with several different metabolisms and that some are involved in Sulfur or Iron biogeochemical cycles. In order to investigate these cycles in the Guaymas Basin hydrothermal system (Gulf of California, Mexico), a multidisciplinary approach was performed to identify the interactions between Geosphere-Biosphere and Hydrosphere in this particular context. To establish links between the structure and the activity of microbial populations with their environment (ie vent mineralogical characteristics and hydrothermal fluid composition), cultural approach in a Gas-Lift bioreactor was chosen (Byrne et al., 2009; Godfroy et al., 2006; Postec et al., 2007). Thus, an enrichment culture was maintained for 23 days at 85°C using an active crushed vent flange as inoculum and diluted hydrothermal fluid as culture medium. During all the culture daily samples, have provided access to active microbial diversity, elements (major and trace), behavior, sulfate, hydrogen sulfide and organic acids concentrations potentially used as substrates. Bacteria were shown to be dominant along the cultivation time. Sequences affiliated toε-proteobacteria, β-proteobacteria and γ-proteobacteria and also archaeal sequences affiliated to Thermococcales,Archeoglobus sp. Geoglobus sp. and Ferroglobus sp., known to be involved in sulfur or iron cycle, were retrieved during all the cultivation time. This experience allowed cultivating thermophilic microorganism populations that were under represented in the crude sample, to cultivate new species and follow community dynamic over the time, depending on growth conditions. Also, the effects of environmental change as pH, on minerals phase dissolution or re-suspension, fluid composition with providence of carbon sources, of electron donors or acceptors on microbial diversity was explored and these different parameters how these modifications allow to influence the structure of the cultured microbial community as this could happen in hydrothermal structure.

References · Byrne N, Lesongeur F, Bienvenu N, Geslin C, Alain K, Prieur D, Godfroy A (2009) Effect of variation of environmental conditions on the microbial communities of deep-sea vent chimneys, cultured in a bioreactor. Extremophiles 13: 595-698. · Godfroy A, Postec A, Raven N (2006) Growth of hyperthermophilic microorganisms for physiological andnutritional studies. In: Rainey FA, Oren A (eds) Methods in Microbiology, Extremophiles. Academic Press, Oxford, England. · Postec A, Lesongeur F, Pignet P, Ollivier B, Querellou J, Godfroy A (2007) Continuous enrichment cultures: insights into prokaryotic diversity and metabolic interactions in deep-sea vent chimneys. Extremophiles 11: 747-757.

100 abstracts book EXTREMOphiles_2012O9 Evidence of species recruitment and development of hypolithic communities along an aridity gradient in the Namib Desert Marla Tuffina, Francesca Stomeoa, Thulani Makhalanyanea, Angel Valverdea, Chris McKayb, Kimberley Warren-Rhodesb, Donna Lacapc, Stephen Pointingc and Don Cowand aInstitute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Cape Town, South Africa bNASA-Ames Research Center, MS 245-3, Moffett Field, CA 94035, USA cSchool of Biological Sciences, the University of Hong Kong, Pokfulam Road, Hong Kong, China dDepartment of Genetics, University of Pretoria, Pretoria 0002, South Africa E-mail: [email protected]

The Namib Desert is considered the oldest desert in the world and is characterized by distinct climatic zonation. The hyper-arid central and western desert zones show little or no evidence of surface colonization such as soil crusts. However, the environmental buffering provided by quartz and other translucent rocks supports extensive hypolithic microbial communities. In this study, open soil and hypolithic microbial communities were investigated along an East-West transect characterised by an inverse fog-rainfall gradient. Multivariate analysis showed that hypolithic and soil communities were structurally distinct. Using variation partitioning, we found that while the soil bacterial community is most likely related to the rainfall gradient, hypolithic communities exhibited a fog-related distribution as indicated by the significant East-West clustering. Sodium content (soil salinity) was also an important environmental factor affecting the composition of both soil and hypolithic microbial communities. Applying the ecological concept of ‘indicator species’, 7 and 9 indicator lineages for hypoliths and soil were detected, respectively. Hypolithons were dominated by cyanobacteria affiliated to Pleurocapsales, whereas actinobacteria were prevalent in the soil. These results are consistent with the concept of species sorting and suggest that quartz rocks provide conditions suitable for the development of discrete and demonstrably different microbial assemblages. However, we found strong evidence for neutral assembly processes, as almost 40 % of the taxa present in the soil were also detected in the hypoliths. These results suggest that hypolithons do not develop independently from microbial communities found in the surrounding soil, but selectively recruit from local populations.

abstracts book 101 O10EXTREMOphiles_2012 New thermophilic Planctomycetes isolated from terrestrial hot springs and deep subsurface environments Galina Slobodkinaa, Margarita Miroshnichenkoa, Olga Kovalevaa, Alexander Slobodkina, Esta van Heerdenb, Alexander Lebedinskya and Elizaveta Bonch-Osmolovskayaa

aWinogradsky Institute of Microbiology RAS, Prospekt 60-letiya Oktyabrya, 117312 Moscow, Russia bUniversity of the Free State, P.O. Box 339,Bloemfontain 9300, South Africa E-mail: [email protected]

Bacteria of the phylum Planctomycetes play significant environmental roles in diverse ecosystems on the Earth: they either degrade organic substrates (aerobically or fermentatively) or perform the unique process of anaerobic ammonium oxidation, «anammox» (Fuerst and Sagulenko, 2011). All Planctomycetes isolates are mesophilic organotrophs, including Isosphaera pallida, which, albeitin found in thermal springs, has its temperature optimum of growth at 40oC (Giovannoni et al., 1987). However, there is molecular evidence of the presence of Planctomycetes in diverse thermal habitats, including anaerobic ones: deep-sea, shallow-water and terrestrial thermal habitats, as well as deep subsurface environments. Two novel Planctomycetes isolates were obtained from a hot spring of Kunashir Island, Kurils (isolate R1-3), and Beatrix gold mine, South Africa (isolate BH1-P). The morphology of both organisms is typical of Planctomycetes: their cells are irregular cocci, and a complicated life cycle with non-motile and motile phases occurs. Both organisms are facultative anaerobes and moderate thermophiles growing optimally at 55-60oC and pH 5.5-6.0. The two isolates differ in their reaction to pH of the medium: the hot spring strain is a moderate acidophile (opt pH 5.5-6.0), while the deep subsurface one is a moderate alkaliphile (opt pH 7.5-8.0). The ranges of temperature and pH at which the growth of new isolates occurs are extremely broad, especially those of isolate R1-3 – from 22 to 83oC and from 4.5 to 9.0. Both isolates grow on various sugars and polysaccharides, but not on peptides, by fermentation or aerobic and anaerobic respiration; in the latter case, nitrate serves as the electron acceptor and is reduced to nitrite. Growth of isolate R1-3 is also stimulated by elemental sulfur which is reduced to hydrogen sulfide. Analysis of 16S rRNA sequences of new isolates showed that they are closely related to an environmental clone from Yellowstone National Park designated as OPB17 (Hugenholz et al., 1998), with homology values of 96% (isolate BH1-P) and 100% (isolate R1-3); they also cluster with numerous environmental clones from thermal habitats all over the world. However, the similarity of 16S rRNAs of the isolates to that of the closest cultivated relative is only 85%. Thus, these first thermophilic isolates of Planctomycetes represent a novel genus and two new species in this phylum and an environmentally significant group widespread in diverse thermal habitats.

References · Furest J.S., Sagulenko E. (2011) Beyond the bacterium: planctomycetes challenge our concept of microbial structure and function. Nature Rev. Microbiol. 9: 403-413. · Giovannoni S., Schabtach E., Castenholz R.W. (1987) Isosphaera pallida, gen. and comb. nov., a gliding, budding eubacterium from hot springs. Arch. Microbiol. 147:276-284. · Hugenholz P., Pitulle C., Hershberger K.L., Pace N.R. (1998) Novel division level bacterial diversity in a Yellowstone hot spring. J. Bacteriol. 190: 366-376.

102 abstracts book EXTREMOphiles_2012O11 Developing novel biophysical tools to explore protein adaptation in stressful environments Toni Hoffmanna,b, Katarzyna Tycha, David J. Brockwellb and Lorna Dougana

aSchool of Physics and Astronomy, University of Leeds, Leeds LS9 2JT, UK bInstitute of Molecular and Cell Biology, University of Leeds, Leeds LS9 2JT, UK E-mail: [email protected]

Proteins are vital for all processes of life. An understanding of how environmental forces shape the structure of a peptide chain is essential to unravel the workings of living systems. Life has adapted to a vast range of different environmental conditions and it is now difficult to find any place on Earth devoid of life. Some conditions are extreme in the sense of being unfavourable to most eukaryotic life forms. The adaption of proteins played a key role in enabling extremophilic organisms to colonise such ecological niches. Understanding the physical properties of proteins from extremophilic organisms and their remarkable preservation capability is not only of fundamental interest, but also pivotal to our ability to rationally engineer biological materials for exploitation. We are developing quantitative biophysical approaches to characterise the physical mechanisms of protein folding and stability in extreme environments. We have built a state-of-the-art force spectroscopy instrument which we use to examine the conformational dynamics of single extremophilic proteins. This technique is used to apply a constant stretching force along a well-defined reaction coordinate, the end-to-end length of the protein, driving proteins to a fully extended unfolded state. By examining single molecules one at a time, the individual dynamics of protein subpopulations can be measured, revealing information which may be crucial for designing ‘artificial’ extremophilic proteins. A single molecule approach provides a new perspective and reveals novel insight into the mechanisms of protein folding particularly with regard to how peptide chains fold in interplay with various environmental parameters. By exploring the structural limitations of extremophilic proteins we aim to uncover unknown universal rules determining how proteins fold. Such knowledge will be invaluable for the de novo design of proteins with desired properties. This work promises to reveal critical information on the physics and function of proteins from extremophilic organisms and to provide the foundations for developing and optimizing biologically enabled and biologically inspired materials. Moreover, this approach could aid in the design of extremophilic proteins which possess specific thermal or mechanical stability, binding specificity and conformational flexibility for exploitation in biotechnology and medicine. Our study will not only help to better understand life on Earth but also its prospects elsewhere.

abstracts book 103 O12EXTREMOphiles_2012 Dehalogenase enzymes from extremophilic organisms Jennifer Littlechild, Halina Novak, Christopher Sayer and Misha Isupov

Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK E-mail: [email protected]

L-haloacid dehalogenases can produce optically pure 2-hydroxyalkonoic acids which can be used in industrial applications and in the production of pharmaceutical intermediates. Several of these enzymes have been studied from different thermophilic and pyschrophilic archaea and bacteria. These enzymes show varying substrate specificity towards short and long chain L-2-haloalkanoic acids with some enzymes showing a preference towards the chlorinated or brominated forms. The thermophilic Sulfolobus tokodaii show activity towards longer chain substrate such as 2-chlorobutryic acid and displays highest activity towards L-chloropropionic acid (Ryeet al., 2009). The enzyme from the psychrophilic bacterium Psychromonas ingrahamii shows highest activity towards monobromoacetic acid and decreasing activity towards monochloroacetic acid, S-chloropropionic acid, S-bromopropionic acid, dichloroacetic acid and 2-chlorobutyric acid respectively. This shows that the P. ingrahamii L-haloacid dehalogenase has highest activity towards substrates with a shorter carbon chain length (≤C3), without preference towards a chlorine or bromine in the α-carbon position. The psychrophilic L-dehalogenase enzyme displays high thermal stability with a melting temperature of 85ºC as determined by thermal shift experiments. It retains 70% of its activity after being incubated at 65°C for 90 min and has an optimal temperature for activity at 45ºC. The enzyme is relatively stable in organic solvents as demonstrated by activity and thermal shift analysis. A homology model of the P. ingrahamii L-haloacid dehalogenase structure has been built and this has been compared with the crystal structure of the thermophilic enzyme to try to understand the basis of the observed stability (H. Novak et al., 2012).

References · C. Rye, M. Isupov and J. Littlechild (2009). Biochemical and structural studies of a L-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii. Extremophiles 13: 179-190. · H. Novak. PhD thesis, University of Exeter (2011). · H. Novak, C. Sayer, J. Panning and J. Littlechild (2012). Characterisation of a thermostable and solvent resistant L-haloacid dehalogenase from the marine psychrophile Psychromonas ingrahamii. Extremophiles, submitted.

104 abstracts book EXTREMOphiles_2012O13 Protein methylation in crenarchaea Yindi Chu, Zhenfeng Zhang and Li Huang

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China E-mail: [email protected]

A number of crenarchaeal proteins are known to undergo posttranslational methylation, a form of modification that has been speculated to confer stability to proteins. However, no enzymes responsible for methylation of these proteins have been found. In our previous study, we found that Cren7, a chromatin protein from the hyperthermophilic archaeon Sulfolobus solfataricus, was methylated at multiple lysine residues. The extent of methylation of the protein was increased as the growth temperature was raised from 75 to 85 oC. Here we report the identification of a novel lysine methyltransferase fromSulfolobus islandicus that is capable of methylating Cren7. The enzyme was active at slightly alkaline pH and over a wide range of temperatures, being optimal at 50-60o C. The methyltransferase required no divalent cations for its activity and was insensitive to relatively high levels of salts. Interestingly, the enzyme showed relaxed substrate specificity and was able to methylate a range of Sulfolobus proteins. Structural requirement for the methylation of a lysine residue in a protein by the enzyme was analyzed. This is the first lysine methyltransferase to be found in crenarchaea. The evolutionary and functional implications of this methyltransferase will be discussed.

abstracts book 105 O14EXTREMOphiles_2012 The DNA-alkyltransferase of Sulfolobus solfataricus: in vivo and in vitro studies on a conserved protein involved in repair of DNA alkylation damage Giuseppe Perugino, Antonella Vettone, Anna Valenti, Mosè Rossi and Maria Ciaramella

Institute of Protein Biochemistry, National Council of Research of Italy, Via Pietro Castellino 111, 80131 Naples, Italy E-mail: [email protected]

DNA-alkyltransferases are ubiquitous proteins, which act by a unique reaction mechanism, leading to irreversible protein alkylation and direct repair of highly mutagenic and carcinogenic DNA alkylation damages. The alkylated form of DNA-alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation (Daniels et al., 2000). We report here in vivo and in vitro studies on the DNA-alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). Transcriptional and post-translational regulation of SsOGT by DNA damage was studied, showing that although alkylating agent treatment induces increase of the transcript level, the protein is degraded by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability. The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. The data obtained on the wild type and a mutant protein impaired in DNA binding activity, clearly showed their higher stability to common chemical and physical denaturing agents as well as protease attack, in comparison with the human AGT (Mollwitz et al., 2012). The utilization of these archaeal catalysts as alternative protein tags to the commercially available SNAP-tag™ (Keppleret al., 2003) in extremophilic model organisms is evaluated.

References · G. Perugino, A. Vettone, G. Illiano, A. Valenti, M.C. Ferrara, M. Rossi, and M. Ciaramella (2012). Activity and regulation of archaeal DNA-alkyltransferase: conserved protein involved in repair of DNA alkylation damage. J Biol Chem 287, 4222-4231. · D.S. Daniels, C.D. Mol, A.S. Arvai, S. Kanugula, A.E. Pegg, and J.A. Tainer (2000) Active and alkylated human AGT structures: a novel zinc site, inhibitor, and extrahelical base binding. EMBO J 19, 1719-1730. · A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, and K. Johnsson (2003). A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21, 86-89. · B. Mollwitz, E. Brunk, S. Schmitt, F. Pojer, M. Bannwarth, M. Schiltz, U. Rothlisberger, and K. Johnsson (2012). Directed evolution of the suicide protein 6O -alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability. Biochemistry 51, 986-994.

106 abstracts book EXTREMOphiles_2012O15 The proteomic response of an extreme acidophile, Acidithiobacillus ferrooxidans, to increased chloride concentrations Timothy J. McCredden, Robert F.L. Steuart, Christopher G. Bryan, Elizabeth L.J. Watkin

School of Biomedical Sciences, Curtin University, GPO Box U1987, Perth 6845, Western Australia, Australia E-mail: [email protected]

High concentrations of chloride ions inhibit the growth of acidophilic iron and sulfur oxidizing microorganisms used in biomining, a problem particularly relevant to Western Australian and Chilean biomining operations. This growth inhibition is due to the chloride ion disrupting homeostatic mechanisms resulting in acidification of the cytoplasm. Acidithiobacillus ferrooxidans is a found widely in biomining systems however, little is known about the mechanisms this microorganism adopts in order to tolerate sub-lethal chloride ion concentrations. This study applied proteomics to elucidate how A. ferrooxidans alters its proteome under increased chloride concentrations.

A. ferrooxidans DSM 14882T was grown in the presence of low (0 g.L-1) and high (8 g.L-1) NaCl concentrations, chosen for optimal and sub-optimal growth rates. Total soluble proteins produced by cells were compared using 2D-polyacrylamide gel electrophoresis. Protein spots that were identified as having statistically different abundance between high and low salt conditions were excised, trypsin digested and analyzed by tandem mass spectrometry. Mascot sequence matching software was used to identify proteins of interest.

Analysis of differential expression showed that A. ferrooxidans adopted several changes in its proteome in response to NaCl stress. The majority of the strategies represented a general stress response mechanism similar to those induced during pH stress. These included major stress related periplasmic chaperones, whose action is essential for outer membrane formation, and proteins involved in central carbon metabolism, energy acquisition, amino acid synthesis and protein fate. A response that was unique to the NaCl stress response was the accumulation of particular amino acids which have osmoprotectant characteristics. Gaining an understanding of the range of mechanisms that acidophilic iron oxidizing microorganisms may use to help the cell function in the presence of elevated concentrations of chloride can be applied to the development of saline biomining operations or improve alterative processes.

abstracts book 107 O16EXTREMOphiles_2012 Mutational loss of the membrane carotenoid of an alkaliphilic Bacillus results in changes in membrane fluidity, cell shape, growth and oxidative stress responses Terry A. Krulwicha, Oliver Fackelmayera, Amyeo Jereena, Amy LaFountainb, Harry Frankb, Ronald Gordonc, Anthony W. Kingstond, John Helmannd and David B. Hicksa

aDepartment of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA bDepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USA cDepartment of Pathology, Mount Sinai School of Medicine, New York, NY 10029, USA dDepartment of Microbiology, Cornell University, Ithaca, NY 14853, USA E-mail: [email protected]

The prevalence of membrane carotenoids in a variety of extremophiles has raised the question of their possible roles in meeting a broad range of stresses, including either very cold or very hot temperature, oxidative stress, elevated salt and extremes of pH (Aono & Horikoshi 1991; Köcheret al., 2009). The question is underscored by observations of important roles of membrane carotenoids in protecting some non-extremophiles against several of these stresses, e.g. protection of pathogenic Staphylococcus aureus against oxidative stress by staphyloxanthin (Clauditz et al., 2006). An intersecting question of interest with respect to membrane carotenoids is how much their role relates to their proposed ability to act as «proto-sterols» in decreasing membrane fluidity (Rohmeret al., 1979). Here, we report that loss of membrane carotenoid results from by deletion of the crtM gene encoding squalene/phytoene synthase in alkaliphilic Bacillus pseudofirmus OF4. The mutant exhibits a pleiotropic phenotype that includes increased fluidity of the membrane that is accompanied by reduced non-fermentative growth, especially at high pH, a somewhat shorter and wider cell shape, elevated resistance to hydrogen peroxide but elevated sensitivity to paraquat. Two different «restored strains», in which the wild-typecrtM gene was restored to its native locus in the alkaliphile had different incomplete reversals of the mutant phenotype, consistent with the possibility that the original deletion necessitated one of several different patterns of additional mutational changes.

References · Aono, R. and K. Horikoshi (1991). Carotenes produced by alkaliphilic yellow-pigmented strains of Bacillus. Agric. Biol. Chem. 55: 2643-2645. · Köcher, S., J. Breitenbach, V. Müller and G. Sandmann (2009).Structure, function and biosynthesis of carotenoids in the moderately halophilic bacterium Halobacillus halophilus. Arch. Microbiol. 191:95-104. · Clauditz, A., A. Resch, K.-). Wieland, A. Peschel and F. Götz (2006). Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infec. Immun. 74:4950-4953. · Rohmer, M., P. Bouvier and G. Ourisson (1979). Molecular evolution of membranes: structural equivalents and phylogenetic precursors of sterols. Proc. Natl. Acad. Sci. USA 76: 847-851.

108 abstracts book EXTREMOphiles_2012O17 Evolutionary analysis of the biosynthesis of glyceryl-glucosides Luís G. Gonçalvesa, Nuno Borgesa, Pedro Fernandesb, Hernán Dopazoc and Helena Santosa

aInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal bInstituto Gulbenkian de Ciência, Portugal c Biomedical Genomics & Evolution Lab, Universidad de Buenos Aires, Argentina E-mail: [email protected]

Mannosylglycerate is a compatible solute encountered in many hyperthermophilic archaeons (Pyrococcus, Thermococcus, Palaeococcus, Archaeoglobus, Aeropyrum, Stetteria), thermophilic bacteria (Rubrobacter, Thermus and Rhodothermus), and in many red algae. The structurally related compound, glucosylglycerate, is also used as a compatible solute in the archaeon Methanohalophilus portucalensis, mesophilic bacteria (Chromohalobacter, Synechococcus, Dicheya, Streptomyces and Prochlorococcus), and in the thermophilic bacterium Persephonella marina. Other glyceryl-glycosides have been found in a restricted number of organisms, namely mannosylglucosylglycerate in Petrotoga miotherma and Petrotoga mobilis, glucosylglucosylglycerate in Persephonella marina, and mannosylglyceramide in Rhodothermus marinus. Interestingly, two pathways have been described for the synthesis of mannosylglycerate and glucosylglycerate: an uncommon one-step pathway that uses glycerate and GDP-mannose and ADP-glucose, respectively; and a two-step pathway that uses 3-phosphoglycerate and the same activated sugars, forming a phosphorylated intermediate, subsequently dephosphorylated by a specific phosphatase to yield the final compounds. The presence of glyceryl-glycosides in phylogeneticaly distant organisms, isolated from distinct environments, raises interest about the evolution and dissemination of the pathways leading to their synthesis. We performed a phylogenetic analysis of the key enzymes involved in the single-step and two-step pathways. The fit of the data to protein and DNA evolutionary models was done using ProTest and ModelTest programs. Phylogenetic trees based on protein as well as on 16S rDNA sequences were constructed using Maximum Likelihood, PhyML program, and Bayesian analysis, Mr.Bayes. Our analysis indicates that the two-step pathways for mannosylglycerate and glucosylglycerate have a common ancestor belonging to the domain Bacteria, and the synthase recognized both the GDP-mannose and ADP-glucose as substrates. No evidence for recent horizontal gene transfer events was found. A comprehensive evolutionary scenario will be presented.

abstracts book 109 O18EXTREMOphiles_2012 Microbial diversity and genome characteristics of thermo-piezophiles Mohamed Jebbara, Pauline Vanniera, Viggo Marteinsson b, Olafur Fridjonssonb, Phil Ogerc, Xiang Xiaod, Xu Jund, Jean Louis Birriena and Daniel Prieura

aUniversité Bretagne Occidentale (UBO), CNRS, Ifremer, IUEM (Institut Universitaire Européen de la mer) – UMR 6197, Technopole Brest-Iroise, Rue Dumont d’Urville, F-29280 Plouzané, France bMatis-Prokaria, Gylfaflot 5, IS-112 Reykjavik, Iceland cUniversité de Lyon, CNRS, Ecole Normale Supérieure de Lyon - UMR 5570, 15 parvis René Descartes BP 7000, F-69342 Lyon, France dSchool of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China E-mail: [email protected]

The deep biosphere, located in the continental underground and in oceans below 1000 m in depth, could represent up to 70% of all cells on Earth, as well as 50% of the primary production of biomass (Oger and Jebbar, 2010). Most of deep biosphere biotopes are oligotrophic and characterized by high hydrostatic pressures (HHP). Indeed, only some HHP-adapted organisms, or piezophiles (from the greek piezo = to press and philo = love, piezophiles have optimal growth rates at pressures greater than 1 atmosphere or 0.1 MPa), have been isolated to date. Since the discovery of deep-sea hydrothermal vents, many mesophilic, thermophilic and hyperthermophilic Bacteria and Archaea have been described. To our knowledge, only a few thermo-piezophilic organisms have been described so far: Thermococcus barophilus, Paleococcus ferrophilus, Marinitoga piezophila, Methanopyrus kandleri and Pyrococcus yayanosii. P. yayanosii sp. nov., is the first obligate piezophilic hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. T. barophilus, P. yayanosii and M. piezophila genomes have been recently completely sequenced (Vannier et al., 2011; Jun et al., 2011) and transcriptomic and proteomic studies are ongoing by comparing data among piezophilic (T. barophilus, P. yayanosii, M. piezophila) versus non piezophilic (T. kodakaraensis, P. furiosus, M. littoralis) related species to decipher the cell adaptive mechanisms to HHP.

References · P. M. Oger and M. Jebbar. (2010).The many ways of coping with pressure. Res Microbiol. 161: 799-809. · P. Vannier, V. T. Marteinsson, O. Fridjonsson O, P. Oger and M. Jebbar. (2011). Complete genome sequence of the hyperthermophilic piezophilic, heterotrophic and carboxydotrophic archaeon Thermococcus barophilus MP. J Bacteriol. 193: 1481-1482 · X. Jun, L. Lupeng, X. Minjuan, P. Oger, W. Fengping, Z. Xiang, M. Jebbar and X. Xiao. (2011). Complete genome sequence of the first obligate piezophilic hyperthermophilic archaeaon Pyrococcus yayanosii CH1. J Bacteriol. 193: 4297-4298

110 abstracts book EXTREMOphiles_2012O19 Membrane homeoviscous adaptation in the archaeon Thermococcus barophilus requires the regulation of unsaturation of accessory, but not core, lipids Anaïs Carioa, Vincent Grossia, Philippe Schaefferb and Philippe Ogera aLaboratoire de Géologie de Lyon, CNRS UMR 5276, Ecole Normale Supérieure de Lyon, Université Lyon 1, 46 Allée d’Italie, 69364 Lyon cedex 0, France bLaboratoire de Géochimie Bioorganique, CNRS UMR 7177, Institut de Chimie de Strasbourg, Université Louis Pasteur, 67200 Strasbourg, France E-mail: [email protected]

Biological membranes, which play an important role as a barrier from the environment and host numerous cellular processes, are highly sensitive to temperature and pressure, which strongly impact membrane fluidity by modifying membrane permeability and membrane-associated functions. To adapt to temperature or pressure fluctuations, bacteria maintain optimal membrane fluidity by altering the degree of unsaturation and the chain length of fatty acids, a mechanism named homeoviscous adaptation. Structural differences between archaeal membrane lipids (containing glycerol ether bonds and phytanyl chains) and bacterial analogues (containing fatty acids esterified to glycerol) raise the question of membrane fluidity regulation in hyperthermophilic and piezophilic archaea. We have studied the influence of sub- and supra-optimal pressures and temperatures on lipid composition of the piezophilic and hyperthermophilic archaeonThermococcus barophilus strain MP. This strain displays optimal pressure and temperature at 40 MPa and 85°C, respectively. Lipids were extracted by the Bligh and Dyer method, analyzed and quantified by GC-FID and GC-MS. Polar headgroups were characterized by TLC and identified using Pyrococcus furiosus strain DSM3638 as a reference. Archaeol (sn-2,3-bis-0-phytanylglycerol) was the major core lipid detected in T. barophilus, and was accompanied by two minor unsaturated homologues (i.e. unsaturated archaeol with one or two double bonds). No tetraethers were detected. The lipid polar headgroups of T. barophilus, characterized using TLC, show similarities with the phosphoglycerol and phosphoinositol headgroups of P. furiosus. The neutral lipids of T. barophilus consist of series of C35 and C40 unsaturated isoprenoid hydrocarbons with a lycopane skeleton. The presence of archaeol as the unique core lipid indicates that T. barophilus possesses a bilayer-type membrane. According to the membrane model of Haines (2001), the hydrocarbon chains could incorporate at the interface between the two layers, in order to regulate the proton gradient across the membrane and, thus, its rigidity. An increase or a decrease of T or P does not affect significantly the pool of core lipids but significantly impacts the unsaturation degree of the hydrocarbon chains. Under high pressure (HP) or cold conditions (LT), the isoprenoid alkenes of strain MP display an increased level of unsaturation (mean number of unsaturations [nu] per molecule of 3.7 and 3.5 for HP and LT, respectively) compared to optimal conditions (2.9 nu). According to the membrane model proposed for T. barophilus, an increase of the unsaturation level of the isoprenoid would lead to reduced membrane rigidity. Inversely, LP or HT lead to lower unsaturation levels (0.9 and 2.2 nu, respectively), indicating an increase of rigidity. This is the first report on the homeoviscous adaptation in a piezophilic, hyperthermophilic archaeon. Interestingly, the adaptation mechanism proposed forT. barophilus is strikingly different from that observed in another hyperthermophilic archaeonMethanocaldococcus janaschii, which regulates its membrane fluidity by modifying the proportions of macrocyclic archaeol and tetraether lipids. T. barophilus constitutes the first example of homeoviscous adaptation that does not involve the modulation of the unsaturation level of the core lipids of the membrane, but that of other structural lipids. Reference · Haines, T.H. (2001) Progress in Lipid Research 40: 299–324.

abstracts book 111 O20EXTREMOphiles_2012 Genomic and metagenomic studies of the abundance of acidic proteins in halophilic microorganisms and hypersaline environments Aharon Oren and Rahel Elevi Bardavid

Department of Plant and Environmental Sciences, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel E-mail: [email protected]

There are two strategies for halophilic microorganisms to achieve osmotic balance. Most halophiles use organic compatible solutes and maintain low intracellular ion concentrations. No drastic modification of cellular proteins is then needed. The Archaea of the family Halobacteriaceae and Salinibacter (Bacteroidetes) accumulate molar concentrations of KCl and they have adapted their intracellular proteome to function at high salt solutions. Their proteins show a large excess of acidic over basic amino acids. We made a survey of the relative abundance of acidic amino acids and of proteins of low isoelectric point in different hypersaline environments based on genomic and metagenomic data. There has been a report of a significantly acid-shifted proteome in a hypersaline (9% salt) microbial mat in Guerrero Negro, Mexico (Kuninet al. 2008). A comparison with the proteins encoded by the genomes of moderately halophilic aerobes (Halomonas elongata, Chromohalobacter salexigens) and marine bacteria (Alteromonas and Vibrio spp.) shows that the average isoelectric point 6.8 reported for the Guerrero Negro mat is not unusually low and does not indicate a salt-adapted proteome (Elevi Bardavid and Oren, 2012a). The proteins encoded by the metagenome of the Dead Sea were highly acidic, as expected based on the dominance of Halobacteriaceae in the community. Halophilic fermentative bacteria (order Halanaerobiales) were earlier shown to accumulate KCl, and therefore an acidic proteome was predicted. Past reports showed an apparent large excess of acidic amino acids in cell hydrolysates. However, genomic analysis did not indicate high contents of acidic amino acids. The earlier reported excess of acidic amino acids is due to a high content of Gln and Asn, which yield Glu and Asp upon acid hydrolysis (Elevi Bardavid and Oren, 2012b).

References · R. Elevi Bardavid and A. Oren (2012a). Acid-shifted isoelectric point profiles of the proteins in a hypersaline microbial mat – an adaptation to life at high salt concentrations? Extremophiles, submitted for publication. · R. Elevi Bardavid and A. Oren (2012b). The amino acid composition of proteins from anaerobic halophilic bacteria of the order Halanaerobiales. Extremophiles, in press. doi: 10.1007/s00792-012-0455-y. · V. Kunin, J. Raes, J.K. Harris, et al. (2008). Millimeter-scale genetic gradients and community-level molecular convergence in a hypersaline microbial mat. Mol Systems Biol 4: 198.

112 abstracts book EXTREMOphiles_2012O21 Comparison of viral assemblages from different hypersaline environments Fernando Santosa, Manuel Martínez-Garcíaa, Judith Villamora, Víctor Parrob, Ramon Rosselló-Mórac and Josefa Antóna

aDepartamento de Fisiología, Genética y Microbiología, Universidad de Alicante, 03080, Alicante, Spain bDepartamento de Evolución Molecular, Centro de Astrobiología (INTA-CSIC), Torrejón de Ardoz, 28850, Madrid, Spain cMarine Microbiology Group, Institut Mediterrani d’Estudis Avançats (CSIC-UIB), 07190 Esporles (Mallorca), Spain E-mail: [email protected]

Hypersaline environments harbor the highest viral densities reported so far for aquatic systems, with concentrations up to more than 109 virus-like particles (VLP) per milliliter (Santoset al., 2012). This abundance, together with the fact that viruses are likely the most important biological agents at very high salinities controlling prokaryotic abundance, make the study of haloviruses a central aspect in the description of halophilic microbial communities. In addition, as also found in marine systems, viruses seem to play a key role in the diversification and evolution of halophilic microorganisms. However, to date only a few dozens of halophilic viruses have been isolated and most of them infect hosts that may not be ecologically relevant in hypersaline environments. In fact, there are not published reports on the isolation of viruses infecting two of the most conspicuous extremely halophilic prokaryotes (the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber). To circumvent the bias introduced by cultivation, we have studied several hypersaline environments using a series of culture independent techniques that include transmission electron microscopy, pulsed field gel electrophoresis and analysis of viral metagenomic libraries. In addition, since isolated virus can be essential tools for testing hypothesis on their interaction with hosts, viruses infecting S. ruber have also been isolated and their genomes compared with the available hypersaline metaviromes. The systems studied (two talassohaline multi pond salterns in SE Spain and Tunisia, and an atalassohaline lake in central Spain) harbour rather different microbial communities whose diversity is mirrored by their viral assemblages, at the different levels analyzed, from the GC content and dinucleotide frequency of their genomes to morphotype distribution. However, there are some traits common to the different viral metagenomes that reveal their «hypersalineness» quality.

References · F. Santos, P. Yarza, V. Parro, I. Meseguer, R. Rosselló-Mora, and J. Antón (2012). Culture-independent approaches for studying viruses from hypersaline environments. Appl Environ Microbiol 78: 1635-1643.

abstracts book 113 O22EXTREMOphiles_2012 Genomic characterization of a novel Nanoarchaeota system from Obsidian Pool, Yellowstone National Park Mircea Podara,b Kira S. Makarovac, David E. Grahama,b, Eugene V. Kooninc and Anna-Louise Reysenbachd

aMicrobiology Department, University of Tennessee, Knoxville TN 37996, USA bBiosciences Division, Oak Ridge National Laboratory, Oak Ridge TN 37831, USA cNational Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20894, USA dBiology Department, Portland State University, Portland OR 97201, USA Email: [email protected]

A decade after its discovery, the proposed archaeal phylum Nanoarchaeota is still represented by a single cultivated species, the marine hyperthermophile Nanoarchaeum equitans. An ectosymbiont/parasite on the surface of another archaeon, the Crenarchaeota species Ignicoccus hospitalis, N. equitans has no apparent primary metabolism and harbors unique genomic features that have spurred many evolutionary questions. While Nanoarchaeota rRNA sequences have been identified from many high temperature marine and terrestrial locations around the globe, the absence of genomic data or cultured isolates has hampered progress in understanding the biology of these organisms and their placing within the Archaea. We now present a genomic characterization of the first representative of a terrestrial Nanoarchaeota and its likely host, obtained from the Obsidian Pool, Yellowstone National Park using single cell isolation and genomic sequencing. Over 600 genes were identified from the Nanoarchaeota cells and over 1200 from the associated, likely host organism. Comparative genomics and evolutionary analyses suggest the Obsidian Pool Nanoarchaeota have retained some primary metabolic functions and offer new perspectives towards understanding the evolution of this group, their interaction with different archaeal hosts as well as strategies for isolation and characterization in pure culture.

114 abstracts book EXTREMOphiles_2012O23 Microbial ecology of endolithic life in the Atacama Desert Courtney Robinsona, Tiffany Souterrea, Jacek Wierzchosb, Jacques Ravelc, Octavio Artiedad Carmen Ascasob and Jocelyne DiRuggieroa

aDepartment of Biology, the Johns Hopkins University, Baltimore, MD 21218-2685, USA bMuseo Nacional de Ciencias Naturales, MNCN-CSIC, 28006 Madrid, Spain cInstitute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA dUniversidad de Extremadura, 10600 Plasencia, Spain E-mail: [email protected]

The Atacama Desert is the oldest and driest desert in the world and its hyper-arid core is described as «the most barren region imaginable». Under these extreme moisture, thermal, and solar radiation stress, habitats inside translucent rocks harbour flourishing microbial communities. We use high throughput sequencing to characterize endolithic communities from several rock substrates collected in the hyper-arid core of the Desert. Chasmoendolithic communities colonizing the fissures of volcanic rhyolite covered by gypsum crust and carbonate sedimentary rocks were uniquely bacterial and dominated by Chroococcidiopsis cyanobacteria (DiRuggiero et al., 2012). Significant differences in community structure and composition were observed between the gypsum and calcite rocks that correlated with geomorphic features of the substrates. The composition of these rock communities supports the idea of a global meta-community of microorganisms adapted to the endolithic environment. In contrast, microbial communities inside salt rocks (halites) were dominated by phototrophs unique to the Atacama Desert and associated with both bacteria and archaea. Here again, community diversity was driven by water availably, and while the Halothece-like cyanobacterium was found in all halites, a completely novel algae was only detected in halites with the highest relative humidity. Endolithic habitats in the Atacama Desert are another example of the diversity of adaptive strategies at the limit for life and can be considered as environmental refuges for life.

References · DiRuggiero J., J. Wierzchos, C. Robinson, T. Souterre, J. Ravel, O. Artieda, V. Souza-Egipsy, and C. Ascaso. (2012). Microbial colonization of chasmoendolithic habitats in the hyper-arid core of the Atacama Desert. Submitted.

abstracts book 115 O24EXTREMOphiles_2012 New tools for functional analysis of genes and meta/genomes David Mead, Phil Brumm, Tom Schoenfeld, Colleen Drinkwater, Jan Deneke, Ronald Godiska and Eric Steinmetz

Lucigen Corp., Middleton WI, USA E-mail: [email protected]

The efficient capture and functional analysis of genes and metabolic pathways is constrained by the choice of available tools. Conventional expression systems based on pET vectors or bacteriophage lambda are surprisingly inefficient at random shotgun expression screening from meta/genomic samples. They also require large amounts of time and reagents for PCR directed expression experiments. A simple system that efficiently captures and expresses ~ 5 kb segments of DNA has been developed and validated through the discovery of hundreds of new carbohydrate metabolizing enzymes (CAZymes) from dozens of genomes and metagenomes. This new tool is ~ 50 fold more efficient for shotgun expression screening than conventional systems. A directed cloning version has been developed that uses in vivo homologous recombination to seamlessly join PCR amplified genes with pre-processed plasmids, eliminating numerous time consuming and expensive reagents and steps. Unlike other cloning systems, no vector preparation, restriction or modifying enzymes, or purification steps are required. The desired insert is simply amplified with primers that include 18 bases of overlap with the ends of a small expression vector, and mixed directly with the plasmid preparation and competent cells. >90% of clones have the target gene inserted in the correct orientation. Using a multiplex assay for endo and exo-cellulases and hemicellulases in a microplate format, we were able to efficiently screen thousands of CAZymes/week without robotics. We describe the expression, purification and characterization of over a hundred different CAZymes from thermophilic, mesophilic and alkaliphilic microbes including Dictyoglomus turgidum, Acidothermus cellulolyticus, Fibrobacter succinogenes, and Bacillus cellulosilyticus. A second tool has been developed that can clone and express ~ 30 kb segments of random DNA or large metabolic pathways. Cloning and expression of large operons with conventional vectors presents numerous challenges due to: 1) Secondary structure associated with repetitive sequences or high AT-content, which can lead to DNA deletions; 2) Unregulated transcription that can select against toxic, but potentially valuable genes; 3) High copy number vectors and gene dosage that creates a large metabolic burden on the cell; 4) Strong promoters can that cause overexpression of deleterious proteins; 5) Gene instability due to superhelical stress seen in large insert circular plasmids; and 6) General decrease in cloning efficiency with larger fragments. We have developed a new pathway capture and engineering tool to clone and express multiple genes from large operons. A linear «pJAZZ» vector for E. coli expression readily holds large inserts (up to 30 kb) of DNA. Its low copy number (2-4/cell) is ideal for high stability protein production. Arabinose-inducible amplification of copy number (5-20 fold) further improves cloning of difficult sequences. The ease with which these clones can be handled and modified will permit the rapid study of a large number of permutations. The system has been validated for expression of the nine gene MEP isoprene pathway. We are now screening for thermostable DNA polymerases and their flanking accessory proteins from hot spring viral metagenomic samples.

116 abstracts book EXTREMOphiles_2012O25 Heterologous gene expressions in Geobacillus kaustophilus HTA426 for generating functional thermophiles Hirokazu Suzukia, Toshihisa Ohshimab and Yutaka Kawarabayasia

aFunctional Genomics of Extremophiles and bDepartment of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, Japan E-mail: [email protected]

Bioprocesses functioning at elevated temperatures have several advantages (Wiegelet al., 1986). We have focused on the thermophile Geobacillus kaustophilus HTA426 as a potential host for various high-temperature bioprocesses and recently established its genetic transformation system. This thermophile, isolated from a deep- sea sediment of the Mariana Trench, is able to grow under aerobic conditions between 42°C and 74°C (optimally at 60°C). The genome sequence and the abundant biological knowledge for the related bacilli allow us to facile predictions of gene functions of strain HTA426. Here we report an endogenous promoter that is inducible and highly expressed in strain HTA426. This was further utilized for functional expressions of heterologous genes in strain HTA426, which generated biomass-degrading thermophiles. Seven endogenous promoters were predicted on the basis of genome information of strain HTA426 and characterized using â-galactosidase reporter assay. This analysis revealed that an upstream region of GK0704 (gk704 promoter) directed high-level expression of the reporter gene in maltose-containing conditions. To test the efficiency of gk704 promoter for gene expressions, we constructed high-copy number plasmids containing heterologous genes under the gk704 promoter. G. kaustophilus were transformed by the plasmids and cultured in the presence of 1% maltose. From the cultured cells, proteins were extracted and purified using Ni2+-affinity chromatography to reveal various genes were successfully expressed in G. kaustophilus. Among genes examined, a thermostable cellulase from Pyrococcus horikoshii was extremely expressed (the productivity, >50 mg/L culture). The transformants were able to be utilized for biomass degradations at elevated temperatures.α -Amylase producer effectively degraded corn starch. Cellulase producer effectively degraded paper filters. These results suggest the possibility that practical higher-temperature bioprocesses may be generated by usingG. kaustophilus host and genes encoding useful thermostable enzymes.

Reference · J. Wiegel and L. G. Ljungdahl (1986). The importance of thermophilic bacteria in biotechnology. Crit Rev Biotechnol 3: 39-108.

abstracts book 117 O26EXTREMOphiles_2012 Mannosylglucosylglycerate biosynthesis in Rhodopirellula baltica,a representative of the ancient phylum Planctomycetes Sofia Cunhaa, Filipa d’Avóa, Ana Mingoteb, Pedro Lamosac, Milton S. da Costaa,d and Joana Costaa,d

aCentro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal bInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal cCentro de Ressonância Magnética António Xavier, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal dDepartment of Life Sciences, University of Coimbra, Apartado 3046, 3001-401 Coimbra, Portugal E-mail: [email protected]

Mannosylglucosylglycerate (MGG) has only been identified as a compatible solute in the genus Petrotoga where the three step biosynthetic pathway has been described (Jorge et al., 2007, Fernandes et al., 2010). Recently, the accumulation of MGG was verified in Rhodopirellula baltica, a member of the monophyletic phylum Planctomycetes, whose members share several peculiar traits, such as a budding mode of reproduction and the lack of peptidoglycan in their cell walls. The most intriguing feature of this group is the existence of a single or double membrane around the chromosome that has been compared to the eukaryotic nucleus. The most recent view is that planctomycetes are indeed a deep-branching phylum, perhaps more ancient than hyperthermophilic phyla such as the Aquificales (Fuerst and Sagulenko, 2012). Homologues for the first two steps of the biosynthetic pathway of MGG inPetrotoga mobilis were detected in R. baltica genome indicating that the same pathway may be active (glucosyl-3-phosphoglycerate synthase (GpgS) and mannosylglucosyl-3-phosphoglycerate synthase (MggA). Additionally, we identified a putative hydrolase that could be involved on the last step of MGG synthesis (MggC). The recombinant GpgS catalyzed the synthesis of glucosyl-3-phosphoglycerate (GPG) from UDP-glucose and D-3-phosphoglycerate; the recombinant MggA catalyzed the synthesis of mannosylglucosyl-3-phosphoglycerate (MGPG), using the product of GpgS activity (GPG) and GDP-mannose as substrates; and finally, MggC catalyzed the specific dephosphorylation of MGPG to MGG. The recombinant GpgS had optimal activity at 60°C while MggA ad MggC had optimal activity at 40°C. In this work, we propose a three-step pathway proceeding through phosphorylated intermediates for the synthesis of the rare compatible solute MGG in R. baltica.

References · C. D. Jorge, P. Lamosa, and H. Santos (2007). α-D-mannopyranosyl-(1’!2)-α-D-glucopyranosyl-(1→2)- glycerate in the thermophilic bacterium Petrotoga miotherma—structure, cellular content and function.FEBS J 274: 3120-3127. · C. Fernandes et al. (2010). Two alternative pathways for the synthesis of the rare compatible solute mannosylglucosylglycerate in Petrotoga mobilis. J Bacteriol 192: 1624-1633. · J. A. Fuerst and E. Sagulenko (2012). Keys to eukaryality: planctomycetes and ancestral evolution of cellular complexity. Front Microbiol 3: 167.

118 abstracts book EXTREMOphiles_2012O27 Genetic and phenotypic analysis of a two-component regulatory system in the moderately halophilic bacterium Halomonas anticariensis Ali Tahrioui, Inmaculada Llamas and Emilia Quesada

Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain E-mail: [email protected]

Two-component systems (TCSs) in bacteria help them to cope with changing environments. Among TCSs, the GacS/GacA system is well preserved in a variety of Gram-negative bacterial genera and it has been reported to regulate an array of phenotypes. Halomonas anticariensisFP35 T is a Gram-negative bacterium isolated from saline soils at Fuente de Piedra, Málaga, in southern Spain (Martínez-Cánovas et al., 2004). We have reported that this bacterium produces a series of quorum-sensing (QS) signal molecules, N-acyl homoserine lactones (AHLs) of many sizes (Llamas et al., 2005) and we have recently identified and characterized the quorum-sensing genes hanR/hanI involved in the production of these AHLs (Tahrioui et al., 2011). During attempts to determine the QS system locus we identified and characterized a possible multi-hybrid sensor histidine kinase GacS and a response regulator GacA, which probably constitute a TCS inH. anticariensis FP35T. To investigate the role of this TCS we generated mutants FP35gacS and FP35gacA, which produced smaller quantities of AHLs and exopolysaccharide than the wild-type strain. Thus the formation of biofilm, as measured by quantitative microtiter plate adherence, was significantly reduced. Furthermore, RT-PCR analysis revealed a considerable decrease in the expression of the quorum-sensing genes hanR and hanI compared to the wild type strain. We show the GacS/GacA TCS to be an all-embracing regulatory system that exerts a positive effect upon the production of AHLs and EPS and the formation of biofilm.

References · A. Tahrioui, E. Quesada, and I. Llamas (2011). The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium. Microbiology 157: 3378-3387. · I. Llamas, E. Quesada, M. J. Martínez-Cánovas, M. Gronquist, A. Eberhard, and J. E. González, (2005). Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide- producing species of Halomonas. Extremophiles 9: 333-341. · M. J. Martínez-Cánovas, V. Béjar, F. Martínez-Checa and E. Quesada (2004). Halomonas anticariensis sp. nov., from Fuente de Piedra, a saline-wetland, wildfowl reserve in Málaga, Southern Spain. Int J Syst Evol Microbiol 54: 1329-1332.

abstracts book 119 O28EXTREMOphiles_2012 Production of rhamnolipid biosurfactants by thermophilic bacteria cultivated on sunflower oil and glycerol Lucie Siříšťováa, Věra Šalplachtováa and Tomáš Řezankab aDepartment of Biotechnology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, 166 28 Prague, Czech Republic bInstitute of Microbiology, 142 20 Prague, Czech Republic E-mail: [email protected]

The aim of our study was to assess the ability of thermophilic bacteria to produce rhamnolipids, which are biosurfactants with great biotechnological potential. We detected rhamnolipids in cultivation media of four thermophilic bacteria belonging to genera Thermus and Meiothermus. Rhamnolipids were extracted, purified by TLC and identified by tandem mass spectrometry. The strains produced both types, mono- and di-rhamnolipids. We revealed 77 rhamnolipid homologues varying in chain length and unsaturation. Thermus thermophilus DSM 579 was selected for further study of the rhamnolipid production under different cultivation conditions. Sunflower oil and glycerol were tested as sole carbon sources at different cultivation temperatures. The spectrophotometric phenol-sulphuric acid method was used for direct measurement of rhamnose concentration in cultivation medium (Dubois et al., 1956). In terms of biomass growth and rhamnolipid production, glycerol was found a more suitable substrate than sunflower oil. Under non-optimal cultivation temperatures the selected strain showed a great decrease in biomass yield as well as in the production of rhamnolipids. New rhamnolipid-producing thermophilic strains were identified as significant sources of rhamnolipids. The rhamnolipids obtained from these strains differ in structure from the most studied ones from Pseudomonas aeruginosa and exhibit excellent surfactant properties.

References · M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and F. Smith (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28: 350-356. · T. Rezanka, L. Siristova and K. Sigler (2011). Rhamnolipid-producing thermophilic bacteria of species Thermus and Meiothermus. Extremophiles 15: 697-709.

120 abstracts book EXTREMOphiles_2012O29 Secondary metabolites from deep sea bacteria: an efficient route for biodiscovery from extreme marine habitats? Antje Labes, Jan Oesterwalbesloe and Johannes F. Imhoff

Kieler Wirkstoff-Zentrum at GEOMAR – Helmholtz Centre for Ocean Research, 24106 Kiel, Germany E-mail: [email protected]

The deep sea is an extreme environment which is still marginally investigated and harbors a great variety of bacteria that have, so far, not been cultivated. Bacteria which live in the deep sea need to adapt to the specific environmental characteristics such as high hydrostatic pressure, low temperature and only occasional nutrient supply. These constraints quite likely determine the phylogenetic diversity of the deep-sea bacterial communities and also affect the secondary metabolite production of these bacteria. However, the potential can be only used and translated to new products having an efficient strategy for biodiscovery from these environments.

Marine members of Actinobacteria are highly potent producers of interesting compounds (Lam, 2006; Jenses et al., 2005; Fiedler et al., 2005) as was already shown for their terrestrial counterparts. Using the rising number of recently isolated deep sea members of Actinobacteria we will show targeted approaches for biodiscovery of new natural products using e.g. selective isolation as well of rational experimental designs for cultivation (Imhoffet al., 2011).

References · Lam, K.S. (2006) Discovery of novel metabolites from marine actinomycetes.Curr Opin Microbiol 9: 245-251. · Jensen, P.R. Mincer, T.J. Williams, P.G. Fenical, W. (2005) Marine actinomycete diversity and natural product discovery. Antonie van Leeuwenhoek 87: 43-48. · Fiedler, H.P. Bruntner, C. Bull, A.T. Ward, A.C. Goodfellow, M. Potterat, O. Puder, C. Mihm, G. (2005) Marine actinomycetes as a source of novel secondary metabolites.Antonie van Leeuwenhoek 87:37-42. · Imhoff, J.F. Labes, A. Wiese, J. (2011) Bio-mining the microbial treasures of the ocean: new natural products. Biotechnology Advances 29: 468-482.

abstracts book 121 O30EXTREMOphiles_2012 Methods to improve productivity and applicability of an in vitro translation system using the lysate of Thermococcus kodakarensis Tamotsu Kanaia,c, Yuki Sakaia, Daijiro Ikegamia, Takashi Endoha , Tadayuki Imanakab,c and Haruyuki Atomia,c

aDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto, 615-8510 Japan bDepartment of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577 Japan cJST, CREST, Sanbancho, Chiyoda-ku, Tokyo, 102-0075 Japan E-mail: [email protected]

We have previously constructed an in vitro translation system using the lysate of a hyperthermophilic archaeon, Thermococcus kodakarensis (Endoh et al., 2006). With this system, protein production was observed within a temperature range of 40–80°C, with an optimum at 60-65°C. To increase the productivity and the applicability of this system, the following improvements were made. First, the lysate preparation procedure and the concentrations of each reaction component were examined. Second, a mutant strain of T. kodakarensis was constructed in which a heat shock transcriptional regulator gene,phr , was disrupted. By using the lysate prepared from the mutant, an elevation in the optimum reaction temperature was observed by 5°C. Third, recombinant archaeal elongation factor 1α (aEF-1α) was added to the reaction mixture.aEF-1α binds to aminoacyl-tRNAs in cells and protects them from hydrolysis. By the addition of 4 ìM aEF-1á in the reaction mixture, the final product concentration increased by about 20%. As for the mRNA added to the system, addition of a stem-loop structure at the 3’-end of mRNA enhanced protein production by several-fold. Furthermore, we have developed a reaction system that is coupled with in vitro transcription. The in vitro transcription system employed transcriptional machinery from the extremely thermophilic bacterium, Thermus thermophilus. With the RNA polymerase and sigma factor from T. thermophilus, an in vitro transcription system was developed that exhibits mRNA synthesis at 65°C. When this system was combined with the T. kodakarensis in vitro translation system, protein synthesis was detected at 65°C using DNA as a template.

References · T. Endoh, T. Kanai, Y. T. Sato, D.V. Liu, K. Yoshikawa, H. Atomi, T. Imanaka (2006). Cell-free protein synthesis at high temperatures using the lysate of a hyperthermophile. J Biotechnol 126: 186-195.

122 abstracts book EXTREMOphiles_2012OL1

posters

abstracts book 123 OL1EXTREMOphiles_2012

124 abstracts book EXTREMOphiles_2012

session 1 diversity and ecology

abstracts book 125 OL1EXTREMOphiles_2012

126 abstracts book EXTREMOphiles_2012P1 Characterization and identification of two hyperthermophilic bacteria isolated from a hot spring in Thailand: a Thermotoga sp. strain FC 1002 and a Fervidobacterium sp. strain FC 2004 Wirojne Kanoksilapathama, Porranee Keawrama, Patlada Pasomsupa, Maria C. Portillob and Juan M. Gonzálezb

aDepartment of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom, 73000, Thailand bIRNAS-CSIC, Avda. Reina Mercedes 10, 41012 Sevilla, Spain Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain E-mail: [email protected]

The aims of this study were to characterize growth kinetics and to classify taxonomic positions of two obligately anaerobic, heterotrophic, hyperthermophilic bacteria, named in this study as «FC 1002» and «FC 2004». Both strains were isolated from sediment samples collected at the water sources (95-100 oC) of Mae Fang Hot Spring, Northern Thailand. Pure cultures of the isolates were obtained using a tube dilution technique conducted successively. Strain FC 1002 was characterized as a Gram-nonreactive, non-endospore forming rod. Ensheathed cells with a terminal toga at each polar arranging singly and doubly were detected. A size range of 0.4-0.6x2.5-5 µm was measured. Polar to subpolar rhizoid filaments were frequently observed under SEM. Growths of strain FC 1002 occurring at temperature range of 75 – 90 oC (opt. temp. 85 oC), pH range of 6 – 9 (opt. pH 7.5), and NaCl concentration range of 0 – 0.5% (opt. conc. 0.05-0.1% NaCl) were determined. The 16S rRNA gene sequence of strain FC 1002 (JF339227) was identified. Blast result revealed highly related to that ofThermotoga neapolitana (98%), T. maritima (97%), T. petrophila (97%), T. thermarum (94%), T. hypogea (92%), T. lettingae (91%), Thermosipho atlanticus (90%), Tsp. geolei (90%), Tsp. japonicus (89%), and Thermopallium natronophilum (87%). The G+C content of chromosome of strain FC 1002 was 41.3 mol%. Strain FC 2004 was characterized as a Gram-negative, non-endospore forming rod. Ensheathed cells with a balloon-like toga at one polar arranging singly and doubly were observed. A size range of 0.5-0.7x1.5-2 µm was measured. Few thick sheathed filamentous cells with the size up to 30 µm long were detected. Growths of strain FC 2004 occurring at temperature range of 65 – 88 oC (opt. temp. 75-85 oC), pH range of 6 – 9 (opt. pH 7.5), and NaCl concentration range of 0 – 0.3% (opt. conc. 0.0-0.1% NaCl) were determined. The16S rRNA gene sequence of strain FC 2004 (JF339224 and JF339225) was identified. Blast result revealed highly related to that of Fervidobacterium pennivorans (96%), F. islandicum (96%), F. gondwanense (93%), F. nodosum (92%), Thermopallium natronophilum (91%) and Tsp. atlanticus (86%). The G+C content of chromosome of strain FC 2004 was 44.0 mol%. Phylogenetic analyses revealed that strain FC 1002 belonged to genus Thermotoga, and strain FC 2004 belonged to genus Fervidobacterium. In addition,16S rRNA gene sequences belonging to Thermotoga tested were categorized into two distinct groups; and strain FC 1002 was positioned in the group asThermotoga petrophila, T. neapolitana and T. maritime; but not Thermotoga thermarum, T. hypogea, T. subterranea, T. elfii, and T. lettingae. Based on the morphological characteristics, the 16S rRNA gene sequences, and phylogenetic analyses, strain FC 1002 was thus identified as a Thermotoga sp. strain FC 1002, and strain FC 2004 as a Fervidobacterium sp. strain FC 2004. These results indicate that strains FC 1002 and FC 2004 are distinct from their close relatives.

abstracts book 127 P2EXTREMOphiles_2012 Characterization of moderately and extremely halophilic microorganisms from salt-pack cured hides Pinar Caglayana,b, Cristina Sanchez-Porroa, Antonio Ventosaa and Meral Birbirb

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bDivision of Plant Diseases and Microbiology, Department of Biology, Faculty of Science and Letters, Marmara University, 34722 Istanbul, Turkey E-mail: [email protected]

The presence of microorganisms in high numbers originated from external and internal sources on hides affect leather quality adversely.The aim of the study was to isolate and identify moderately halophilic bacteria and extremely halophilic archaea which may cause damage on the salt-pack cured hides and evaluate quality of the salted hide. The hides used in the present study were imported from England and Australia. Total numbers of halophilic, proteolytic and lipolytic halophilic isolates on the salted hide samples were examined and hydrolytic activities of these isolates were determined. Total numbers of halophilic, proteolytic and lipolytic halophilic isolates on the salted hide samples imported from England were found as 106-107 cfu/g, 105 cfu/g and 105 -106 cfu/g, respectively, while total numbers of halophilic and proteolytic halophilic isolates on the salted hide samples imported from Australia were on the range 102 cfu/g, 101 -102 cfu/g, respectively. According to phenotypic characteristics and comparative partial 16S rRNA sequence analysis, 13 moderately halophilic bacterial strains, isolated from the salt pack-cured hides imported from England (9 isolates) and Australia (4 isolates), were identified as Salimicrobium album, Salimicrobium halophilum, Halomonas eurihalina, Salimicrobium luteum, Halomonas koreensis, Halomonas elongata, Halomonas halmophila, Halomonas alimentaria, Marinococcus halophilus, Chromohalobacter salexigens, Oceanobacillus picturae, Thalassobacillus devorans and Alkalibacillus salilacus. In addition, 5 extremely halophilic archaeal strains, isolated from the hides imported from England (3 isolates) and Australia (2 isolates), were identified asHalorubrum saccharovorum, Halorubrum tebenquichense, Halorubrum lacusprofundi, Natrinema pallidum and Natrinema gari. In this study, hydrolytic activities have been detected in most moderately halophilic isolates. Results of protease, amylase, lipase, pullulanase, xylanase, caseinase, urease and DNase tests revealed that the isolated strains produced hydrolytic enzymes, which may cause deterioration of the salt-pack cured hides. We concluded that the isolates on the salted hides were active to damage the hide substance. The determination and characterization of proteolytic and lipolytic halophilic isolates and their hydrolytic activities provides important information of the quality of salted hides. This study is the first research that taxonomically characterizes moderately halophilic bacteria and extremely halophilic archaea on the salted hides.

128 abstracts book EXTREMOphiles_2012P3 Polyphasic characterization of a new extremely halophilic bacterium isolated from an Iranian hypersaline lake Seyed Abolhassan Shahzadeh Fazelia,f, Mohammad Ali Amoozegara,b, Ali Makhdoumi-Kakhkia,c, Mohadeseh Ramezania, Peter Schumannd and Antonio Ventosae

aMicroorganisms Bank, Iranian Biological Resource Centre (IBRC), ACECR Tehran, Iran bExtremophiles Lab., Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran cDepartment of Biology, School of Sciences, Ferdowsi University of Mashhad, 91775-1436 Mashhad, Iran dDSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany eDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain fDepartment of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran E-mail: [email protected]

Halophilic bacteria are represented by heterogeneous physiological and taxonomic groups. Studies of such bacteria are of great importance, as they may produce compounds of industrial interest, such as extracellular hydrolytic enzymes that have diverse potential use in biotechnological processes. The aim of the study was the taxonomic characterization of an extremely halophilic bacterium isolated from Aran-Bidgol hypersaline lake in Iran. A polyphasic approach including phenotypic, chemotaxonomic and phylogenetic characterization was used for the identification of the new extremely halophilic bacterium. During the study of the microbial population in hypersaline lakes in Iran, an aerobic, Gram-staining-negative, non-pigmented, rod shaped, extremely halophilic bacterium, designated strain IA16T, was isolated and characterized. Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed that strain IA16T is a member of the family Rhodospirillaceae; however its similarity was as low as 91.6 %, 88.9 % and 88.7 % to the most closely related taxa including Rhodovibrio, Pelagibius and Fodinicurvata type species, respectively. Growth occurred between 2.5 and 5.2 M NaCl and optimally at 3.4 M NaCl. The optimum pH and temperature for growth of strain IA16T were pH

7.0 and 40 °C, respectively. The major cellular fatty acids of the isolate were C19:0 cyclo ω7c and C18:0. The polar lipid pattern of strain IA16T consisted of diphosphatidylglycerol, phosphatidylglycerol, two phospholipids and two glycolipids. The G+C content of the genomic DNA of this strain was 67.0 mol%. On the basis of polyphasic evidence from this study, IA16T is suggested as representative of a new species in a new genus within the family Rhodospirillaceae, for which the name Limimonas halophila gen. nov., sp. nov. is proposed.

abstracts book 129 P4EXTREMOphiles_2012 Thermosulfurimonas dismutans gen. nov., sp. nov. a novel extremely thermophilic sulfur- disproportionating bacterium from a deep-sea hydrothermal vent Alexander Slobodkina, Anna-Louise Reysenbachb, Galina Slobodkinaa, Roman Baslerovc, Nadezhda Kostrikinaa, Icaak Wagnerb and Elizaveta Bonch-Osmolovskayaa

aWinogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, 117312 Moscow, Russia bDepartment of Biology and Center for Life in Extreme Environments, Portland State University, PO Box 751, Portland, OR 97207-0751, USA cBioengineering Center, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/1, 117312 Moscow, Russia E-mail: [email protected]

Microbial sulfur disproportionation may represent a previously unrecognized process of primary organic matter production in thermal environments. An extremely thermophilic, anaerobic, chemolithoautotrophic bacterium (strain S95T) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Center, Pacific Ocean at a depth of 1910 m. Cells of strain S95T were oval to short Gram-negative rods, 0.5 to 0.6 μm in diameter and 1.0 to 1.5 μm in length, growing singly or in pairs. Cells were motile with a single polar flagellum. The temperature range for growth was 50-92oC, with an optimum at 74oC. The pH range for growth was 5.5-8.0, with an optimum at 7.0. Growth of strain S95T was observed at NaCl concentrations ranging from 1.5 T to 3.5% (w/v). Strain S95 grew anaerobically with elemental sulfur as an energy source and bicarbonate/CO2 as a carbon source. Elemental sulfur was disproportionated to sulfide and sulfate. The growth was enhanced in the presence of poorly crystalline Fe(III) oxide (ferrihydrite) as a sulfide-scavenging agent. Strain S95T was also able to grow by disproportionation of thiosulfate and sulfite. Sulfate was not used as an electron acceptor. Analysis of the 16S rRNA gene sequence revealed that the isolate belongs to the phylum Thermodesulfobacteria. On the basis of its physiological properties and results of phylogenetic analyses, it is proposed that the new isolate represents the sole species of a novel genus, Thermosulfurimonas dismutans gen. nov., sp. nov. with the type strain S95T (=DSM 24515T =VKM В–2683T). Thermosulfurimonas dismutans is a first described thermophilic microorganism that disproportionates elemental sulfur.

130 abstracts book EXTREMOphiles_2012P5 Deferrisoma camini gen. nov., sp. nov. a novel moderately thermophilic dissimilatory Fe(III)- reducing bacterium from a deep-sea hydrothermal vent that forms a distinct phylogenetic branch in Deltaproteobacteria Galina Slobodkinaa, Anna-Louise Reysenbachb, Angela Panteleevac, Nadezhda Kostrikinaa, Isaac Wagnerb, Elizaveta Bonch-Osmolovskayaa and Alexander Slobodkina

aWinogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, 117312 Moscow, Russia bDepartment of Biology and Center for Life in Extreme Environments, Portland State University, PO Box 751, Portland, OR 97207-0751, USA cBioengineering Center, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/1, 117312 Moscow, Russia E-mail: [email protected]

A moderately thermophilic, anaerobic, dissimilatory Fe(III)-reducing bacterium (strain S3R1T) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Center, Pacific Ocean at a depth of about 2150 m. Cells of strain S3R1T were ovals to short rods with a single polar flagellum, Gram-stain-negative, 0.5 to 0.6 µm in diameter and 0.8 to 1.3 µm in length, growing singly or in pairs. The temperature range for growth was 36-62oC, with an optimum at 50oC. The pH range for growth was 5.5- 7.5, with an optimum at 6.5. Growth of strain S3R1T was observed at NaCl concentrations ranging from 1.0 to 5.0% (w/v) with an optimum at 2.0-2.5% (w/v). The isolate used acetate, fumarate, malate, maleinate, succinate, propanol, palmitate, stearate, peptone and yeast extract as the electron donors for growth and Fe(III) reduction.

All electron donors were completely oxidized to CO2 and H2O. Fe(III) (in the form of ferrihydrite, Fe(III) citrate or Fe(III)-NTA) and S0 were the electron acceptors supporting growth. The DNA G+C content was 64.4 mol% Results of 16S rRNA gene sequence analysis showed that the novel bacterium was related to the representatives of the orders Desulfuromonadales and Syntrophobacterales with 84-86% sequence similarity and formed a distinct phylogenetic branch in Deltaproteobacteria. On the basis of its physiological properties and results of phylogenetic analyses, it is proposed that the new isolate represents the sole species of a novel genus, Deferrisoma camini gen. nov., sp. nov. with the type strain S3R1T (=DSM 24185T =VKM В-2672T).

abstracts book 131 P6EXTREMOphiles_2012 Natrinema salaciae sp. nov., a halophilic archaeon isolated from the deep, hypersaline anoxic Lake Medee in the Eastern Mediterranean Sea Luciana Albuquerquea, Marco Tabortdab, Violetta La Conoc, Michail Yakimovc and Milton S. da Costab,d

aCenter for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal bMicrobiology Unit, BIOCANT Biotechnological Park, 3060-197 Cantanhede, Portugal cInstitute for Coastal Marine Environment, CNR, Spianata S. Raineri 86, 98122 Messina, Italy dDepartment of Life Sciences, University of Coimbra, 3001-401 Coimbra, Portugal E-mail: [email protected]

Two halophilic archaea, strains MDB25T and MDB20, were isolated from a sample of the brine from Medee Lake, at a depth of 3050 meters, in the Mediterranean Sea. Cells of the organisms were Gram-negative, non-motile and pleomorphic, and colonies were red pigmented. Strains MDB25T and MDB20 showed optimum growth at 45 °C, in 2.6-3.4 M NaCl and at pH 7.0-8.0. The major polar lipids of the two strains were phosphatidylglycerol (PG1 and PG2), phosphatidylglycerol phosphate methyl ester (PGP-Me) and mannose-2,6-dissulfate (1g2)- glucose glycerol diether (S2-DGD). Menaquinone MK-8 and MK-8(H2) were the major respiratory quinones. The DNA G+C content of strain MDB25T was 63.0 %. The strains were facultatively anaerobic but grew better under aerobic conditions, nitrate served as electron acceptor. Analysis of the almost complete 16S rRNA gene sequence indicated that the strains MDB25T and MDB20 represented a member of the genus Natrinema in the family Halobacteriaceae. Both strains formed a distinct cluster and were most closely related toNatrinema ejinorense JCM 13890T and Haloterrigena longa JCM 13562T (98.0% and 97.9% sequence similarity, respectively). Based on 16S rRNA gene sequence analysis, physiological and biochemical characteristics we describe a new species represented by strain MDB25T (=DSM 25055T =JCM 17869T) for which we propose the name Natrinema salaciae sp. nov.

132 abstracts book EXTREMOphiles_2012P7 Characterization of novel Natronococcus strains and their polar lipidomic analysis Paulina Corrala, Roberto Angelinib, Patrizia Lopalcoc, Carmen Gutiérreza Thane Papked, Angela Corcellib,e and Antonio Ventosaa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bDepartment of Basic Medical Sciences, University of Bari «Aldo Moro», 70124 Bari, Italy cIMM-CNR, Institute for Microelectronics and Microsystems, National Research Council, 73100 Lecce, Italy dDepartment of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA eIPCF-CNR, Institute for Chemical-Physical Processes, National Research Council, 70124 Bari, Italy E-mail: [email protected]

A novel extremely haloalkaliphilic archaeon, strain CG-1T, belonging to the genus Natronococcus was isolated from sediment of the soda lake Chagannor in Inner Mongolia, China. The strain CG-1T was most closely related to the type strains Natronococcus amylolyticus Ah-36T and Natronococcus occultus SP4T, with which it shared 98.4 % and 95.7 % 16S rRNA gene sequence similarity, respectively. The DNA G+C content of strain CG-1T was 62.1 mol%. DNA–DNA hybridization with the type strain ofNatronococcus amylolyticusDSM 10524T, phylogenetically the most closely related species, was 39 %; this value showed that strain CG-1T constituted a different genospecies. The comparison of 16S rRNA gene sequences, detailed phenotypic characterization, polar lipid profile and DNA-DNA hybridization studies revealed that strain CG-1T belongs to the genus Natronococcus, and constitutes a novel species for which the name Natronococcus roseus sp. nov. is proposed. In addition, we analized the lipidome of two members of the genus Natronococcus: Ncc. occultus and Ncc. amylolyticus by means of combined thin-layer chromatography and MALDI-TOF/MS analysis in order to extends the knowledge on the presence and the structures of cardiolipins in archaeal membranes. The detailed investigation of lipid profiles confirmed the presence of phosphatidylglycerol and phosphatidylglycerophosphate methyl ester as main lipid components and some novel minor or trace phospholipids and glycolipids. The results indicate the presence of cardiolipin variants, lipids constituted by different combinations of isoprenoid chains of different lenghts, never before described in the archaea and whose role may be related to adaptation to alkaline pH gradients and high salt concentrations.

References · P. Lopalco, S. Lobasso, M. Baronio, R. Angelini and A. Corcelli (2011). Impact of lipidomics on the microbial world of hypersaline environments. In Halophiles and Hypersaline Environments, A. Ventosa, A. Oren and Y. Ma (eds.). Current Research and Future Trends, pp. 123-135. Springer: Heidelberg.

abstracts book 133 P8EXTREMOphiles_2012 Characterization of an extremely thermophilic bacterium, Coprothermobacter sp. PM9-2, isolated from an undersea petroleum reservoir Wataru Urushibataa, Tetsu Funayamab and Masaaki Morikawaa

aDivision of Biosphere Science, Graduate School of Environmental Science, Hokkaido University, Japan bDepartment of Material and Life Science, Graduate School of Engineering, Osaka University, Japan E-mail: [email protected]

Coprothermobacter sp. PM9-2 was isolated from an undersea petroleum reservoir in the South China Sea. The strain PM9-2 was an extremely thermophilic and anaerobic bacterium that have an optimum growth temperature at 65ºC and pH 6.5. Phylogenetic analysis of the 16rRNA gene placed PM9-2 on one of the deepest branch in the domain Bacteria. The petroleum reservoir was located at 500 meters depth from the sea level. Elemental composition of water in the reservoir was Na (140 ppm), Mg (23.3 ppm), Ca (2.33 ppm low), and Si (46.8 ppm high) that was different from the sea water. This fact indicated that the reservoir water was isolated from the sea. In order to shed light on the ecological position of PM9-2 in undersea petroleum reservoir, its metabolisms were analyzed. PM9-2 grew well in the medium containing sugar or protein as carbon source. PM9-2 produced acetate,

H2 and CO2 as major metabolites. However, the cells growth was inhibitied by H2. Since methane was produced in the resorvoir, PM9-2 was suggested to have syntrophic relationship with methanogen that utilize H2 and CO2 or acetate. We are planning to co-culture PM9-2 and methanogen to verify this hypothesis.

References · B. M. Ollivier, R. A. Mah, T. J. Ferguson, D. R. Boone, J. L. Garcia and R. Robinson (1985). Emendation of the Genus Thermobacteroides: Thermobacteroides proteolyticus sp. nov., a Proteolytic Acetogen from a Methanogenic Enrichment. International Journal of Systematic Bacteriology 35: 425-428. · C. Etchebehere, L. Muxi (2000). Thiosulfate reduction and alanine production in glucose fermentation by members of the genus Coprothermobacter. Antonie van Leeuwenhoek 77: 321-327.

134 abstracts book EXTREMOphiles_2012P9 High genetic diversity and novelty of planktonic microeukaryotes inhabiting inland and coastal hyperhaline waters Xavier Triadó-Margarit and Emilio O. Casamayor

Department of Continental Ecology, Center for Advanced Studies of Blanes-CEAB, Spanish Council for Research-CSIC, 17300 Blanes, Spain E-mail: [email protected]

We analyzed the genetic diversity of microbial eukaryotes (size range 0,2-40µm ) along the salinity gradient in 21 different coastal and inland salt ponds by DGGE fingerprinting and 18S rRNA gene sequencing. A wide range of environmental conditions was covered: two orders of magnitude in the salinity gradient (from 0.38 to 38.4%), different biomes with contrasted salt composition (semi-arid inland endorheic regions and coastal salterns), and different water (permanent and temporal), and connectivity (isolated pools and connected solar salterns ponds) regimes. The genetic diversity was high and the sequences were spread throughout nine high-rank taxonomic groups and 29 eukaryal classes. Most of the sequences affiliated with Viridiplantae (35%) - mainly Chlorophyta -, Alveolata (28%), Stramenopiles (16%), Opisthokonta (12%) - mostly Choanoflagellida and Fungi -, and Rhizaria (5%, cercomonads). The novelty level was also high; >40% of the sequences showed <97% identity to any previously reported sequence, and <90% identity was observed in 15% of the whole sequences dataset. Opisthokonta and Rhizaria had the highest novelty and Chlorophyta and Alveolata the lowest. Semi-arid inland endorheic regions showed higher level of novelty, phylogenetic diversity and phylogenetic species variability than coastal sites. We found similar level of novelty both at the highest (15% salinity to saturation) and at the lowest salinities (0,3-5% and 5-15% salinity), indicating that culturing effort should be equally applied along the whole salinity gradient. At the most extreme conditions (>15% salinity) sequences of Choanoflagellida, Chlorophyceae, Trebouxiophyceae, Bicosoecida, Bacillariophyceae, Cercozoa, Dinophyceae, Heterotrichea, Spirotrichea, and Euamoebida were found. Their closest cultured counterparts were Proterospongia sp. (81% identity), Salpingoeca sp. (83%), Dunaliella sp. (>99%), Picochlorum sp. (100%), Cafeteria minima (86%), Cylindrotheca sp. (100%), Lotharella globosa (88%), Scrippsiella sp. (89%), Fabrea salina (100%), Euplotes sp. (98%), and Flabellulidae sp. (98%). Overall, this study reveals that eukaryotes inhabiting extreme saline areas are by far more diverse than previously expected, and highlights saline endorheic environments as important reservoirs of microbial eukaryotic biodiversity.

References · X. Triadó-Margarit and E.O. Casamayor (2012). High genetic diversity and novelty of planktonic microeukaryotes inhabiting inland and coastal haline waters. Protist (submitted).

abstracts book 135 P10EXTREMOphiles_2012 Genus Salinivibrio: biodiversity in aquatic hypersaline habitats Clara López-Hermosoa, Cristina Sánchez-Porroa, Rafael R. de la Habaa, R. Thane Papkeb and Antonio Ventosaa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bDepartment of Molecular and Cell Biology, University of Connecticut, 06269 Storrs, CT, USA E-mail: [email protected]

Smith (1938) described the species Vibrio costicola, isolated from rib bones of bacon, which was latterly transferred to the genus Salinivibrio (Mellado et al., 1996); this genus currently includes 4 species. They are Gram-negative, facultatively anaerobic moderately halophilic bacteria that have been isolated from various hypersaline environments, salted food and saline brines. In order to study the biodiversity of the genus Salinivibrio in hypersaline environments and the phylogenetic relationships among species of this genus by comparing highly conserved genes using a multilocus sequencing analysis (MLSA) we are working on the isolation of strains from different environments. To date we have isolated 45 strains of the genus Salinivibrio from salterns in Huelva, Alicante and Mallorca (Spain). We have used different media and culture conditions, taking into account the physiological characteristic of the species of the genus Salinivibrio, which are facultative anaerobes. Thus, the inoculated plates from the various samples were incubated in anaerobic chambers and then the resulting strains were isolated in aerobic conditions. The isolates were characterized by partial sequencing of the 16S rRNA gene, to determine whether they were related to the genus Salinivibrio. Currently, we are characterizing these strains based on phenotypic and genotypic features, in order to determine their taxonomic position and if they might constitute new species. The last objective of this work is to obtain a large collection of strains belonging to different species of Salinivibrio, isolated from various saline environments, in order to carry out a MLSA study.

References · E. Mellado, E.R.B. Moore, J.J. Nieto and A. Ventosa (1996). Analysis of 16S rRNA gene sequences of Vibrio costicola strains: description ofSalinivibrio costicola gen. nov., comb. nov. Int J Syst Bacteriol 46: 817-821 · F.B. Smith (1938). An investigation of a taint in rib bones of bacon. The determination of halophilic vibrios (n spp.). Proc. Roy. Soc. Queensland 49: 29-52

136 abstracts book EXTREMOphiles_2012P11 Isolation and identification of polyextremophilic bacteria from Gomishan Wetland, the alkaline thalassohaline lake in North of Iran Mohammad Ali Amoozegara,b, Azadeh Shahinpeib, Seyed Abolhassan Shahzedeh Fazelib and Abbase Akhavan-Sepahic aExtremophiles Laboratory, Department of Microbiology, Faculty of Biology, College of Sciences, University of Tehran, Tehran, Iran bMicroorganism Bank, Iranian Biological Resource Centre (IBRC), ACECR, Tehran, Iran cDepartment of Microbiology, Faculty of Biology Science, Islamic Azad University NorthTehran Branch, Tehran, Iran E-mail: [email protected]

Iran consists of numerous hypersaline environments including salt mines, deserts, and hypersaline lakes and playas. Despite high abundance and diversity of hypersaline ecosystems in Iran, little information is currently available regarding the biodiversity of halophilic bacteria in hypersaline lakes of Iran (Makhdoumi-Kakhki et al., 2012; Rohban et al., 2009 ). International Gomishan wetland is a natural ecosystem with an extension of about 20000 hectares is located along the eastern shore of Caspian Sea. Most salts in this wetland are NaCl and

MgSO4. pH value varies between 8.0-9.5 and salinity reaches to 100 g/l. In the present study, twice sampling from 3 different geographic positions in dry and rainy seasons, led to the isolation of 224 isolates. For 57 isolates, halophilic and halotolerant behavior and also optimum and growth range in different pH and temperature were determined. Most of the moderately halophilic and halotolerant strains were capable to grow optimally in media with pH 8.5-9, and presented the temperature range for growth from <4 to 40ºC. The isolates were examined for hydrolytic enzymes production. Most of the isolates presented lipase activities and a total of 15, 7 and 3 strains produced amylases, proteases and DNases, respectively. 16S rRNA phylogenetic analysis were carried out for 55 strains and these strains were placed in four phyla, Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes and 22 different genera: Achromobacter, Aeromicrobium, Altererythrobacter, Bacillus, Caenispirillum, Cyclobacterium, Erythrobacter, Halobacillus, Halomonas, Idiomarina, Jonesia, Marinobacter, Martelella, Nesiotobacter, Paenibacillus, Planococcus, Pseudomonas, Rheinheimera, Saccharospirillum, Stappia, Thalassospira and Vibrio with similarity between 91.1-99% to closest known strains. 23% of these strains were haloalkalophilic bacteria and belonged to Bacillus, Halobacillus, Halomonas, Idiomarina and Marinobacter.

References · A. Makhdoumi-Kakhki, M. A. Amoozegar, B. Kazemi, L. Pašic and A. Ventosa (2012). Prokaryotic diversity in Aran-Bidgol salt lake, the largest hypersaline playa in Iran. Microbes Environ. 27: 87-93. · R. Rohban, M. A. Amoozegar and A. Ventosa (2009). Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. J. Indust. Microbiol. Biotechnol. 36: 333-340.

abstracts book 137 P12EXTREMOphiles_2012 Eukaryotic diversity in the deep sedimentary rock from Horonobe, Japan Yoshimoto Saitoha, Toru Nagaokaa and Yuki Amanob aEnvironmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 270-1194 Abiko, Chiba, Japan bGeological Isolation Research and Development Directorate, Japan Atomic Energy Agency, 098-3224 Horonobe, Hokkaido, Japan E-mail: [email protected]

To gain insight into the eukaryotic community composition in deep subsurface environments, we extracted DNA from terrestrial sedimentary rocks obtained from -250 m subsurface environment at the Horonobe Underground Research Laboratory (Horonobe URL) in Hokkaido and performed culture-independent community profiling of 18S (approximately 500 bp) rRNA gene fragments. The resultant clone library contained algal, protistal, and fungal sequences, with no significant differences in the numbers of protistal, algal, and fungal clones. The algal clones were grouped into 2 operational taxonomic units (OTUs). Both the OTUs were proposed to be Trebouxiophyceae species and were closely related to Chloroidium saccharophilum (100% sequence homology) and Myrmecia incise (100% sequence homology), respectively. The protistal clones were also classified into 2 OTUs. One OTU was proposed to be an Acanthamoebidae species and was related to Protacanthamoeba bohemica (94% sequence homology). Another OUT was suggested to be a Heterolobosea species, showing homology with only Euplaesiobystra hypersalinica (82% sequence homology), isolated from a high-salinity environment. The fungal clones were grouped into 5 OTUs. Four OTUs were proposed to be Basidiomycetes species, i.e. Malassezia pachydermatis (96% sequence homology), Scytinostroma renisporum (92% sequence homology), Cryptococcus humicolus (92% sequence homology) species and an ascomycetous species, i.e. Claviceps fusiformis (99% sequence homology); M. pachydermatis, C. humicolus and C. fusiformis are yeast or dimorphic yeast species. The other OTU had no significant homology with any known fungal species but presented high homology with the sequence that belongs to the novel clade of fungi described in previous studies (Lefèvre et al., 2007). These results indicate that diverse eukaryotic communities including algae, protists, and fungi are found in the deep subsurface environment at Horonobe (URL).

References · E. Lefevre, C. Bardot, C. Noel, J.-F. Carrias, E. Viscogliosi (2007). Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake. Environ Microbiol 9: 61-71.

138 abstracts book EXTREMOphiles_2012P13 New species of the genus Marinobacter isolated from hypersaline habitats Cristina Sánchez-Porro, María Jose León, Carmen Infante, Clara López-Hermoso and Antonio Ventosa

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain E-mail: [email protected]

The genus Marinobacter was established with the description of Marinobacter hydrocarbonoclasticus in 1992 (Gauthier et al., 1992). At the time of writing, another 29 species had been described. These species are tolerant to various conditions and they were isolated from diverse locations, i.e. from sediments, the water column, coastal, deep-sea waters, the Antartic, the Red Sea, oil-contaminated areas, hot springs and saline environments. They belong to the Gammaproteobacteria, order Alteromonadales, family Alteromonadaceae. Cells are Gram-negative, nonendospore-forming and motile and they have absolute requirements for sodium ions. In 2011 an exhaustive screening was carried out in diverse hypersaline areas of Spain, specifically in Huelva, Alicante and Mallorca. Water and sediment samples were collected and processed. More than 500 strains have been isolated. Initially, their complete 16S rRNA gene sequence was obtained and compared with those from the databases. Of them 20 strains showed the highest similarity (93-97%) with species of the genus Marinobacter. We are carrying out a polyphasic taxonomic study based on a phenotypic, genotypic, phylogenetic and chemotaxonomic analisis of these strains in order to determine if they could constitute new species in the genus Marinobacter.

References · M. J. Gauthier, B. Lafay, R. Christen, L. Fernandez, M. Acquaviva, P. Bonin and J.C. Bertrand (1992). Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Bacteriol 42: 568-576.

abstracts book 139 P14EXTREMOphiles_2012 Ornatilinea apprima gen. nov., sp. nov., a first cellulolytic representative of class Anaerolineae

Olga Podosokorskayaa, Elizaveta Bonch-Osmolovskayaa, Andrey Novikovb, Tatyana Kolganovac and Ilya Kublanova

aWinogradsky Institute of Microbiology, Russian Academy of Sciences, Prospekt 60-Letiya Oktyabrya 7/2, 117312 Moscow, Russia bGubkin Russian State University of Oil and Gas, Leninskiy Prospect 65, 117485 Moscow, Russia cBioengineering Center, Russian Academy of Sciences, Prospekt 60-Letiya Oktyabrya 7/1, 14 117312 Moscow, Russia E-mail: [email protected]

At present phylum Chloroflexi consists of 6 classes of microorganisms with extremely diverse morphology and physiology. Class Anaerolineae is one of the recently discovered groups (Yamada et al., 2006). All cultured representatives of this class are multicellular Gram-negative, non-motile filamentous microorganisms. They are neutrophilic, strictly anaerobic chemoorganotophs able to utilize sugars and polysaccharides (starch, pectin, xylan). However, growth on cellulose or its derivatives so far has not been reported for this group.

Sample of microbial mat was collected from the surface of wooden chute, filled with hot water (pH 7.5, 47°C) emerging from a 2775-m-deep oil-exploration borehole (Tomsk region, Russia). Isolate P3M-1T was obtained using the modified Widdel freshwater medium with microcrystalline cellulose (MCC). Phylogenetic analysis of 16S rRNA gene sequences of this strain and related microorganisms showed that its closest relative isLevilinea saccharolytica(92.8 %) (Yamada et al., 2007). Morphology and physiology of the isolate P3M-1T in general was similar to that of other members of class Anaerolineae. However, there were a number of distinct features, differentiating the new organism: growth on microcrystalline cellulose, production of ethanol, biofilms formation and slightly alkaline pH optimum (7.5-8.0). Furthermore, the doubling time of the new strain was significantly shorter than that of other members of Anaerolineae (6 hours for P3M-1T vs. 45 hours for Bellilinea caldifistulae previously considered the fastest). Thus, based on the phylogenetic position and phenotypic characteristics of the strain P3M-1T we have proposed a novel genus and species Ornatilinea apprima(Podosokorskaya et al., in press).

References · Yamada et al. (2006). Int J Syst Evol Microbiol 56: 1331-1340. · Yamada et al. (2007). Int J Syst Evol Microbiol 57: 2299-2306. · Podosokorskaya et al. (2012). Int J Syst Evol Microbiol, in press

140 abstracts book EXTREMOphiles_2012P15 Detection of putatively thermophilic methanotrophic archaea by using new primer systems Alexander Y. Merkela, Julie A. Huberb and Alexander V. Lebedinskya

aWinogradsky Institute of Microbiology, Russian Academy of Sciences, Prospekt 60-Letiya Oktyabrya 7/2, 117312 Moscow, Russia bJosephine Bay Paul Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA, USA E-mail: [email protected]

It is assumed that one of the major obstacles to the isolation of methanotrophic archaea is their slow growth, caused by the very low energy yield of anaerobic oxidation of methane (AOM) (Nauhaus et al, 2007). In this regard, thermophilic process of AOM may be of great interest, since, according to theoretical calculations, ΔG of sulfate-dependent oxidation of methane increases with increasing temperature (Holler et al., 2011). Our analysis of all sequences of the 16S rRNAs of archaeal isolates available from the RDP database with checking the Topt of the corresponding microorganisms in relevant publications showed that no mesophilic archaeal isolates have been reported with a 16S rRNA gene G+C content above 61.0 mol% and no moderately thermophilic archaeal isolates have been reported with a 16S rRNA gene G+C content above 63.0 mol%. We have analyzed over 900 16S rRNA gene sequences of seven ANME groups: 1a, 1b, 1AT, 1GB, 2ab, 2c and 3. As a result, it was found that the G+C content of the 16S rRNA genes is 63,49±0.74% in the group ANME-1GB, while it does not exceed 58% in all other groups. This allows us to predict that microorganisms of the ANME-1GB group are adapted to thrive at a high temperature (most probably, about 70ÚC). We designed highly specific 16S rRNA gene-targeted primers able to detect all phylogenetic groups within the ANME-1 cluster, including ANME-1GB group. These primers have been successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected: those from the Mississippi Canyon 118 (MC118) in the Gulf of Mexico and from Guaymas Basin in the Gulf of California. Further, these primers were used for the detection of microorganisms of ANME-1 cluster in diffuse vent fluid samples from 42 basalt-hosted deep-sea hydrothermal vents at seven sites in the Pacific Ocean: the Axial Volcano and the Endeavour Segment, both on the Juan de Fuca Ridge, and five volcanoes along the Mariana Arc. Phylotypes of the ANME-1 cluster were detected in four individual vents. These phylotypes belonged to the ANME-1GB, ANME-1a and ANME-1AT groups. The putatively thermophilic methanotrophic archaea of the ANME-1GB group had previously been detected only in hydrothermal sediments in the Guaymas Basin. Our findings provide evidence for a much wider occurrence of this group.

References · K. Nauhaus et al. (2007) Environ Microbiol 9: 187-196. · T. Holler et al. (2011) ISME J 5:1946-1956.

abstracts book 141 P16EXTREMOphiles_2012 Isolation of new groups of halophilic microorganisms based on metagenomic studies of a marine saltern Carmen Infantea, Mª José Leóna, Clara López-Hermosoa, Ana B. Fernándeza, Cristina Sánchez-Porroa, Francisco Rodríguez-Valerab and Antonio Ventosaa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bEvolutionary Genomics Group, Division of Microbiology, University Miguel Hernandez, 03550 Alicante, Spain E-mail: [email protected]

Hypersaline environments are excellent models of extreme habitats in which for the past decades there have been studies that have allowed the isolation and characterization of a large number of halophilic microorganisms. However, these environments are habitats with high unknown biodiversity, due to the difficulty of cultivation of many microorganisms under laboratory conditions. The results obtained by metagenomic studies carried out recently on marine salterns in Santa Pola (Alicante, Spain) have revealed the presence of new groups of prokaryotes that have not been isolated until date (Ghaiet al., 2011; Fernández et al., unpublished results). Thus, using culture media with different concentrations of total salts, pH values, different concentrations of carbon sources and yeast extract, we have obtained a total of 627 isolates. The initial phylogenetic study based on the comparison of the 16S rRNA gene sequence shows that the isolates are related mainly to the genera Halomonas, Idiomarina, Pseudomonas, Salicola, Salinivibrio, Marinobacter and Halorubrum. Those isolates showing a 16S rRNA percentage below 97% (Stackebrandt & Goebel, 1994) have been selected to carry out a detailed study. References · R. Ghai, l. Pašiæ, A.B. Fernández, A.B. Martin-Cuadrado, C.M. Mizuno, K.D. McMahon, R.T. Papke, R. Stepanauskas, B. Rodriguez-Brito, F. Rohwer, C. Sánchez-Porro, A. Ventosa and F. Rodríguez-Valera (2011). Sci Rep. 1: 135. · E. Stackebrandt and B.M. Goebel (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology.Int J Syst Bacteriol 44: 846-849.

142 abstracts book EXTREMOphiles_2012P17 Spatial distribution of microbial communities along salinity gradient in six saline and hypersaline lakes from Transylvania (Central Romania) Andreea Baricza, ªtefan Adrian Andreia, Vasile Munteana, Cristian Comanb, Zsolt Gyula Keresztesa, Mircea Alexec and Horia Banciua

aDepartment of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeº-Bolyai University, 400006 Cluj-Napoca, Romania bNational Institute of Research and Development for Biological Sciences (NIRDBS), Institute of Biological Research, 48 Republicii Street, 400015 Cluj-Napoca, Romania cFaculty of Geography, Babeº-Bolyai University, 400006 Cluj-Napoca, Romania E-mail: [email protected]

Most of the neutral, thalassohaline lakes located in the Transylvanian Basin (Central Romania) originate from salt mining activity. Some Transylvanian salt lakes date back to the Roman Age, while others were formed in modern times. Six salt lakes from three different locations (Turda - Cluj County, Ocna Sibiului-Sibiu County, and Sovata-Mureº County) were sampled along the water column during the winter season of 2011-2012. Five out of six sampled lakes are of meromictic nature, with a steep chemocline (halo- and oxycline) and thermocline. Their salinities varied from around 4%, in surface, up to 35%, near the bottom. Two lakes (Lake Brâncoveanu and Lake Fãrã Fund) are hypersaline (>20%) starting from the surface. The aim of the present study was to asses the relationship between the spatial distribution of the prokaryotic diversity and the environmental factors (salinity, dissolved oxygen, temperature) by a polyphasic approach. Physical and chemical parameters, such as pH, oxidation/reduction potential (ORP), temperature, conductivity and dissolved oxygen, were measuredin situ by using a field multiparameter. Water samples were collected at each 0.5 m down to a depth of 4 meters, and at each 1 m down to the closeness of the bottom. Chemical analyses of water samples have been employed in order to determine total organic carbon (TOC), + + - 2- total nitrogen (TN) and concentration of inorganic anions and cations (Na , K , Cl , SO4 ). Total cell count was performed by epifluorescence microscopy using DAPI staining. The total number of bacterial cells has been estimated by qRT-PCR, using specific bacterial primers. Total DNA extracted from filter- immobilized cells has been used to explore the prokaryotic diversity by PCR-DGGE on 16S rRNA genes and ARDRA. Culture-based methods, using direct plating of water samples on high-salt media allowed to obtain tens of isolates. Molecular approaches have been further used to identify these prokaryotic isolates. Our preliminary results indicated the prevalence of bacteria and phytoplankton in the upper layer (0-3 m) of the studied lakes. Apparently, the archaeal abundance increases with depth (and, implicitly, with the salinity), in a similar trend with the total number of cells counted by DAPI staining, TOC and TN. Interestingly, the presence of Dunaliella-like cells has been recorded in the mixolimnion of all studied lakes.

abstracts book 143 P18EXTREMOphiles_2012 Prokaryotic community structure and diversity at a shallow hydrothermal vent off Panarea Island (Italy) by Illumina high-throughput sequencing Valeria Lentinia, Concetta Gugliandoloa, Boyke Bunkb, Jorg Overmannb and Teresa L. Maugeria

aDepartment of Animal Biology and Marine Ecology, Faculty of Science, University of Messina, 98166 Messina, Italy bLeibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 38124, Braunschweig, Germany E-mail: [email protected]

The development of molecular techniques has greatly advanced our knowledge of the diversity of natural microbial communities. Previous molecular fingerprinting of prokaryotic communities at shallow hydrothermal vents off Panarea Island (Tyrrhenian Sea, Italy) demonstrated a high richness and biodiversity of Bacteria that exceeded those of Archaea, and revealed the presence of previously unknown prokaryotic phylotypes (Maugeri et al., 2009, 2010). In the present study we examined the structure and diversity of these hydrothermal vent prokaryotic communities over several years using the high throughput Illumina sequencing technology. Millions of analyzed sequences (5,233,501) revealed that Bacteria constituted a major component (99.1%) of prokaryotic community at this vent location. Synthetic diversity and non parametric richness indices revealed that the prokaryotic community structure varied among samples. The majority of bacterial sequences was affiliated to the phylum Proteobacteria followed by Firmicutes, Actinobacteria and Bacteroidetes. The most abundant classes were Gamma- and Epsilon- proteobacteria similar to deep-sea hydrothermal vents. Among Archaea, Euryarchaeota constituted the predominant phylum followed by Crenarchaeota and Korarchaeota. Members of Euryarchaeota and Crenarchaeota were related to hyperthermophilic genera already isolated from different extreme habitats. A huge number of low-abundant and rare phylotypes have been demonstrated in this shallow hydrothermal vent. These phylotypes may be considered as a potentially inexhaustible reservoir of genomic variability, that might rapidly respond to environmental stressors by increasing the abundance of more specific populations. A considerable number of bacterial and archaeal phylotypes still remained phylogenetically unaffiliated. These findings allow us to consider the shallow hydrothermal vent off Panarea Island as a hotspot of prokaryotic diversity.

References · T.L. Maugeri et al. (2009). Bacterial and archaeal populations at two shallow hydrothermal vents off Panarea Island (Eolian Islands, Italy). Extremophiles 13: 199-212. · T.L. Maugeri et al. (2010). Microbial diversity at a hot shallow thermal vent in the Southern Tyrrhenian Sea (Italy). Geomicrobiol J 27: 1-11.

144 abstracts book EXTREMOphiles_2012P19 Isolation and characterization of a new arsenate- reducing bacterium, which produces an arsenic sulfide ore from the Salar de Ascotán, Chile: Fusibacter sp. 3D3 Lorena V. Escuderoa,b, Cinthya Tebesa, Jonathan Bijmana, Olga Encaladaa and Cecilia Demergasso Ca,b

aCentro de Biotecnología «Alberto Ruíz», Universidad Católica del Norte, Antofagasta, Chile bCentro de Investigación Científica y Tecnológica para la Minería (CICITEM) E-mail: [email protected]

In the Northern Chile, there are several environments with high contents of arsenic (As). Salar of Ascotán with its morfometric, climatic and physicochemical characteristics is an excellent place for the studying microbial implications in the biogeochemical cycle of As and nature source of bacterial strains able to carry out As transformations. A novel arsenate reducing Gram-positive halotolerant bacterium is reported in this work from phylum Firmicutes and genus Fusibacter. This microorganism is be able to metabolize As (V), reducing it and forming a sulfur mineral of As. Fusibacter ascotense 3D3 is a rod, Gram-positive, spore-forming, anaerobic, mesophile (it grows within 20°C y 30°C), neutrophile-alcalophile (it grows within pH 6-9), halotolerant (it grows within 0 and 5% w/v NaCl), heterotrophic (it uses lactate, glucose, tryptone and yeast extract) and reducing arsenate form 0,075 to 7,5 g L-1 and precipitated arsenic sulfur. Phylogenetically, the strain 3D3 belonged to the Firmicutes group, within the genus Fusicbacter, close to Fusibacter puacivorans strain SEBR4211. Scanning microscopy and X-ray diffraction have shown that precipitate has molar rate As/S 1.05 likely to rejalgar mineral (AsS). Also, the characterizations by energy-dispersive X-ray spectroscopy (EDS) of the biogenic mineral obtained from the cultures of Fusibacter ascotense indicate that is realgar. Characteristics of this mineral were studied through confocal microscopy and Raman for measure the size of arsenic particule and the measure obtained from 20 to 100 nm , because rejalgar mineral can be used like nanoparticules in medical treatments. Production of biogenical minerals of As through new microorganism from phylum Firmicutes is relevant for a better understanding of biogeochemical cycle of arsenic in the Salar of Ascotán. Furthermore, these findings could contribute to predict the shape and size of realgar nanoparticles, produced by microbial biogenesis, helping to establish this technology for pharmaceutical applications and other nanotechnological industrial processes in our country.

abstracts book 145 P20EXTREMOphiles_2012 Analysis of extremophilic bacterial community from Kamchatka hot oil field Igor Babkina, Konstantin Kutasheva, Vera Morozovaa, Alla Bryanskayab, Elena Lazarevac, Sergei Zhmodikc, Irina Babkinaa, Andrei Matveeva and Nina Tikunovaa

aInstitute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia bInstitute of Cytology and Genetics SB RAS, Novosibirsk, Russia cInstitute of Geology and Mineralogy SB RAS, Novosibirsk, Russia E-mail: [email protected]

The microbiota of the Kamchatka hot geothermal fields has been little studied. In the present study, the structure of the bacterial community in hot-oil-field (Kamchatka) was investigated using 16S rRNA gene clone libraries. Environmental samples for this study were obtained from hot-oil-field (geothermal field, Uzon volcanic caldera, Kamchatka). DNA was isolated and then amplified with primers specific to 16S ribosomal RNA gene fragment with 1230 nucleotides of estimated length. Purified amplicons were cloned intoE.coli plasmids. Since monoclonal libraries were grown, inserted fragments were sequenced by Sanger. All sequenced clones were chimera checked by Mallard (Cardiff University, UK) and Bellerophon v.3 (GreenGenes project) software packages. Some sequences were identified as chimeras. Phylogenetic analysis was performed by RDP software (http:// rdp.cme.msu.edu) with 80% confidence threshold. The most abundant microbial phylotypes were estimated.

146 abstracts book EXTREMOphiles_2012P21 Comparison of bacterial communities from microbial mats accumulating germanium in high and low concentration, Barguzin hot springs of Baikal Rift Zone Nina Tikunovaa, Alla Bryanskayab, Elena Lazarevac, Vera Morozovaa, Igor Babkina, Aleksandr Kurilshikova, Artem Tikunova, Irina Babkinaa, Andrei Matveeva and Sergei Zhmodikc

aInstitute of Chemical Biology and Fundamental Medicine SB RAS, 630090 Novosibirsk, Russia bInstitute of Cytology and Genetics SB RAS, 630090 Novosibirsk, Russia cInstitute of Geology and Mineralogy SB RAS, 630090 Novosibirsk, Russia E-mail: [email protected]

Barguzin basin in the Baikal Rift Zone with its hot springs is one of locations where minerals forming have been observed in microbial mats. Maximal germanium accumulation was found in the Uro spring cianobacterial mat, while germanium content in the Alla spring microbial mat was low. Bacterial communities in the Uro and Alla microbial mats were compared based on 16S rRNA gene sequences. The 16S rRNA gene was amplified by PCR and two clone libraries were constructed. DNA fragments of 1230 n were sequenced, and the sequences were analyzed by RDP software http://rdp.cme.msu.edu( ). All sequences were chimera checked by Mallard (Cardiff University, UK) and Bellerophon v.3 (GreenGenes project) software packages. As a result, taxonomic units belonging to Cianobacteria, Bacteriodetes, Actinobacteria, Firmicutes and Fusobacteria were detected only in the Uro cianobacterial mat.

abstracts book 147 P22EXTREMOphiles_2012 Diversity of microorganisms isolated from saline lakes of Novosibirsk region Vera Morozova, Irina Saranina, Vladimir Repin, Igor Babkin and Nina Tikunova

Laboratory of Molecular Microbiology, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia E-mail: [email protected]

The microbiota of the saline lakes of Novosibirsk region has been little studied. We collected the samples of water and sediments of some saline lakes during the expeditions in 2008-2010. Forty two halotolerant bacterial isolates were cultivated. Strains had distinct morphology and grew optimally at° 37 C, pH 7.0-7.5 and in the presence of 2-10 % NaCl. In order to determine the taxonomic position of these isolates, DNA was isolated and then amplified with the primers specific to 16S rRNA gene fragment with 1340 nucleotides of estimated length. The 16S rRNA gene sequences were analyzed and taxonomic affiliation was determined using the naïve Bayesian rRNA Classifier of the RDP. Phylogenetic analyses based on the 16S rRNA gene sequence comparison were carried out.

148 abstracts book EXTREMOphiles_2012P23 An update of the Multilocus Sequence Analysis (MLSA) of the Halobacteriales Rafael R. de la Habaa, Andrea Makkayb, Kamisha Byasb, Chelsea Giffordb, Antonio Ventosaa and R. Thane Papkeb

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bDepartment of Molecular and Cell Biology, University of Connecticut, 06269 Storrs, CT, USA E-mail: [email protected]

Members of the order Halobacteriales are obligate extreme halophiles that belong to the phylum Euryarchaeota, within the domain Archaea. Up to May 2012, the order comprises a total of 38 genera and 132 species. Currently, the classification of the Halobacteriales relies on a polyphasic approach, which integrates phenotypic, genotypic, chemotaxonomic and phylogenetic features. However, the most utilized genetic marker for phylogeny, the 16S rRNA gene, has multiple drawbacks for use with theHalobacteriales : the species of many genera exhibit large intragenic differences between multiple ribosomal RNA operons; the gene is too conserved to discriminate reliably at the species level; and it appears to be the most frequently transferred gene among closely related species (Papke, 2009). In addition, theHalobacteriales is a rapidly expanding group due to recent successes at cultivating novel strains from a diverse set of hypersaline environments. Therefore, a fast, reliable, inexpensive, portable molecular method for discriminating species is required. Recently, Papke et al., (2011) have demonstrated that multilocus sequence analysis (MLSA) is a rapid and effective tool that can be used to differentiate individual strains, to reliably group strains into species and species into genera and to identify potential novel species and also family-like relationships within theHalobacteriales . In this study, we have extended this MLSA to a larger number of strains (including all the type strains of the species in the order Halobacteriales) analyzing 6 housekeeping genes: atpB, EF-2, glnA, ppsA, radA, and rpoB’. On the other hand, an online database is available at https://sites.google.com/a/uconn.edu/papkelab/mlsa, which provides free and fast access to MLSA sequences of the Halobacteriales, with the aim of spreading the use of this molecular method by the scientific community.

References · R. T. Papke (2009). A critique of prokaryotic species concepts. Methods Mol Biol 532: 379-395. · R. T. Papke, E. White, P. Reddy, G. Weigel, M. Kamekura, H. Minegishi, R. Usami and A. Ventosa (2011). A multilocus sequence analysis approach to the phylogeny and taxonomy of the Halobacteriales. Int J Syst Evol Microbiol 61: 2984-2995.

abstracts book 149 P24EXTREMOphiles_2012 «Melioribacter roseus» gen. nov., sp. nov., a novel facultatively anaerobic moderately thermophilic cellulolytic bacterium and proposal of «Ignavibacteriae» phyl. nov. Ilya V. Kublanova, Olga Podosokorskayaa, Vitaly Kadnikovb, Sergey Gavrilova, Andrey Mardanovb, Alexander Merkela, Olga Karnachukc, Nikolay Ravinb and Elizaveta Bonch-Osmolovskayaa

aWinogradsky Institute of Microbiology; bBioengineering Center, Russian Academy of Sciences, Prospekt 60-Letiya Oktyabrya 7/2 (fora ) and 7/1 (for b), 117312 Moscow, Russia cDepartment of Plant Physiology and Biotechnology, Tomsk State University, Prospekt Lenina 36, 634050 Tomsk, Russia E-mail: [email protected]

A novel moderately thermophilic, facultatively anaerobic heterotrophic bacterium «Melioribacter roseus» strain P3M-2T was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46oC) coming out of a 2775 meter-deep oil explorating well (Tomsk region, Siberia, Russia). Together with its closest relative (90.8% 16S rRNA sequence identity),Ignavibacterium album strain Mat9-16T (Lino et al., 2010), the new organism represents a deep phylum-level branch, most close to Chlorobi phylum. Strain P3M-2T is a moderate thermophile and facultative anaerobe growing on simple and complex carbohydrates either by fermentation or by respiration using various electron acceptors (see Gavrilov et al., this issue). The organism differs from Chlorobi by its motility, obligately organotrophic mode of life, the absence of chlorosomes, and inability to grow phototrophically. Analysis of cellular fatty acids showed a distant relationship between strain P3M-2T and Bacteroidetes but not Chlorobi. Phylogenetic analysis of strain P3M-2T based on 16S, 23S rRNA and RecA genes, as well as chemotaxonomic and physiological studies of strain P3M-2T and I. album indicated that these two organisms represent a novel phylum «Ignavibacteriae» (Podosokorskaya et al., submitted). Analysis of the distribution of environmental 16S rRNA gene clones falling within the novel phylum confirmed a wide distribution of its representatives, comparable with that of Bacteroidetes and far exceeding that of Chlorobi, which were found in a limited number of anoxic aquatic habitats. Three of these clones previously represented candidate division ZB1, proposed by Elshahed et al. in 2003. The position of ZB1 within the bacterial phylogenetic tree correlates with that of «Ignavibacteriae», supporting our proposal of the novel phylum. This work was supported by Federal Ministry of Education and Science grants # P646 and 16.512.11.2152.

References · M.S. Elshahed et al. (2003) Appl Environ Microbiol 69: 5609–5621. · T. Lino et al. (2010) Int J Syst Evol Microbiol 60: 1376–1382. · O. Podosokorskaya et. al. Environ Microbiol, submitted.

150 abstracts book EXTREMOphiles_2012P25 Biodiversity analysis of archaeal and bacterial communities from two hot springs in Galicia (northwestern Spain) Roberto Gonzáleza, Olalla López-Lópezb, Clara Fuciñosa, Natalia Estéveza, A. Cristina Rodriguesa, Martín Mígueza, Pablo Fuciñosa and M. Luisa Rúaa

aDepartment of Analytical and Food Chemistry, University of Vigo, Campus of Ourense, As Lagoas, 32004 Ourense, Spain bDepartment of Cell and Molecular Biology, University of A Coruña, 15071 A Coruña, Spain E-mail: [email protected]

The seasonal variation in biodiversity and physico-chemical parameters was evaluated in water samples from Río Caldo (pH 9.0, 77 °C) and As Burgas (pH 7.5, 68 °C), two hot springs located in Ourense (Galicia, northwestern Spain). Metagenomic DNA was isolated using a Rapid Water DNA Isolation Kit (Mo-Bio Laboratories). The 16S rRNA gene fragments were PCR amplified from total community DNA using Archaea and Bacteria-specific primers, and analysed by denaturing gradient gel electrophoresis (DGGE; Dorigo et al., 2005) according to Akarsubasi et al. (2005). Mineral content in As Burgas water (678.3 mg/L) was significantly higher than in Río Caldo (313.3 mg/L). However, arsenic and total sulfur content was higher in Río Caldo samples. DGGE fingerprints for both Archaea and Bacteria populations also showed significant differences between Río Caldo and As Burgas samples. Prominent bands were excised from the DGGE gels. DNA was released from the polyacrilamide matrix, re- amplified and purified for sequence determination. Sequences obtained were searched against NCBI databases with BLAST (Altschul et al., 1990). In the case of Archaea, patches of similarity were found, for instance, with Sulfurisphaera ohwakuensis TA-1 (88% 16S rRNA sequence maximum identity and 65% of query coverage) in Río Caldo, and with Thermoclaudium modestius IC-125 (82% maximum identity and 92% query coverage) in As Burgas. In the case of Bacteria, examples of high similarity were found, for instance, with Thermodesulfovibrio hydrogeniphilus Hbr5 (86% and 94%) in Río Caldo, and with Burkholderia sabiae Br3407 (90% and 94%) in As Burgas.

References · A.T. Akarsubasi, O. Ince, B. Kirdar, N. Oz, D. Orhon, T.P. Curtis, I.M. Head and K. Bahar (2005). Effect of wastewater composition on archaeal population diversity.Water Research 39: 1576-84. · U. Dorigo, L. Volatier and J.F. Humbert (2005). Molecular approaches to the assessment of biodiversity in aquatic microbial communities. Water Research 39: 2207-18. · S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. and Lipman (1990). Basic local alignment search tool. J Mol Biol 215: 403-410. Available at: http://www.ncbi.nlm.nih.gov.

abstracts book 151 P26EXTREMOphiles_2012 Survey of microbial diversity, abundance and activity in anaerobic sediments of Tinto river: a natural acid and high heavy metals content environment Irene Sánchez-Andreaa,b, Nuria Rodriguezc, Ricardo Amilsa,b,c and Jose Luis Sanza

aUniversidad Autónoma de Madrid, Departamento de Biología Molecular, 28049 Madrid, Spain bCentro de Biología Molecular Severo Ochoa, UAM-CSIC, 28049 Madrid, Spain cCentro de Astrobiología, INTA-CSIC, Ctra. Ajalvir-Torrejón de Ardoz, 28850 Madrid, Spain E-mail: [email protected]

Tinto river (Huelva, Spain) is a natural acidic rock drainage (ARD) environment produced by the bio-oxidation of metallic sulfides from the Iberian Pyritic Belt. So far, and despite their/its ecological interest, the underlying sediments had been studied only very sparsely and no complete studies undertaken. In this study, we present an extensive survey of the Tinto River anaerobic sediment microbiota combining culture independent and dependent methods following the «full-cycle rRNA approach». A geomicrobiological model of the different microbial cycles operating in the sediments has been developed through molecular biological methods. Culture independent methods targeting the small subunit ribosomal RNA (SSU rRNA) such as denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene sequencing (cloning) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) have been used (1, 2). Microorganisms involved in the iron (Acidithiobacillus ferrooxidans, Sulfobacillus spp., etc.), sulfur (Desulfurella spp., Desulfosporosinus spp., Thermodesulfobium spp., etc.), carbon (Acidiphilium spp., Bacillus spp., Clostridium spp., Acidobacterium spp., etc.) and nitrogen (Alcaligenes spp., Pseudochrobactrum spp., etc.) cycles were identified and their distribution correlated with physicochemical parameters of the sediments. Where the pH and redox potential were closer to that of the water column (pH 2.5 and +400 mV), the most abundant organisms were identified as iron-reducing bacteria:Acidithiobacillus spp. and Acidiphilium spp., probably related to the higher iron solubility at low pH. At higher pH (4.2-6.2) and more reducing redox potential (50, -210 mV) members of sulfate-reducing genera Syntrophobacter, Desulfosporosinus and Desulfurella were dominant. Additionally, we used targeted enrichment incubations to validate this model and prove the existence of the potential anaerobic activities detected in the acidic sediments of Tinto River. Methanogenic, sulfate- reducing, denitrifying, iron-reducing and hydrogen-producing enrichments yield positive results (3). Finally, classical techniques for bacterial isolation were applied and some microorganisms were isolated such as acidophilic sulfate-reducing bacteria (two new species and a new genus, far affiliated to Desulfosporosinus/ Desulfitobacterium cluster), fermenters (a new genus related with Paludibacter) and hydrogen-producers (Clostridium spp.). References · I. Sánchez-Andrea, K. Knittel, R. Amann, R. Amils, and J. L. Sanz (2012). Quantification of Tinto River sediment microbial communities: the importance of sulfate-reducing bacteria and their role in attenuating acid mine drainage. Appl.Environ Microbiol doi: 10.1128/AEM.00848-12. · I. Sánchez-Andrea, N. Rodriguez, R. Amils, and J. L. Sanz (2011). Microbial diversity in anaerobic sediments at Rio Tinto, a naturally acidic environment with a high heavy metal content. Appl Environ Microbiol 77: 6085-6093. · I. Sánchez-Andrea, P. Rojas, R. Amils, and J. L. Sanz (2012). Screening of anaerobic activities in sediments of an acidic environment: Tinto River. Extremophiles, accepted.

152 abstracts book EXTREMOphiles_2012P27 Roles of an extreme thermophile, Calditerricola satsumensis, in high-temperature compost Takahiro Yoshii, Takafumi Sugihara, Risa Yamabi, Yoshihiro Furui, Hiroko Yano, Toshiyuki Moriya and Tairo Oshima

Institute of Environmental Microbiology, Kyowa-kako Co., Machida, Tokyo 194-0035, Japan E-mail: [email protected]

High-temperature compost in which the internal temperature exceeds 90°C or even 100°C decomposes several organic wastes quickly. We have previously isolated new extreme thermophiles Calditerricola satsumensis YMO81T and C. yamamurae YMO722T from a high-temperature compost pile (Moriya et al., 2011). C. satsumensis is able to grow up to 82°C, much higher than those of members of the genus Geobacillus. This study aims to identify the potential role of C. satsumensis in high-temperature composting process. We have investigated changes of the microbial community and physicochemical parameters in high- temperature composting process. The results of 16S rDNA PCR-denatured gradient gel electrophoresis (DGGE) indicated that the bands of the genus Calditerricola were detected clearly up to day 25. The sequence analyses revealed that these predominant bands can be assigned to C. satsumensis. However, other thermophiles became dominant organisms at and after the middle stage of the composting. To analyze the relative abundance of C. satsumensis during the composting process, we have carried out a 16S rDNA-targeted quantitative PCR. The abundance ratio of C. satsumensis per whole eubacterial cells was more than 20% at day 5. It decreased gradually and was not able to detect at day 20 and the later stages. The addition of gelatine, keratin, tristearin, stearic acid or starch into the compost resulted in an increase of abundance ratio of C. satsumensis. Based on these findings, we tentatively concluded that C. satsumensis was one of the most dominant species at the beginning of compost fermentation and may utilize proteins, fatty acids and starch as nutrient sources in early stages of high-temperature compost.

References · T. Moriya, T. Hikota, I. Yumoto, T. Ito, Y. Terui, A. Yamagishi and T. Osima. (2011). Calditerricola satsumensis gen. nov., sp. nov. and Calditerricola yamamurae sp. nov., extreme thermophiles isolated from a high-temperature compost. Int J Sys Evol Microbiol 61: 631-636.

abstracts book 153 P28EXTREMOphiles_2012 Investigation of bacterial community dynamics in PAH-contaminated soil by PCR-DGGE Zahra Khomarbaghia, Mohammad Ali Amoozegara, Mahmoud Shavandib, Seyed Mohammad Mehdi Dastgheybb and Hasan Tirandazb aExtremophiles Lab., Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran bBiotechnology Research Center, Research Institute of Petroleum Industry,Tehran,Iran E-mail: [email protected]

The present study is designed to investigate the dynamics of bacterial communities during bioremediation of oil contaminated soils of Siri Island in the Persian Gulf. For this purpose a quantity of clean soil was contaminated artificially with a defined mixture of polycyclic aromatic hydrocarbons (PAHs) including anthracene, phenanthrene, fluoranthene, pyrene (each 100 ppm) and benzo[a]pyrene (20 ppm) as sole carbon source to prepare the microcosms. To supply the nitrate and phosphate source, Ammonium nitrate and potassium phosphate salts were added based on C:N:P ratio of 100:5:1 And 20 % (v/w) deionized water was added to the microcosms to provide required moisture. The microcosms were kept in room temperature during a six-month survey period. Sampling was done every week for microbial analysis including both culturable and unculturable techniques. HPLC analysis was also carried out every two weeks in order to monitor PAH degradation by soil microorganisms. Results of culture dependent technique indicated a logarithmic increase of bacterial count during the first two weeks. Afterwards, it seemed that they reached to stationary phase. According to HPLC results, the amounts of all amended PAHs were reduced to less than 10 ppm during 14 weeks. To compare the efficiency of the culture dependent technique, dominant colonies were selected for phylogenetic analyses based on the 16S rRNA gene sequencing. To analyze the metagenome of the microcosms, DNA was extracted from soil samples every week, and a 560 bp fragment of 16S rRNA gene was amplified using a DGGE-specific pair of universal primers. Analysis and comparison of bacterial diversity in soil will be investigated using Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing.

154 abstracts book EXTREMOphiles_2012P29 The geochemistry of the deep Iberian Pyrite Belt (IBP) subsurface supports multiple microbial metabolisms Victor Parroa, Fernando Puente-Sáncheza, Miriam García-Villadangosa, Mónica Sánchez-Romána, Mercedes Moreno-Paza, Patricia Cruz-Gila, Pablo Fernándeza, Luis A. Rivasa, Yolanda Blancoa, Francisco López de Saroa, Monike Oggerina, Enoma Omoregiea, Nuria Rodrígueza, Antonio Molinaa, Sagrario Arias-Rivasb, José Antonio Rodríguez-Mafredia, Kenneth Timmisb, David Fernández-Remolara and Ricardo Amilsa

aCentro de Astrobiología (INTA-CSIC), Carretera de Ajalvir km 4, Torrejón de Ardoz, 28850, Madrid, Spain bHelmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany E-mail: [email protected]

Terrestrial subsurface geomicrobiology is a matter of growing interest not only to understand the life in the absence of light but also as a model for searching for life in other planetary bodies where the superficial conditions preclude any form of life. The Río Tinto fluvial basin is an acidic system in continuous geological evolution dating back from more than 2 million of years. It emerges in the Rio Tinto Anticline region, southwestern Spain, which is a complex geological structure taking part of the Iberian Pyrite Belt (IBP). Its formation is associated to the building up of a Carboniferous volcano-sedimentary complex exposed to a late intensive event of hydrothermal activity that mineralized the volcanic systems. During million of years, surface and underground microbial communities sustained on sulfur and iron chemolithotrophy have been using the ancient hydrothermal materials as energy sources to grow. Here we will present the first results of the IPBSL project (Iberian Pyrite Subsurface Life) funded by an European Research Council (ERC) Advanced Grant. The main scientific objective is the understanding of the bio-geochemical processes for sustaining the iron and sulfur driven life in this special subsurface habitat. The geochemical analysis of more than 200 core samples from two drilling, one to a depth of 340 m and another one to 612 m deep, revealed the presence of all the nutrients required for different anaerobic metabolisms. We detected small organic acid like propionate, acetate or formate that can be used by microbes as electron donors, and compounds like sulfate, nitrate, nitrite, and ferric iron as electron acceptor for anaerobic respiration. We used immunological techniques, a 450 antibody- containing microarray for the detection of microbial biomarkers in near real time (Rivas et al., 2008; Parro et al., 2011). We identified different microbial biomarkers at different depths revealing the presence of certain groups of prokaryotes. Environmental DNA is being extracted for metagenomic and molecular phylogenetic studies in some of the samples. Altogether the data will allow us to decipher the operating metabolisms in the deep Iberian Pyrite Belt Subsurface.

References · L.A. Rivas, M. García-Villadangos, M. Moreno-Paz, P. Cruz-Gil, J. Gómez-Elvira, and V. Parro (2008) A 200-antibody microarray biochip for environmental monitoring: searching for universal microbial biomarkers through immunoprofiling. Anal Chem 80: 7970-9. · V. Parro, G. de Diego-Castilla, M. Moreno-Paz, Y. Blanco, P. Cruz-Gil, J.A. Rodríguez-Manfredi, D. Fernández- Remolar, et al. (2011) A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars. Astrobiology 11: 969-96.

abstracts book 155 P30EXTREMOphiles_2012 The development of cyanobacteria on volcanic ashes L.M. Gerasimenko, G.A. Karpov, V.K. Orleansky and G.T. Ushatinskaya

Winogradsky Institute of Microbiology, Moscow, Russia E-mail: [email protected]

The possibility of growth of two different oscillatorian cyanobacteria on the water suspension of volcanic ashes was studied. First cyanobacteria – neutrophilic Oscillatoria terebriformis – was isolated from thermal spring of caldera Uzon (Kamchatka) and second one – alcaliphilic Phormidium sp. – from soda lake Hilganta (Burjatia). Powders of ashes were took immediately after eruptions of Kamchatka active volcano: Karimsky (eruption of 2003 and 2008 years) and Bezymjanny (eruption of 2006 year). The ashes were differed on the size of particles and their fraction ratio. There were some differences on the concentration macro and micro elements in the ashes. Dynamic of cyanobacteria growth was different on various ashes. It was connected from physical a chemical characteristics of ashes and pH medium. The ashes of volcano Bezymjanny were appeared toxic for cyanobacteria and both species have dead. The dinamic of the growth of both cyanobacteria on the ashes of Karimsky volcano was different.Oscillatoria terebriformis developed 10 days, after that it growth became more slowly and ended during two months. Phormidium sp. developed very slowly during all time (two months). Several elements were lixivated from asches to the medium and they could as to stimulate, as to inhibate the growth of cyanobacteria. The significant alterations in chemical composition of ashes took place in interaction with alcaliphilic Phormidium (comparing with neutrophilic Oscillatoria): carry out Al from 2 to 5.5%; Si from 7 to 17%; Ca from 6 to 9%. Cyanobacteria formed the powerfull glycocalix on the trichomes and between them under action of ashes. Elements went out to the medium and were adsorbed on the mucous covers, sometimes they mineralized trichomes. External polysaccharides of cyanobacteria cells promoted to bind particles of ashes and to change them. The ability of oscillatorian cyanobacteria to the moving , the formation of mucous glycocalix, cementing particles of ashes, led to formation of lamellar mats. These examples permit us to propose the analogy between processes in recent volcanic regions with processes of biogenetic weathering on volcanic area in the earliest Earth evolution.

156 abstracts book EXTREMOphiles_2012P31 Iron-tolerant cyanobacteria from thermal spring in Volcanic Complex of Furnas (Sao Miguel Island, Azores) Olga Samylina and Liudmila Gerasimenko

Winogradsky Institute of Microbiology RAS, Moscow, Russia E-mail: [email protected]

Cyanobacteria from iron-rich hot springs are a unique group of extremophilic prokaryotes. They represent good models for investigating the mechanisms of early organism’s evolution to survive in this type of habitats common on early Earth in the beginning of life. Three active stratovolcanoes are situated on the Sao Miguel Island: Sete Cidades, Fogo and Furnas. Furnas is the easternmost volcanic complex which consists of a number of calderas of different age with total area 8×5 km. This wide territory contains crater lake Lagoa das Furnas and many sulphide- and iron-rich thermal springs (Guest et al., 1999). Cyanobacterial communities inhabiting iron-rich thermal springs are wide-spread on the territory of Volcanic Complex of Furnas. They represent floating or bottom biofilms in thermal slow waters or attached biofilms on the rocks and stones in or near the stream. The samples of cyanobacterial community were taken during the 8th International Congress on Extremophiles held on the Sao Miguel Island (Azores) in September, 2010. These samples were characterized by various morphotypes of cyanobacteria with the most abundant Calothrix sp., Planktothrix sp., Chroococcus sp., Cyanothece sp., Synechococcus/Thermosynechococcus sp. Also ferruginized thickened cyanobacterial cell walls were abundant. The aim of this work was an investigation of cyanobacterial diversity from iron-rich thermal spring in Furnas using modern polyphasic approach, which includes a combination of molecular methods with studying of phenotypic and ecological features (Komárek, 2010). This work is supported by RFFI No. 11-05-00462-a. References · J.E. Guest, J.L. Gaspar, P.D. Cole, G. Queiroz and A.M. Duncan (1999). Volcanic geology of Furnas Volcano, São Miguel, Azores. J of Volcanology and Geothermal Research 92: 1-29. · J. Komárek (2010). Recent changes (2008) in cyanobacteria taxonomy based on a combination of molecular background with phenotype ane ecological consequences (genus and species concept). Phytoplankton 639: 245-259.

abstracts book 157 P32EXTREMOphiles_2012 Halocin-producing archaea are persistent in Algerian hypersaline ecosystems Alyssa Carré-Mloukaa, Manon Vandervenneta, Nacéra Imadalou-Idresb, Houa Yahiaouib, Said Benallaouab, Jean Peduzzia and Sylvie Rebuffata

aLaboratoire Molécules de Communication et Adaptation des Microorganismes, UMR7245 CNRS-MNHN, Muséum National d’Histoire Naturelle, CP 54, 57 rue Cuvier, 75005 Paris, France bLaboratoire de Microbiologie Appliquée/ Biochimie Microbienne, Université d’Abderahmane, Mia Béjaia Targua Ouzemour Béjaia 06000, Algeria E-mail: [email protected]

Twenty-four archaeal strains were isolated from three Algerian hypersaline environments, including the solar salterns in Ichekaben, and two natural salt lakes at Ouargla and Oran. Full-length 16S rRNA genes were amplified using archaeal-specific primers, sequenced, and phylogenetically analyzed. The ability of these strains to produce antimicrobial compounds of proteinaceous nature termed halocins (Rodríguez-Valeraet al., 1982) was examined. Three isolates, all from the salt lake of Ouargla, were affiliated to Halorubrum sp., while six isolates from Ichekaben were Natrinema sp. The remaining strains belonged to the genus Haloarcula, which was identified in the three Algerian sampling sites. A phylogenetic tree of Haloarcula sp. including the two copies of the 16S rRNA gene generally encountered for this genus, showed that two strains group with Haloarcula hispanica, while the other thirteen strains cluster with OHF-1 and OHF-2 isolated from French commercial salt (Usami et al., 2003), and would potentially constitute a new species of Haloarcula, widespread in Algeria. Both Natrinema sp. and Haloarcula sp., which were isolated from different samplings obtained between 2007 and 2009, appeared persistent at Ichekaben. For the six Natrinema sp. isolates, this correlated with their capacity to produce antimicrobial compounds, and a DNA fragment with sequence similarities to the halocin C8 gene (Sun et al., 2006) was amplified for five strains. In addition, oneHaloarcula strain from Ouargla displayed a significant inhibitory activity against archaeal strains, a feature hitherto unknown for Haloarcula sp.

References · F. Rodríguez-Valera, G. Juez, D. J. Kushner (1982) Halocins: salt-dependent bacteriocins produced by extremely halophilic rods. Can J Microbiol 28:151-154. · R. Usami, T. Fukushima, T. Mizuki, A. Inoue, Y. Yoshida and K. Horikoshi (2003) Organic solvent tolerance of halophilic Archaea. Biosci Biotechnol Biochem 67:1809-1812. · C. Sun, Y. Li, S. Mei, Q. Lu, L. Zhou and H. Xiang (2005) A single gene directs both production and immunity of halocin C8 in a haloarchaeal strain AS7092. Mol Microbiol 57:537-549.

158 abstracts book EXTREMOphiles_2012P33 Phylogenetic diversity of subseafloor aerobic microorganisms isolated from Suruga Bay in Japan Takeshi Miuraa, Hiroaki Minegishib, Tohru Kobayashic and Koki Horikoshic

aFaculty of Life Sciences, Toyo Uuniversity, Japan bBio-Nano Electronics Research Center, Toyo University, Japan cBiogeos, JAMSTEC, Japan E-mail: [email protected]

Using constructed deep-sea drilling vessel «Chikyu», we obtained 65 m of sediment cores from Suruga Bay in Japan at a water depth of 756 m. The dilution series of core sediments suspension with sterilized 3% NaCl (adjusted to pH4, 7 and 10) were spread onto Marine Agar 2216 (pH7) plates, and they were incubated at 30oC for 2 days. The subsequent quantitative cultivation on Marine agar represented remarkably high numbers of aerobic, heterotrophic microbial populations throughout all core sediments. The results were in agreement with the extracellular enzyme activities detected in all core sediments using a kit for detection of enzyme activities (Apizym). In the extra-sediment fraction of the core samples, the most cultivable population was obtained 1.2×107 cells/g at 59.7 mbsf. The next higher populations (~6.9×106 cells/g) were found at 4.65 and 50 mbsf. Meanwhile, the minimum population was 5.7×102 cells/g at 54.7 mbsf. Based on the sequence analysis of partial 16S rRNA genes of 191 purified isolates, the isolates were classified into four phyla, namely Actinobacteria, Bacteroidetes, Firmicutes, and Alpha- and Gamma-Proteobacteria. In shallower sediments (<4.7 mbsf), the members of Citreicella, Thalassospira, Halomonas, and Pseudomonas belonging to the phylum Alpha- and Gamma-Proteobacteria dominated the cultivable population. Furthermore, these members as well as members of Sulfitobacter, Paracoccus, and Roseovarius inhabited through core sediments until 59.7 mbsf. The members ofDietzia, Rhodococcus, and Micobacterium residing within the phylum Actinobacteria predominantly inhabited from 25.3 to 64.8 mbsf. Especially, the genus Dietzia was abundantly dominated the cultivable component. The only member ofBacillus belonging to the phylum Firmicutes inhabited from 10.4 to 64.8 mbsf and accounted for 10-20% of the total population in respective core sediments. The members of Marinobacter within the phylum Bacteroidetes were scattered in several core sediments although the population was very few.

abstracts book 159 P34EXTREMOphiles_2012 Taxonomic diversity of culturable and unculturable moderately halophilic and halotolerant bacteria in Urmia Lake, second biggest hypersaline lake in the world Maliheh Mehrshada, Mohammad ali Amoozegara,b, Bagher Yakhchalic and Seyed Abolhassan Shahzedeh Fazelib

aExtremophiles Laboratory, Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, I.R. Iran bMicroorganisms Bank, Iranian Biological Resource Center (IBRC), ACECR, Tehran, I.R. Iran cDepartment of Industrial and Environmental biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, I.R. Iran E-mail: [email protected]

Biodiversity of western coastal line of Urmia Lake has been investigated by culture dependent and culture independent methods. Urmia Lake, located in the northwest of Iran, is the largest permanent lake in Iran and one of the three permanent salt lakes in the world. Microbial sampling from four different regions of the lake was done and biodiversity of the samples were studied by culture dependent and culture independent methods. Direct plating, dilution plating and long incubation period were used to isolate microorganisms on MH, SWN, MHLN and SWNLN media, each with different pH condition. Isolation of different bacterial types was performed based on colony features, Gram staining and also primary biochemical tests (KOH, catalase and oxidase). Within 237 purified isolates, 52 strains were selected for 16S rRNA gene sequencing. In culture independent methods, genomic content of environmental samples (water and soil) were extracted and subjected to 16S rRNA PCR amplification using universal primers and 16S rDNA library was constructed and sequenced. Representatives ofHalobacillus, Halomonas, Planococcus, Gracilibacillus, Bacillus, Pontibacillus, Paracoccus, Marinobacter, Providencia, Staphylococcus, Alkalibacterium, Sanguibacter, Lysobacter, Kocuria, Pontibacter, Salicola, Micrococcus, Oceanobacillus, Brevundimonas, Thalassobacillus, Microbacterium and Piscibacillus genera were identified by culture dependent methods. In culture independent method 20% of the 16S rDNA clones were selected for sequencing. Selected isolates belonged to Bacteroidetes category within three different genera Salinibacter¡ Adhaeribacter and Cesiribacter. In culture dependent methods Representatives of Firmicutes, Actinobacteria, Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes categories were identified. 18 strains show less than 98.7% sequence similarity to the closest known strains and are representative of new endemic species of Urmia Lake. Bacteroidetes are the most abundant group in culture independent methods.

160 abstracts book EXTREMOphiles_2012P35 Environmental antibody microarray immunoassays reveal metabolic versatility of South African deep- mine biofilms Luis A. Rivasa, Yolanda Blancoa, Antonio García-Moyanob, Jacobo Aguirrea, Patricia Cruz-Gila, Arantxa Palacínc, Esta Van Heerdenb and Víctor Parroa aDepartment of Molecular Evolution, Centro de Astrobiología (INTA-CSIC), Carretera de Ajalvir km4, Torrejón de Ardoz, 28850, Madrid, Spain bTIA/UFS Metagenomics Platform, Department of Biotechnology, University of the Free State, P. O. Box 339, Bloemfontein 9300, South Africa cCentre for Plant Biotechnology and Genomics (UPM-INIA), Campus de Montegancedo, Autopista M40, km 38, 28223 Pozuelo de Alarcón, Madrid, Spain E-mail: [email protected]

We have conducted a comprehensive microbial community culture-independent analysis of four biofilms sampled in the walls of the ultra-deep gold Beatrix mine in South Africa. Whereas the biogeochemical characteristics and prokaryotic diversity in fracture and service waters from South African mines have been extensively studied, the biofilms located on the mine walls have remained relatively under-studied. The 66-antibody microarray immunoassay analyses have been compared with those results obtained by 16S rRNA gene sequencing. Moreover, we applied a previously reported deconvolution method (Rivas et al., 2011) to the immunoassay experimental data to obtain reliable qualitative information about the microbial composition of the biofilms that contained analytes that were not used as immunogens for producing the antibodies used for the immunoassay. Deconvolution analysis and 16S rRNA gene sequencing analysis agreed on the presence of sulphur-oxidizer, methanotrophic and heterotrophic bacterial communities. In addition, in good agreement with the environmental characteristics of the samples, no positive signals were detected from antibody spots related to psychrophilic, halophilic and hyperthermophilic environments. Deconvolution analysis also assured the detection of ferritins and DPS proteins only in one biofilm. ICP-MS analysis revealed that total Fe concentration in that biofilm was 10 and 100 –fold higher than the Fe concentration in the rest of them. This correlation could be due to the fact that those proteins are involved in iron metabolism; whereas ferritins are Fe2+ scavenging and storage proteins, DPS proteins are involved in Fe2+ detoxifying and DNA-protecting under starving conditions.

References · L.A. Rivas, J. Aguirre, Y. Blanco, E. González-Toril and V. Parro (2011) Graph-based deconvolution analysis of multiplex sandwich microarray immunoassays: applications for environmental monitoring.Environ Microbiol 13: 1421-1432.

abstracts book 161 P36EXTREMOphiles_2012 Microbial diversity of lithic microbial ecosystems in Miers and Garwood Valleys (McMurdo Dry Valleys, Continental Antarctica) Asunción de los Ríosa, Sergio Pérez-Ortegaa, Rüdiger Ortiz a, Steve Pointingc, Alan Greenb and Carmen Ascasoa

aDepartment of Environmental Biology, Museo de Ciencia Naturales (CSIC), 28006 Madrid Spain bDepartment of Plant Biology II, Universidad Complutense de Madrid, 280040 Madrid, Spain. cSchool of Biological Sciences, The University of Hong Kong, Hong Kong E-mail: [email protected]

Mc Murdo Dry Valleys (78°C) are ice-free valleys in Antarctica located within Victoria Land west of McMurdo Sound. They display one of the most remote and harshest environments for terrestrial life. Nevetheless, a range of terrestrial microbial ecosystems are represented, associated to the colonization of the lithic substrate. The evolutionary history and geographical isolation of the McMurdo Dry Valleys have produced a unique environment, inhabited by species adapted to its extreme conditions. During a McMurdo Dry Valleys expedition in December of 2010 to the area of Miers and Garwood Valleys, colonized granite samples from different locations were collected. They harbour endolithic microbial communities dominated by cyanobacteria, and epilithic and endolithic lichen communities. The endolithic microbial communities were characterized by electron microscopy and identified by DGGE and amplification and sequencing of the main bands. Seven predominant cyanobacterial operational taxonomic units (OTUs) (using 97% similarity cut off during sequence clustering) and three corresponding to non-phototrophic bacteria were detected. The cyanobacterial OTUs were related to unidentified hypolithic and endolithic microorganisms from extreme environments related Nostoc, Leptolymgbya, Acaryochloris and Chroococcidiopsis genera. Heterotrophic bacteria from Proteobacteria, Bacteriodetes, Actinobacteria and Acidobacteria divisions were also detected in these communities. A decrease in cyanobacterial diversity with increasing altitude was detected. Correct identification of lichen-forming fungi from extreme ecosystems is complicated by the presence of numerous sterile and extremely modified thalli. We detected 26 species of lichen-forming fungi and 5 putative species of photobionts in the studied area, using DNA-based species delimitation methods for the identification of the mycobiont species and ITS sequences for the the photobionts. No spatial structure was found in the distribution of photobionts in the studied area (Pérez-Ortegaet al., 2012).

References · S. Pérez-Ortega, R. Ortiz, T.G. Alan Green and A. de los Ríos (2012). Myco- and photobiont diversity and their relationships at the edge of life (McMurdo Dry Valleys, Antarctica). FEMS Microb Ecol (in press).

162 abstracts book EXTREMOphiles_2012P37 Polyphasic characterization ofBacillus persicus, a novel slightly halophilic bacterium from Aran-Bidgol salt lake, Iran Maryam Didaria, Mohammad Ali Amoozegara,b, Maryam Bagherib, Maliheh Mehrshada, Peter Schumannc, Cathrin Spröerc, Cristina Sánchez-Porrod and Antonio Ventosad aExtremophiles Lab., Department of Microbiology, School of Biology, College of Science University of Tehran, Tehran, Iran bMicroorganism Bank, Iranian Bioresource Centre (IBRC), ACECR, Tehran-Iran cDSMZ-German Collection of Microrganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany dDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain E-mail: [email protected]

Through a survey on halophilic bacterial diversity on the hypersaline Lake Aran-Bidol in central desert of Iran, we isolated several halophilic and halotolerant bacteria, some representing new taxa. The aim of this study was the taxonomic characterization and description of strain B48, a slightly halophilic strain isolated from hypersaline soil of Aran-Bidgol salt lake. For the polyphasic characterization biochemical and physiological tests, chemotaxonomic characterization and phylogenetic analysis were performed according to proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria (Logan et al., 2009). Strain B48 is a Gram-positive, aerobic, rod-shaped, non motile, endospore-forming bacterium. Phylogenetic analyses based on 16S rRNA sequence comparisons indicated that strain B48 was most closely related to Bacillus foraminis CV53T (97.4 %). The genomic DNA G+C content of strain B48 was 40.1 mol%. The major cellular fatty acids of strain B48 were iso-C15:0, and anteiso-C15:0, and its polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an aminophospholipid and two unknown phospholipids. The only quinone present was menaquinone 7 (MK-7). The peptidoglycan containedmeso -diaminopimelic acid as the diagnostic diamino acid. All these features confirm the placement of isolate B48T within the genus Bacillus. DNA-DNA hybridization experiments revealed a low level of relatedness between strain B48 and Bacillus foraminis IBRC-M 10628T (8.1 %). On the basis of polyphasic evidence from this study, a new species of the genus Bacillus, Bacillus persicus sp. nov. is proposed, with strain B48T (= IBRC-M 10115T = DSM 25386T) as the type strain.

Reference · N.A. Logan, O. Berge, A.H. Bishop, H.J. Busse, P. De Vos, D. Fritze, M. Heyndrickx, P. Kämpfer, L. Rabinovitch and other authors (2009). Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59: 2114-2121.

abstracts book 163 P38EXTREMOphiles_2012 Changing the microbial community at the Japan Trench, after the East Japan Earthquake of March 11th, 2011

Chiaki Katoa, Takayoshi Sekiguchia,b, Sayumi Kinoshitaa,c, Chinatsu Zamaa,b, Norio Miyamotoa, Hiromi Watanabea, Masahiro Itoc, Makiko Enokib and Katsunori Fujikuraa

aInstitute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan bDepartment of Marine Science, Tokyo University of Marine Science and Technology, Tokyo, Japan cDepartment of Life Science, Toyo University, Itakura, Japan E-mail: [email protected]

On March 11th, 2011, we had a huge earthquake (M9.0) at North-East Pacific Ocean Coast in Japan. After that, the largest Tsunamis attacked to the coast several times, and we got so serious damages. This earthquake, called the East Japan Earthquake, was a typical plate boundary type earthquake, and we got several on last thousand years. Focal region of this earthquake was at the bottom of Japan Trench land slope, thus, we were considering so many changes happen there. To investigate the changing of Japan Trench, my institute, JAMSTEC, organized several cruises from April to August 2011, using JAMSTEC research vessels and submersibles. The cruises, YK11-E04 and YK11-E06, were particularly organized for the studying the bio-diversity changing, and we obtained the sediment samples from the bottom of the Japan Trench, closely located from the central point of the focal region (Kawaguchi et al., 2012). Using those sediment samples, we have investigated about the microbial diversity and the isolation of piezophilic microorganisms. The results of the diversity studies indicated that the methane relating archaeal and the sulfate reducing bacterial (SRB) communities became abundant in the surface sediment. Originally, such microbial community was located sub-surface environment at plate boundary region. Therefore, these results suggested that the abundant of methane (seep?) appear from the deep subsurface environment together with the abundant of methane-SRB microbial consortium, affected by this very huge earthquake. At the same time, we have succeeded to isolate several piezophilic bacteria from the same sediment, the genus, Shewanella, Moritella, and Photobacterium. Those isolates were closely related with the known piezophiles, S. benthica, M. japonica, and P. profundum. But one of isolate, Ptobacterium sp. BJT#73 MB1, were far from known species, and grow well at 50 MPa pressure conditions. Those results also suggested that microbial diversity involved piezophiles were widely spread at the bottom of Japan Trench, compare with the sediment before the earthquake.

References · S. Kawaguchi et al. (2012). Disturbance of deep-sea environments induced by the M9.0 Tohoku Earthquake. Sci. Rep. 2: 270; DOI: 10.1038/srep00270.

164 abstracts book EXTREMOphiles_2012P39 Microbial diversity and enzyme characteristics of the unique ikaite columns; a permanently cold and alkaline environment in SW Greenland Mikkel A. Glaringa, Jan K. Vestera, Jeanette Lylloffa, Waleed A. Al-Soudb, Søren J. Sørensenb and Peter Stougaarda

aDepartment of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Denmark bDepartment of Biology, Faculty of Science, University of Copenhagen, Denmark E-mail: [email protected]

The ikaite columns in the Ikka Fjord, SW Greenland, constitute a permanently cold (4-6°C), alkaline (pH 10.4), and low salinity (0.9%) environment. The columns grow from the bottom of the shallow inner fjord and are composed of the mineral ikaite, an unusual metastable hexahydrate of calcium-carbonate. The columns are formed where alkaline, carbonate-rich groundwater seeping up from the fjord bottom meets the cold, calcium-rich marine waters of the Ikka Fjord. Investigations of the ikaite columns have revealed a microbial community adapted to this extreme ecological niche. We have carried out a comprehensive investigation of the microbial diversity in the ikaite columns and the surrounding fjord by pyrosequencing of the V3 and V4 hypervariable regions of 16S rDNA (pyrotag sequencing). Analysis of the more than 500,000 high-quality tags generated have revealed a diverse environment with significant inter- and intra-column heterogeneity dominated byAlpha - and Gammaproteobacteria, Bacteroidetes, and Firmicutes. Several of the most common species found in the ikaite columns are related to alkaliphilic or psychrophilic species isolated from similar environments, such as alkaline soda lakes. Analyses of a collection of cultivable isolates show a clear growth-preference for the cold and alkaline conditions characteristic of the ikaite columns. Many of the isolates also produce extracellular enzymes active at low temperatures and high pH making the ikaite columns a promising source of future industrial enzymes.

abstracts book 165 P40EXTREMOphiles_2012 Seasonal variability in microbial mat composition along a temperature gradient in Northern Patagonia geothermal systems Roy Mackenziea, Beatríz Díezb and Carlos Pedrós-Alióa

aInstituto de Ciencias del Mar, CSIC Barcelona, Spain bDepartamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Chile E-mail: [email protected]

The microbial communities from three pristine Chilean hot springs were analyzed using a culture-independent method assessing the 16S ribosomal RNA genes. These hot springs had diverse bacterial communities that shared similarities to other hot springs elsewhere with similar environmental characteristics. In this study, microbial communities were dominated mainly by phyla Cyanobacteria and Bacteroidetes, and order Thermales. Also, bacterial groups such as Proteobacteria, Acidobacteriales and Non-Sulfur Bacteria were detected in small amounts in some samples. The dominant Cyanobacterial populations were the heterocyst- containing Mastigocladus, followed by the filamentous non-heterocyst Leptolyngbya and Oscillatoria. Both main groups were detected in both seasons and showed clusters related to marine and thermal organisms. Overall, SSU 16S rRNA fingerprinting of samples from several microbial mats showed high bacterial community similarities within each hot spring, and slight differences between seasons. Comparison among the hot springs showed higher resemblance between the two geographically close springs than with the more distant one.

166 abstracts book EXTREMOphiles_2012P41 Isolation of halophilic and halotolerant microorganisms from core samples of North Kanto area in Japan Tomonori Takashina, Atsuro Watanabe, Takeshi Miura and Akira Inoue

Faculty of Life Sciences, Toyo University, Itakura, Gunma, 374-0193 Japan E-mail: [email protected]

We evaluated a variety of extracellular enzyme activities of several core samples collected by drilling under ground in North Kanto area in Japan. These activities were expected to be produced by microorganisms existed in the core samples. It is well-known that unconsolidated loose sands of the late Pleistocene Paleo- Tokyo Bay deposited in North Kanto area. Therefore we attempt to isolate aerobic halophilic and halotolerant microorganisms from the core samples and detect enzyme activities produced by isolated microorganisms. Total 29 core samples were collected by drilling under ground to 45m depth in North Kanto area in Japan. A portion of each core samples were subjected to JCM 168 liquid medium containing 10 and 20 % of NaCl. These were incubated at 37°C in 4 weeks and growth of microorganisms was observed by light-microscopy. For further isolation of microorganisms from the cores, above cultures were spread onto a JCM 168 agar medium containing 10 and 20 % of NaCl w/o certain substrates for assay for enzyme-producing such as protease, lipase, amylase, xylanase and cellulase. The agar plates were incubated at 37°C for 4 weeks. The colony number was counted and enzyme activities were detected. The cell growth of microorganisms in the liquid culture containing 10% of NaCl was observed in almost all core samples. However the growth was observed only in the core samples collected at the depth of 2m and around 35m in the medium containing 20% of NaCl. In the agar plate culture under 10% NaCl medium condition, several colonies were observed in almost all core samples. Protease, lipase, amylase, xylanase and cellulose activities were detected in the colonies isolated from almost all core samples. The count of viable cells was between 2.0x101 and 2.6x108 (cells/ml). On the other hand, in the agar culture under 20% NaCl medium condition, the colonies were observed only in the core samples collected at 2m depth and around 35m depth. Amylase, cellulase and lipase activities were detected in core sample at 2m depth and amylase and lipase activities were detected in core sample at around 35m depth. The cultivable cell numbers were 9.5x103 and 1.5x105 (cells/ml), respectively. It was suggested that there was difference in biological condition such as salt concentration in the geological formation.

abstracts book 167 P42EXTREMOphiles_2012 Influence of environmental variables on bacterial community composition in Rambla Salada, a hypersaline environment in south-eastern Spain Nahid Oueriaghli, Victoria Béjar, Emilia Quesada and Fernando Martínez-Checa

Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja s/n, 18071 Granada, Spain E-mail: [email protected]

We have studied the bacterial diversity in a hypersaline habitat in Murcia (south-eastern Spain) using a range of microbial molecular techniques, including 16S rDNA sequencing from denaturing gradient gel electrophoresis (DGGE) and catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH). We then used multivariate statistical analysis to identify the relationships between the bacterial community and environmental variables. Water and sediments from three different zones, covering a salinity range from 1.1% (riverbed zone) to 15.8% (upwelling zone) were sampled and analysed in June 2006, February 2007 and November 2007. DGGE showed changes in the composition of the bacterial community from season to season; the communities obtained in February and November 2007 were similar in terms of richness and evenness, and also showed the highest diversity. An analysis of the DNA sequences recovered from the DGGE patterns revealed that the bacterial community was composed predominantly of members of the Proteobacteria and Bacteroidetes phyla. We also detected sequences related to Firmicutes, Cyanobacteria and Actinobacteria. These results differ from those obtained previously by Luque et al. (2012), who applied culture methods to samples from the same rambla. CARD-FISH revealed that the total microbial population in Rambla Salada was around 6.67 × 108 cells/ml, with the bacterial community ranging from 54% to 79% of the DAPI counts. The bacterial count was highest in February 2007 at 4.81 × 108 cells/ml. Canonical correspondence analysis (CCA) showed that salinity and oxygen were the key factors driving the changes in bacterial community composition.

References · M. Marzorati, L. Wittebolle, N. Boon, D. Daffonchio and W. Verstraete (2008). How to get more out of molecular fingerprints, practical tools for microbial ecology. Environ Microbiol 10: 1571-1581. · A. Pernthaler, J, Pernthaler and R, Amann. (2002). Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria.Appl. Environ Microbiol 68: 3094-3101. · R. Luque, V. Béjar, E. Quesada and I, Llamas. Diversity of cultivable halotolerant and halophilic bacteria isolated from Rambla Salada, Murcia (Spain) (unpublished).

168 abstracts book EXTREMOphiles_2012P43 Halophile diversity of the Darling Salt Pans of the Western Cape, South Africa Brian E. Jonesa, William D. Grantb, Shaun Bondb, Marla Tuffinc and Don Cowanc

aGenencor International B.V., Archimedesweg 30, Leiden, 2333CN, The Netherlands bDepartment of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN, UK cInstitute for Microbial Biotechnology and Metagenomics, Department of Biotechnology, University of the Western Cape, Modderdam Road, Bellville, Cape Town 7535, South Africa E-mail: [email protected]

Darling is a small agricultural town located 72 km north of Cape Town in the West Coast region of the Western Cape. The dozen or so salt pans situated a few km NW of the town in an area called «Swartland» are athalassohaline in origin but are separated from the nearby Atlantic Ocean by the Darling Hills and coastal sand dunes. The pans were probably formed initially during the early Holocene (Smith & Compton, 2004). The area experiences a Mediterranean type climate with hot, dry, windy summers (October to March) when arid conditions prevail and evaporation significantly exceeds the annual [mainly winter] rainfall of about 400 mm/yr. The salt pans are fed by coastal rainwater, groundwater and ephemeral steams, and were sites of commercial salt production until very recently. The microbial composition of two salt pans (Burgherspan and Rooipan) was investigated using a culture- based and a metagenomic approach. From the first sampling in Nov. 2003 at the beginning of the austral summer, 16S rRNA genes were directly amplified by PCR from environmental DNA and different genes selected by RFLP analysis. Archaeal genera; Haloquadratum, Halorubrum, Haloarcula and Halobacterium were detected as well as Bacterial phylotypes related to Firmicutes, Bacteroidetes and Gammaproteobacteria. However, none of the metagenomic sequences matched any of the sequences obtained from the cultured bacteria or Archaea. A re-sampling of Rooipan was performed in March 2012 at the end of the austral summer and at Burgherspan in April 2012 after the first winter rains. The 16S rRNA gene sequences from cultured bacteria were compared with sequences obtained in 2003 to determine the level to which population shifts occur in these highly selective saline environments.

References · Smith M and Compton JS (2004) Origin and evolution of major salts in the Darling pans, Western Cape, South Africa. Applied Geochemistry 19: 645-664.

abstracts book 169 P44EXTREMOphiles_2012 Microbial diversity of Namib Desert Salt Pans Melissa Du Plessisa, AngelValverdea, Marla Tuffina and Don Cowanb aInstitute for Microbial Biotechnology and Metagenomics (IMBM), Department of Biotechnology, University of the Western Cape, Cape Town, South Africa bDepartment of Genetics, University of Pretoria, Pretoria, South Africa E-mail: [email protected]

Salt pans are characteristic to desert landforms and are thought to be particularly complex and generally dominated by halophilic micro-organisms. Although saline pools are found frequently within the hyper-arid Namib Desert, the microbial communities of these saline sites have been scarcely investigated. The aim of the present study was to characterise the bacterial diversity inhabiting these extreme saline pools by using three culture-independent molecular techniques (DGGE, T-RFLP and 16S rRNA clone libraries). Eleven sediment samples were collected from two salt pans (Gobabeb and in the Swakopmund regions) in the Namib Desert. The conductivity readings recorded from the Gobabeb and the Swakopmund sites was averaged between 103.0 mS/cmand 165.0 mS/cm respectively. Results obtained from DGGE and TRFLP MDS plots were in agreement indicated that the two salt pan microbial communities are significantly different from each other (ANOSIM Global R=0.443, p<0.01). Furthermore, statistical analysis from TRFLP operational taxonomic units (OTUs) obtained implicates the less saline site of Gobabeb to be more diverse with a mean of 10.0 compared to the Swakopmund site with a mean of 7.8. Phylogenetic data obtained from 16S rRNA clone libraries found similar phylotypes that are normally associated with salt rich sites, such as the bacterial families Rhodothermaceae (Sphingobacteria), Flammeovirgaceae (Sphingobacteria), Idiomarinaceae (Gammaproteobacteria) and Puniceicoccaceae (Opitutae). This study highlights the presence of a rich microbial diversity and it also establishes a platform in which future investigation on the Namib Desert salt pans can emanate.

170 abstracts book EXTREMOphiles_2012P45 Characterisation of halotolerant and halophilic Actinobacteria: Phylogeny and enzymatic activities Gabriela Scarlett Alonso Carmonaa, Félix Aguirreb, Horacio Sandoval Trujillob, Ninfa Ramírez Durána and Hugo Ramírez Saadb aLaboratorio de Microbiología Médica y Ambiental, Facultad de Medicina, Universidad Autónoma del Estado de México. Paseo Tollocan y Jesús Carranza s/n Colonia Moderna de la Cruz, Toluca, C.P. 50180 México bDepartamento de Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco. Calzada del Hueso 1100, Colonia Villa Quietud, Coyoacán, C.P. 04960 México, D.F. E-mail: [email protected]

Halophilic bacteria are extremophile microorganisms with biotechnological potential, not only for producing compounds of great industrial interest, such as biopolymers, compatible solutes, and mainly as enzyme producers. The term extremozyme has been used to refer to enzymes that can be used in processes carried out under extreme conditions. Most of the extra-and intracellular haloenzymes isolated and characterized so far are from halophilic bacteria, however, little interest has been given to haloenzymes from halophilic and halotolerant actinobacteria. The aim of this study was to characterize a collection of Actinobacteria isolated from saltern ponds in southern Mexico. The isolates were first classified as halotolerant or halophilic; then amylase, protease and lipase activity was tested in 20 strains of halophilic Actinobacteria, while lipase and protease activity was assayed in 20 strains of halotolerant Actinobacteria. All isolates were grouped by DGGE fingerprinting and 16S rRNA gene sequencing was performed to selected members of each formed group. Salt requirements for growth were tested in medium with 0, 5, 15, 15, 20, 25 and 30%. All the strains are able to grow from 5 to 15% NaCl, with an optimum at 10% NaCl. Proteolytic activity was determined on milk- agar as proposed by Sanchez Porro (2005). Lipolytic activity was tested on MH medium amended with 1% olive oil and 3% rhodamine (Kouker & Jaeger 1987). Determination of amylase was done in MH base medium supplemented with 1% potato starch. The collection consisted in 40 actinobacterial strains, 20 were determined as halotolerant, while 20 were considered halophilic on different degrees. All isolates were genotyped by means of DGGE and six strains representative from each formed group were selected for 16S rRNA gene sequencing. Sequence analysis determined that our halophilic and halotolerant actinobacteria belonged to the genus Saccharomonspora. A further study was performed to distinguish phenotypic-metabolic characteristics of microorganisms in each group. The enzymatic activities survey on 20 halotolerants pointed 5 lipolytic strains, 5 protelytic strains and one with both activities. From the halophilic strains; 14 showed proteolytic activity, 3 were lipolytic and 17 positive for amylase, 9 strains showed two activities.

References · G. Kouker and K.E. Jaeger (1987). Specific and sensitive plate assay for bacterial lipases. Applied and Environmental Microbiology 53: 211-213. · Sánchez-Porro, C. (2005). «Caracterización bioquímica y molecular de la haloproteasa CP1 producida por Pseudoalteromonas ruthenica». (Tesis doctoral). Universidad de Sevilla.

abstracts book 171 P46EXTREMOphiles_2012 Comparative analysis of yellow microbial communities growing on the walls of geographically distinct caves indicates a common core of microorganisms involved in their formation Estefania Porcaa, Valme Juradoa, Darja Žgur-Bertokb, Cesareo Saiz-Jimeneza and Lejla Pasicb

aInstituto de Recursos Naturales y Agrobiologia, IRNAS-CSIC, 41012 Sevilla, Spain bDepartment of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia E-mail: [email protected]; [email protected]

Morphologically similar microbial communities that often form on the walls of geographically distinct limestone caves have not yet been comparatively studied. Here, we analyzed phylotype distribution in yellow microbial community samples obtained from the walls of distinct caves located in Spain, Czech Republic and Slovenia. To infer the level of similarity in microbial community membership, we analyzed inserts of 474 16S rRNA gene clones and compared those using statistical tools. The results show that the microbial communities under investigation are composed solely of Bacteria. The obtained phylotypes formed three distinct groups of operational taxonomic units (OTUs). About 60% of obtained sequences formed three core OTUs common to all three sampling sites. These were affiliated with actinobacterial Pseudonocardinae (30–50% of sequences in individual sampling site libraries), but also with gammaproteobacterial Chromatiales (6–25%) and Xanthomonadales (0.5–2.0%). Another 7% of sequences were common to two sampling sites and formed eight OTUs, while the remaining 35% were site specific and corresponded mostly to OTUs containing single sequences. The same pattern was observed when these data were compared with sequence data available from similar studies. This comparison showed that distinct limestone caves support microbial communities composed mostly of phylotypes common to all sampling sites (Porca et al., 2012).

References · E. Porca, V. Jurado, D. Žgur Bertok, C. Saiz-Jimenez and L. Pasic (2012). Comparative analysis of yellow microbial communities growing on the walls of geographically distinct caves indicates a common core of microorganisms involved in their formation. FEMS Microbiol Ecol DOI: 10.1111/j.1574-6941.2012.01383.x

172 abstracts book EXTREMOphiles_2012P47 Diversity and activity of the denitrification population of Halomonas in Rambla Salada Carmen María González-Domenecha, Nahid. Oueriaghlia, Fernando Martínez-Checaa, Emilia Quesadaa, Melanie Mormileb and Victoria Bejara

aDepartamento de Microbiología, Facultad de Farmacia, Campus Universitário de Cartuja s/n., 18071 Granada, España bDepartment of Biological Sciences, Missouri University of Science and Technology, 400 W. 11th St., 105 Schrenk Hall, Rolla, MO 65409-1120, USA E-mail: [email protected]

A molecular study of the Halomonas population isolated in Rambla Salada (Murcia, S.E. Spain) revealed that H. ventosae was the predominant species [1]. One of the characteristics ofH. ventosae is its ability to denitrify, so we decided to focus our studies on attempting to identify the denitrifying community Halomonasof in this habitat. We also investigated the denitrification activity of the species found. Our initial study into the bacterial community was conducted using PCR-DGGE. We employed oligonucleotides designed on the basis of earlier experiments in which we sequenced the complete nirS and nosZ genes in the denitrifying species of this genus [2]. The conserved regions (about 500bp) were then employed in our ecological studies using PCR-DGGE. The results showed that the diversity of the denitrifying Halomonas community was quite wide, including at least 6 different species: H. cerina, H. desiderata, H. fontilapidosi, H. koreensis, H. nitroreducens and H. ventosae. We also went on to study phenotypic diversity among the denitrifying bacteria represented in Rambla Salada by comparing their rates of respiration on nitrate and nitrite. To this end we used saline media supplemented with various sources of carbon for growth. This study shows that respiration with nitrite depends on the bacterium in question rather than the carbon source. Among the denitrifying species ofHalomonas represented in Rambla Salada, we found the highest denitrification activity in the type strain Halomonasof desiderata, whilst H. nitroreducens 11ST correlated biomass/nitrite respiration best.

References · N. Oueriaghli, C.M. González-Domenech, F. Martínez-Checa, E. Quesada, G. Muyzer and V. Béjar (2012). Molecular study of the diversity of the genus Halomonas in Rambla Salada (Murcia) by means of DGGE, CARD- FISH and multivariate analysis. (under revision). · C.M. González-Domenech, F. Martínez-Checa, V. Béjar and E. Quesada (2010). Denitrification as an important taxonomic marker within the genus Halomonas. Syst Appl Microbiol 33: 85-93.

abstracts book 173 P48EXTREMOphiles_2012 Partial characterization and activity of extremely halophilic microorganisms isolated from an Argerian salt lake (Chott el Beida) Houa Yahiaoui, Samy Brahimi, Nabil Bouali and Said Benallaoua

Applied Microbiology Laboratory, Department of Microbiology, Faculty of Nature and Life Sciences, University A.MIRA of Bejaia, Targa Ouzemour, Bejaia 06000, Algeria E-mail: [email protected]

Chott El beida is a saline lake located in El Eulma, situated in the Algerian Province of Setif. The lake is not exploited and ecologically unpolluted. Thirty samples from soil, salt and water were isolated. Twelve of them exhibit inhibitory activities against one or more strains of haloarchaea, including strains isolated from other Algerian hypersaline environments such as Natrinema from solar saltern of Ichakaben (Carré Mlouka et al., 2012) , strain Halobacterium salinarum DSM 3754 and Haloferax mediterranei DSM 1411, a strain known to produce halocin H4 (Rodríguez-Valera et al.,1982). These twelve isolates are potentially producers of halocins in agreement with the definition of halocins, which are generally active on phylogenetically related strains. Diameters of halos inhibition obtained for five isolates when challenged againstHbt. salinarum are important and can range up to 45mm for one strain isolated from water. Characterization of the isolates was performed using phenotypic, morphological and some biochemical approaches. Sensitivity of the halophilic isolates to antibiotics was checked likewise. Electronic microscopy was also performed for five of the more active isolates. The isolates are aerobic (pH 7), Gram negative, mainly mesothermic, requiring 250 g/l of NaCl, and could grow at alkaline pH (pH 9) and at 300 g/l of NaCl. The isolates are pigmented, pink or red, except for two strains that remain white .They also have different morphological forms, some are pleomorphic, others are rods or spherical.

References · F. Rodríguez-Valera, G. Juez, D. J. Kushner (1982) Halocins: salt-dependent bacteriocins produced by extremely halophilic rods. Can J Microbiol 28:151-154. · Alyssa Carré-Mlouka, Manon Vandervennet, Nacéra Imadalou-Idres, Houa Yahiaoui, Said Benallaoua, Jean Peduzzi and Sylvie Rebuffat (2012). Halocin-producing archaea are persistent in Algerian hypersaline ecosystems (abstract submitted to th9 International Congress on Extremophiles).

174 abstracts book EXTREMOphiles_2012P49 Microbial community in a Zinc impacted mining site Hugh Morgan and Lin Tan

Department of Biological Sciences, University of Waikato, Hamilton, New Zealand E-mail: [email protected]

A disused gold mine site at Mt Te Aroha, New Zealand is one of the countries most polluted areas. The mine spoil tailings particulates contain 2.4% Zn++ and immediate runoff waters have pH values between 2.5 and 3.5 and contain up to 55 mg Zn++/litre as well as lesser quantities of lead, copper and mercury. In the generally aerobic top sediment layer the dominant heterotrophic organism is a Zinc tolerant Acidocella sp. This isolate showed good growth in the presence of 500mM Zn++ and was similar to the previously reported isolate Acidocella GS19h (which has now been lost) with the exception that no resistance plasmid was detected in the Te Aroha isolate. Three methods of DNA extraction were trialled on the different sediment layers in the soil tailings. While all three methods extracted amplifiable DNA, only the Mol Biol Soil Extraction kit produced measurable quantities of DNA. 16S rDNA clone libraries of extracted DNA have been used to define the bacterial members of these sediments for comparison with cultured heterotrophic isolates.

References · S. Gosh, N.R. Mahaptra and P.C. Banerjee (1997) Metal resistance in Acidocella strains and plasmid- mediated transfer of this characteristic toAcidiphilium multivorum and Escherichia coli. Appl Environ Microbiol 63: 4523-4527.

abstracts book 175 P50EXTREMOphiles_2012 Roseivivax salinus and Roseovarius halophilus, two new species from a multi-pond saltern Mª José Leóna, Ana B. Fernándeza, Cristina Sánchez-Porroa, Francisco Rodríguez-Valerab and Antonio Ventosaa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bEvolutionary Genomics Group, Division of Microbiology, University Miguel Hernandez, 03550 Alicante, Spain E-mail: [email protected]

Despite advances in knowledge of halophilic microorganisms, most studies have been carried out using pure cultures and in many cases this is not the dominant microbiota in the hypersaline environments. It is claimed that only a minimal proportion of the existing microbiota, between 0.1 and 1 % of the total is cultured under laboratory conditions, due to the ignorance of the nutritional requirements of microorganisms inhabiting these environments. Based on the metagenomic analysis of a multi-pond solar saltern located in Santa Pola, Alicante, Spain we have designed different culture media in order to isolate those groups of organisms that constitute an important part of the biodiversity of these environments and have not been isolated yet. For this study we have selected seven strains for a detailed taxonomic characterization: strains R7, R8, R9, R10 and strains RP8, RP10 and RP15 that could constitute new species of the generaRoseivivax and Roseovarius, respectively. The phylogenetic study of the strains R7, R8, R9 and R10 showed that they are related to the genusRoseivivax, specifically to the speciesRoseivivax lentus, with 16S rRNA gene sequence similarity values ranging from 93.1 to 93.3 %. On the other hand, the comparison of the 16S rRNA gene sequence of the strains RP8, RP10 and RP15 with the 16S rRNA gene databases revealed that they are related to the species Roseovarius tolerans, with similarity values ranging from 96.0 to 96.2 %. These data indicate that the isolates could constitute two new species. For their taxonomic characterization we used a polyphasic approach, based on a combination of phenotypic, phylogenetic, genotypic and chemotaxonomic characteristics. The results obtained confirm that strains R7, R8, R9 and R10 constitute a new species of the genus Roseivivax, for which we propose the name Roseivivax salinus sp. nov., and the strains RP8, RP10 and RP15 constitute a new species of the genusRoseovarius, for which we propose the name Roseovarius halophilus sp. nov.

176 abstracts book EXTREMOphiles_2012P51 The effect of carbon input on the evolution of the soil microbial community in the central hyper-arid Namib Desert Alacia Armstronga, Jean-Baptiste Ramonda, Marla I. Tuffina and Don A. Cowanb

aInstitute for Microbial Biotechnology and Metagenomics (IMBM). University of the Western Cape, Bellville 7535, South Africa bDepartment of Genetics, University of Pretoria, Pretoria 0002, South Africa E-mail: [email protected]

The Namib Desert, situated along the south western coast of Africa, is considered an extreme environment due to the aridity and daily fluctuating temperatures (below 0 °C and over 50 °C) (van der Wateren & Dunai, 2001). Vegetation is sparse and seasonal grass contributes the majority of carbon to the nutrient-deficient soils. The impact of the carbon input and the role of the soil microbial communities in the carbon turnover in this environment are not at all known.

In order to begin investigating the ecology of this desert, a plot (8100 m2, separated into 10x10m plots ) colonized by seasonal grass and two adjacent control sites (100 m2) with no apparent grass colonization, were selected near the Gobabeb Research Station in the Namib desert. Eight surface soil (0-3 cm depth) samples were collected from randomly selected plots on day 0, 3, 7, 15, 30, 45 and 60 for analysis. Analyses included: (i) Fluorescein diacetate (FDA) and ATP determinations to assess soil microbial activity and biomass evolutions respectively, (ii) Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of the microbial communities, and (iii) soil chemistry monitoring. Environmental factors such as moisture and temperature were also monitored. Such a temporal study should begin to illustrate the community structure variation in response to a variety of environmental contributions.

References: • F.M. Van der Wateren and T.J. Dunai (2001). Late Neogene passive denudation history cosmogenic isotope measurements from the central Namib Desert. Global and Planetary Change 30: 271-307.

abstracts book 177 P52EXTREMOphiles_2012 Sulfolobus mongibelli sp. nov., an acidophilic hyperthermophilic archaeon isolated from Etna fumarole Tatyana Sokolovaa, Michail Yakimovb, Nikolai Chernyha, Alexander Lebedinskya and Elizaveta Bonch- Osmolovskayaa

aWinogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia bIstituto per l’Ambiente Marino Costiero, Sezzione di Messina – CNR, Messina, Italy E-mail: [email protected]

An isolate, strain SYETT, was obtained from fumarole samples collected near the main crater of Etna Volcano,

Italy. The strain was enriched and isolated in microaerobic conditions, under nitrogen mixed with 2% 2O , 10% CO and 10% CO2. Initial enrichments were made in medium with pH 6.0 and were incubated at 75 and 85°C. Cells of strain SYETT were cocci, 1-1.5 µm in diameter. The isolate grew optimally at 80 °C, pH 3.5. CO consumption by growing culture was observed. Phylogenetic analysis based on 16S rRNA gene sequence comparison indicated that strain SYETT was an archaeon of Crenarchaeota phylum, where it clustered within the branch constituted by species ofSulfolobus. The most closely related species was Sulfolobus islandicus (96% 16S rRNA sequence similarity). CoxL gene was detected in strain SYETT DNA by direct PCR with specific primers. The genome of strain SYETT was sequenced and analysed and the occurrence of Cox genes cluster was confirmed. On the basis of the phenotypic and phylogenetic features, a novel speciesSulfolobus mongibelli is proposed.

178 abstracts book EXTREMOphiles_2012P53 Microstructure and prokaryotic diversity of microbialites formed around a man-made geothermal spring from Romania Cristian Comana, Bogdan Drugãa, Adriana Hegedusa, Lucian Barbu-Tudoranb, Nicolae Dragosa,c and Cosmin Sicorad aInstitute of Biological Research, Cluj-Napoca, branch of The National Institute for Research and Development in Biological Sciences (NIRDBS), Bucharest, Romania bElectron Microscopy Center, Babes-Bolyai University, Cluj-Napoca, Romania cBiology and Geology Faculty, Babes-Bolyai University, Cluj-Napoca, Romania a,dBiological Research Center, Jibou, Romania E-mail: [email protected]

Contemporary microbial mats are believed to hold the key for the past by providing insight into the role of microbes in mineral precipitation. Understanding microbe-mineral interactions is critical for interpretation of the rock record. To see how certain conditions influenced the development of the fossils it is very important to look for sedimentary structures that can be considered modern analogues («living stromatolites») of fossils. In the Western Plain of Romania, around a man-made geothermal spring, modern, laminated sedimentary structures were discovered. Three morphologically distinct samples (Z1, Z3 and Z4) were taken for further investigations. Light microscopy images of thin sections showed a laminated, multi-layered structure, the thickness of the layers ranging from a few μm to hundreds of μm, in all 3 samples. Based on EDX analysis, we can say that the sedimentary structures are most likely formed by CaCO3 precipitation in the form of calcite/aragonite groups: calcite - CaCO3; magnesite - MgCO3; siderite - FeCO3; strontianite - SrCO3; witherite -BaCO3).

The bacterial biofilm has a paralell orientation in regards to the CaCO3 crystals, sometimes wrapped around them. The cyanobacterial filaments are around 2.5μm in diameter and tens of μm in length, sometimes forming a fibrous net. Together with cyanobacteria, other bacteria were observed, some filamentous other rod-shaped. The archaeal clone libraries are dominated by Methanmicrobia, followed by Thermoprotei, Archaeoglobi and Thermoplasmata groups. In the case of Z1 bacterial clone library, the following groups were identified: Actinobacteria, Oscillatoriales, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Sphingobacteria, Bacteroidetes/Chlorobi and Chloroflexi. In Z3 bacterial clone library, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Epsilonproteobacteria, Sphingobacteria, Chloroflexi, candidate division OP9, Bacteriodetes and Clostridia groups were observed. The Z4 bacterial clone library was composed of Deferribacterales, Alphaproteobacteria, Betaproteobacteria, Thermotogales, Clostridia, Bacillales, Thermotogae and Chloroflexi.

abstracts book 179 P54EXTREMOphiles_2012 Diversity of an hydrolytic bacterial community isolated from heavy-metal-contaminated soils of the Atacama Desert María de Lourdes Morenoa, Francine Piubelib, Maricy R.L. Bonfáb, María Teresa Garcíaa, Lucia R. Durrantb and Encarnación Melladoa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bDepartament of Food Science, University of Campinas-UNICAMP, Rua Monteiro Lobato, 80 CEP 13083-862, Campinas, SP, Brazil E-mail: [email protected]

The Atacama Desert constitutes an arid to semi arid environment dominated by the presence of many different saline deposits. As a result of the desert nature of the area, there is no agricultural activity and the only industrial activity is mining. This study investigates the cultivable community of hydrolase producers (amylase, protease, lipase, DNase, xylanase and pullulanase) inhabiting this extremophilic and heavy-metal contaminated habitat by using cultivation-dependent techniques. A total of 25 bacterial strains showing hydrolytic activities have been selected including halotolerants, extremely halotolerants and moderate halophiles. Most hydrolase producers were assigned to the family Bacillaceae, belonging to the genera Bacillus (9 strains), Halobacillus (7 strains) and Thalassobacillus (5 strains) and four isolates were related to members of the families Pseudomonadaceae, Halomonadaceae and Staphylococcaceae, included in the genera Pseudomonas, Halomonas and Salinicoccus, respectively. The selected strains were then characterized for their tolerance pattern to 6 heavy metals, measured as minimal inhibitory concentrations (MICs). The highest toxicities were found with Cd and Zn. The majority of strains (56%) were tolerant to nickel. Strains related to Halobacillus hunanensis were the most sensitive to the heavy metals tested. In contrast, the strains related to Bacillus subtilis and Bacillus stratosphericus were the most tolerant, showing high tolerance to the metal ions of Fe, Co, Ni and Cu and moderate tolerance to Cd and Zn. Our research suggests that the extremophilic hydrolytic bacterial community heavy-metal resistant from the Atacama Desert is a promising resource for bioremediation of heavy-metals and may be excellent candidates of novel enzymes with biotechnological applications.

180 abstracts book EXTREMOphiles_2012P55

Phylogenetic diversity of xylan-degrading thermophilic bacteria from hot springs in Benguet, Philippines Socorro Martha Meg-ay V. Daupana, Phuc Thi Hac, In-Seop Changc and Windell L. Riveraa, b

aInstitute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines bNatural Sciences Research Institute, University of the Philippines Diliman, Quezon City 1101, Philippines cSchool of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Korea E-mail: [email protected]

Xylan is a major component of hemicellulose and its hydrolysis has great application in the paper and pulp, fabric, and biofuel industries (Beg et al., 2000; Somerville, 2007). Xylan hydrolysis by thermophilic bacteria is of particular interest because these microbes exhibit optimum enzyme activity at elevated temperatures (Haki and Rakshit, 2003). Although the Philippines is geographically situated in the Pacific Ring of Fire and is subject to geothermal environment, there is limited information on the thermophilic bacterial community and compositions in Philippine geothermal sites as there are few reports (Lanticanet al., 2011). Moreover, in such reports bacterial community was assessed based only on some molecular techniques and 16S rRNA cloning (Lantican et al., 2011; Hongmei et al, 2005). In this study, we isolated and characterized putative xylan-degrading thermophilic bacteria in Benguet hot springs, Philippines. The phylogenetic relationship among these and other known xylan-degrading bacteria was constructed. A total of 53 thermophilic, mostly Gram negative bacteria were isolated and 18 showed positive results for xylan degradation using plate assay. The phylogenetic tree generated with the 16S rRNA sequences of the xylan-degrading bacterial isolates showed that they are distributed to three genera, namely Geobacillus (83%), Anoxybacillus (11%) and Brevibacillus (6%). The thermophilic bacteria Meiothermus and Thermonema have also been isolated but showed no xylanolytic activity. Interestingly, all isolates that showed 99-100% sequence similarity with Geobacillus spp. revealed most positive activity for xylan degradation.

References: · Q. Beg, B. Bhushan, M. Kapoor and G. Hoondal (2000). Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp.Enz Micro Technol 27: 459-466. · C. Somerville (2007). Biofuels. Curr Biol 17: R115-9. · G. Haki and S. Rakshit (2003). Developments in industrially important thermostable enzymes: A review. Biores Technol 89: 17-34. · N. Lantican, M. Diaz, J. Cantera, F. de los Reyes and A. Raymundo (2011). Microbial community of a volcanic mudspring in the Philippines as revealed by 16S rDNA sequence analysis and fluorescence in situ hybridization. World J Microbiol Biotechnol 27: 859-867. · J. Hongmei, J. Aitchison, D. Lacap, Y. Peerapornpisal, U. Sompong, S. Pointing (2005). Community phylogenetic analysis of moderately thermophilic cyanobacterial mats from China, the Philippines, and Thailand. Extremophiles 9: 325-32.

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182 abstracts book EXTREMOphiles_2012

session 2 adaptation to extreme environments

abstracts book 183 OL1EXTREMOphiles_2012

184 abstracts book EXTREMOphiles_2012P56 Improvement in organic solvent-tolerance by double mutations of acrR and marR genes in Escherichia coli Rei Watanabea,b and Noriyuki Doukyua,b,c

aGraduate School of Interdisciplinary New Science, Toyo University, 2100, Kujirai, Kawagoe, Saitama, 350-8585, Japan bBio-Nano Electronic Research Center, Toyo University, 2100, Kujirai, Kawagoe, Saitama, 350-8585, Japan cDepartment of Life Science, Toyo University, 1-1-1 Izumino, Itakura-machi, Gunma, 374-0193, Japan E-mail: [email protected]

Hydrophobic organic solvents can be toxic to organisms. Environments containing high concentrations of organic solvents represent one definition of extreme environments for organisms. Microorganisms are also harmed by various organic solvents (Inoue and Horikoshi, 1989). E. coli strain JA300 is highly sensitive to cyclohexane (Aono, 1998). In the present study, eight cyclohexane-tolerant Escherichia coli JA300 mutants were isolated and examined by DNA sequencing for organic solvent-tolerance-associated mutations in acrR, marR and soxR. No mutant carried a mutation in soxR. Every mutant carried a mutation in either acrR or marR. Two mutants had mutations in both acrR and marR. Among all mutants, strain CH7 exhibited the highest organic solvent-tolerance levels. Strain CH7 carried an insertion of IS5 in acrR and a nonsense mutation in marR. To clarify the involvement of these mutations in improving organic solvent-tolerance, these two mutations were introduced into E. coli JA300. The resulting strain was named JA300 acrRIS marR. The organic solvent- tolerance level and AcrAB-TolC efflux pump-expression level in JA300 acrRIS marR were similar to those of CH7. Thus, it was shown that synergistic effects of mutations in both acrR and marR can significantly increase the AcrAB-TolC efflux pump-expression level and organic solvent-tolerance in E.coli. The organic solvent-amount accumulated in E. coli cells was examined after incubation in an organic solvent-aqueous medium two-phase system. The intracellular organic solvent levels in JA300 acrRIS marR and CH7 were equally kept lower than that of the parent strain JA300. An acrA-disruptant of JA300 acrRIS marR became as sensitive to organic solvents as JA300ΔacrA, indicating that the AcrAB-TolC pump is essential for JA300 acrRIS marR to acquire high-level organic solvent-tolerance.

References · A. Inoue and K. Horikoshi (1989). A Pseudomonas thrives in high concentrations of toluene. Nature 338: 264-265. · R. Aono (1998). Improvement of organic solvent tolerance level of Escherichia coli by overexpression of stress-responsive genes. Extremophiles 2: 239-248.

abstracts book 185 P57EXTREMOphiles_2012 Genetic analysis of the genes involved in archaeosine biosynthesis of hyperthermophilic archaeon Thermococcus kodakarensis Hiroki Higashibataa,b and Tadayuki Imanakac

aGraduate school of Life Sciences, Toyo University, Ora-gun, Gunma, 374-0193, Japan bBio-Nano Electronics Research Centre, Toyo University, Kawagoe, Saitama, 350-8585, Japan Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan E-mail: [email protected]

Archaeosine, found at position 15 in the D-loop of archaeal tRNA, has an unusual 7-deazaguanosine structure and is converted from Guanosine 5’-triphosphate (GTP) in six steps. In the biosynthetic pathways to deazapurine- containing metabolites from GTP, four enzymes (GCHI, QueD, QueE, and QueC) are necessary for the production of 7-cyano-7-deazaguanine (preQo). The preQo is inserted into tRNA by tRNA-guanine transglycosylase (TGT), and then converted to archaeosine by ArcS. Computational analysis suggests that archaeosine stabilize the tertiary structure of tRNA. However, it is still unclear whether archaeosine stabilizes tRNA structure in high-temperature environments. In order to clarify the physiological significance of archaeosine in hyperthermophilic archaeon Thermococcus kodakarensis, gene disruption experiments were performed. Each gene (queD, queE, queC, tgt and arcS) was successfully disrupted, indicating that these genes are not essential for cell viability. The growth profiles ofΔ tgt and ΔarcS mutants were almost identical with that of host strain at 85oC (the optimum growth temperature) and 93 oC (the upper limit of growth temperature). The growth profiles of the other mutants (ΔqueD, ΔqueE, and ΔqueC) were also measured at 85 and 93 oC, respectively.

References · R. M. McCarty, Á. Somogyi, G. Lin, N. E. Jacobsen, and V. Bandarian (2009). The deazapurine biosynthetic pathway revealed: in vitro enzymatic synthesis of PreQ(0) from guanosine 5’-triphosphate in four steps. Biochemistry 48: 3847-3852. · G. Phillips, V. M. Chikwana, A. Maxwell, B. El-Yacoubi, M. A. Swairjo, D. Iwata-Reuyl, and V. de Crécy-Lagard (2010) Discovery and characterization of an amidinotransferase involved in the modification of archaeal tRNA. J Biol Chem 285: 12706-12713. · R. Oliva, A. Tramontano, and L. Cavallo (2007) Mg2+ binding and archaeosine modification stabilize the G15-C48 Levitt base pair in tRNAs.RNA, 13: 1427-1436.

186 abstracts book EXTREMOphiles_2012P58 Elemental and intact polar lipid compositions of the hyperthermophilic archaeon Thermococcus kodakarensis grown under control and phosphate-reduced conditions Travis B. Meadora, Emma J. Gagenb, Michael Loscarb, Michael Thommb and Kai-Uwe Hinrichsa

aOrganic Geochemistry Group, Marum Center for Environmental Sciences, University of Bremen, Bremen, Germany bDepartment of Microbiology, University of Regensburg, Regensburg, Germany E-mail: [email protected]

In the absence of pure culture isolates, biomarkers are invaluable tools for identifying and investigating microorganisms in nature. Intact polar lipids (IPLs) -membrane lipids with an attached polar headgroup, such as phospholipids and glycolipids- are one such biomarker, presently being employed to study uncultured microorganisms, particularly Archaea, in deep sedimentary ecosystems (e.g. Lippet al., 2008). IPLs are considered to be a marker for viable prokaryotes because lipid headgroups are quickly hydrolysed after cell death (White et al., 1979). While IPL analysis can provide information about the identity and putative metabolic strategies of microorganisms in sedimentary ecosystems (e.g. Hinrichs et al. 1999) little is known about how environmental parameters influence the diversity and distribution of IPLs. In the present study Thermococcus kodakarensis was used as a model organism to investigate the effect of altered phosphate concentrations on the diversity of IPLs in an archaeon. Elemental and IPL compositions were determined for T. kodakarensis cells grown fermentatively on pyruvate under control, reduced yeast extract (YE), and reduced phosphorus (P) conditions. Control cells increased in percent carbon (%C) and nitrogen (%N) as the culture entered stationary phase but we observed no similar increase for cells grown under reduced conditions. In general, phosphatidyl diethers were the dominant IPL class found in all cells. However control cells incubated for an extended period (>40 h) after reaching stationary phase, exhibited a marked increase in the percentage of IPLs that were tetraethers (>50%) compared to cells harvested at earlier growth stages (<10%). Principal component analysis of IPL composition revealed distinct groupings of cultures according to control and reduced growth conditions. Hexose moieties attached to the PA headgroup in diether lipids were observed under reduced P conditions, illustrating an archaeal membrane adaptation to reduced phosphorus conditions. A diether lipid with an acetylated hexoseamine moiety connected to a phosphatidic acid (PA) headgroup is described for the first time and was primarily found in cells that were harvested during stages of more rapid cell division, under all growth conditions.

References · Lipp et al. 2008. Significant contribution of Archaea to extant biomass in marine subsurface sediments, Nature 454: 991-994. · Hinrichs et al. 1999. Methane-consuming archaebacteria in marine sediments, Nature 398: 802-805. · White et al. 1979. Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oecologica 40: 51-62.

abstracts book 187 P59EXTREMOphiles_2012 Morphology of alkaliphilic Bacillus halodurans C-125 Shun Fujinami and Masahiro Ito

Bio-Nano Electronics Research Centre, Toyo University Toyo University, 2100 Kujirai, Kawagoe Saitama 350-8585, Japan E-mail: [email protected]

The facultative alkaliphile Bacillus halodurans C-125 can grow in a pH range from 6.8 to 10.8. The morphology of the cells grown at a pH value above 7.5 is typically rod-shaped, whereas, that gown at pH values less than 7.5 is coiled. Cytoplasmic membrane staining revealed that this coiled morphology was formed not by one filamentous cell but by many chained bent/non-bent cells. The transcription levels of several bacterial cytoskeleton genes (ftsZ, mreB and mreBH) were hardly affected by growth pH, however, that of thembl gene at pH7.0 was about one fifth of that at pH10. The expression level of the Mbl protein at pH7.0 was about one forth of that at pH10. Immunofluorescence microscopy (IFM) showed that the Mbl protein was localized as a helical structure in the rod-shaped cell grown at pH10, however, a clear localization was not observed in a coiled cell grown at pH7.0. These data suggested that a decrease in the expression level of the Mbl protein can influence the morphology of B. halodurans C-125 grown at pH 7.0. The morphology of related alkaliphilic Bacillus pseudofirmus OF4 is hardly affected by growth pH. However, deletion of thecsaB gene, required for the anchoring of surface layer proteins with the SLH domain to the cell wall, caused coiled morphology similar to B. halodurans C-125 grown at pH 7.0. The Bacillus pseudofirmus OF4 genome encodes three putative autolysins with the SLH domain. Unanchored autolysins may cause the coiled morphology. In B. halodurans C-125, it is possible that the expression level, catalytic activity, or localization of an autolysin which is involved in morphology may be decreased by a decrease in the expression level of the Mbl protein. This work was supported by the Inoue Enryo Memorial Foundation for Promoting Sciences to S.F.

References · S. Fujinami, T. Sato and M. Ito (2011). The relationship between a coiled morphology and Mbl in alkaliphilic Bacillus halodurans C-125 at neutral pH values. Extremophiles 15: 587-96. · S. Mesnage, T. Fontaine, T. Mignot, M. Delepierre, M. Mock, A. Fouet (2000). A Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation. EMBO J 19: 4473-84.

188 abstracts book EXTREMOphiles_2012P60 Evidence of arsenate reducing bacterium in sediment from salar de Gorbea, an acidic salt flat in northern Chile L.V. Escuderoa,b, J. Bijmanb, O. Encaladaa, G. Chonga, J. Pueyo and C. Demergassoa,b

aCentro de Investigación Científica y Tecnológica para la Minería (CICITEM), Antofagasta, Chile bCentro de Biotecnología, Universidad Católica del Norte, Antofagasta, Chile E-mail: [email protected]

Acid saline lake systems are an uncommon type of natural extreme environment occurring in Northern Chile. As other environments in Northern Chile those are very unique and threatened systems that are worth to be studied..In addition these environments can also serve as a terrestrial analog to certain ancient Martian terrains. The Salar de Gorbea and Ignorado are located in the Chilean altiplano. Salar de Gorbea is characterized by low pH to 1.2, as well by salinity to 110 g/L, high aluminum to 5 g/L(12-2500 mg/L) and arsenic to 11 mg/L contents. The Ignoradoextreme lake is characterized by low pH 3.59, salinity (1%), aluminium (142 mg/L) and arsenic (0.22 mg/L) .The aim of the study in these lakes is to investigate the microbial communities and specifically the presence of arsenic-reducing bacteria (AsRB) has not been reported in acid saline systems. If activity for dissimilatory arsenate reduction is found, it could lead to isolations of novel microbes (and their enzymes/genes) that can achieve this feat. Because the Salar de Gorbea is both very saline and acidic, it can serve as Mars analog considering what was uncovered by the recent Opportunity Rover mission (Meridiani Planum; Tosca et al. 2008. Water activity and the challenge for life on early Mars. Science 320: 1204 -1207). Brine and sediment samples from the 2011 field campaign in Gorbea and Ignorado were studied. Sediment slurries were prepared by mixing sediments with minimal medium with the salar water supplemented with different electron donors (lactate, H2 and Na2S) and arsenate as electron acceptor (2 mM). The microbial assemblages were analyzed by DGGE of 16S rRNA gene fragments. In addition, the arsenic genes arrA, arsC and acr were screened with degenerated primers. The bacterial community in the most acidic brine samples (pH 1.8 to 2.5) was dominated by Gammaproteobacteria closely related to Acidithiobacillus ferrooxidans, Firmicutes closely related to Geobacillus sp. and the Actinobacteria Mycobacterium sp. and Actinobacterium sp. In the water samples with pH 4.7 the bacteria community was dominated by Deltaproteobacteria closely related to the genus Desulfomicrobium sp. and an uncultured bacterium. Amplification of thearsC and ACR3 (arsenic resistant) gene fragment was observed only in water sample and enrichment culture from GOR-5 (pH 4.7, salinity 1.0 % and arsenic 0.25 mg/L) and IGN. Culture enrichment that respire and reduce As (V) have been only obtained from sediment sample GOR-5 using lactate and H2 as electron donors and arsenate as electron acceptor and

IGN using lactate and Na2S as electron donors and arsenate as electron acceptor. The evidence of the arsenic reduction was the arsenic sulfide precipitation of yellow color that was not observed in the abiotic controls. Microbial assemblage of the AsRB cultures was closely related to the Gammaproteobacteria Shewanella putrefaciens. The arsC and ACR3 gene were detected in all the enrichment cultures. The evidence of AsRB in the acid saline lake at pH 4,7 allows to complete the study of environments in acid, neutral and alkaline saline environments. Species of Shewanella have been reported to reduce arsenic such as Shewanella sp. HN-41 (Lim et al., 2008) a facultative and versatile iron reducing bacterium that can reduce As (V) and sulfate, and form As sulfide, but isolates in batch and columns were only test at pH 7.5 to 8. The distribution of AsRB in that environment suggests the critical pH for arsenic respiratory reduction. The isolate culture is of great interest as a model organism to study how the microbial ecology and geochemical interact in controlling the occurrence of arsenic in saline environments.

abstracts book 189 P61EXTREMOphiles_2012 Specific jarosite biomineralization by acidophilic fungi from Río Tinto Monike Oggerina, Nuria Rodrígueza, Catalina del Moralb, Mónica Sánchez-Romána and Ricardo Amilsa,b

aDepartment of Planetology and Habitability, Astrobiology Centre (INTA-CSIC), 28850 Torrejón de Ardoz, Madrid, Spain bCentre of Molecular Biology Severo Ochoa (UAM-CSIC), 28049 Madrid, Spain E-mail: [email protected]

Rio Tinto (Huelva, Spain) is considered as the largest natural extreme acidic ecosystem on Earth surface. Its extremely acidic pH (~2.3) facilitates the dissolution of metals, making the river highly enriched in heavy metals, with an elevated concentration of ferric iron and sulfate present in its waters (Amils et al. 2002). All this makes Río Tinto as a suitable scenario with the essential geochemical conditions for the precipitation of ferric sulfate minerals (e.g., jarosite, copiapite) related to acid mine drainages (AMD). Jarosite can be only formed under acidic conditions and recently, it has been discovered on sedimentary outcrops at Meriadiani Planum and Mawrth Vallis (Klingelhöfer et al., 2004) on Mars surface, which makes Rio Tinto a good geochemical Mars analogue on Earth. The formation of jarosite, in natural acidic environments, has been related to microbial activity and there are many studies about its bacterial precipitation in laboratory culture experiments and in natural environments (Ziegler et al., 2009) in a process called biomineralization. But what makes Río Tinto a unique acidic environment is the unexpected degree of eukaryotic diversity (Aguilera et al. 2006). We report in this work, for the first time, jarosite formation by a fungal strain isolated from Río Tinto. The strain has been identified as Purpureocillium lilacinum, a wide world soil-borne filamentous fungi isolated from many and varied substrates associated to areas with high concentrations of heavy metals. Growth of P. lilacinum in Río Tinto water conditions promoted the formation of a yellow-ocher colored precipitate. X-Ray Diffraction (XRD), High-resolution Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and geochemical study of the precipitating solution were used to show that the fungal induced mineral was jarosite. Our results show that the mineral starts to nucleate on the fungal cell wall in alive or dead cells and also on the extracellular polymeric substances (EPS), which may act as heterogeneous crystallization nuclei for jarosite, promoting the local ion oversaturation for mineral crystallization in a metabolism independent process. These results indicate that P. lilacinum may play an important role in the formation of jarosite deposits in acidic ecological systems such as Río Tinto, providing information about the possible astrobiological interest of this mineral as a biosignature.

References · Aguilera A., Manrubia SC., Gómez F., Rodríguez N., Amils R. (2006). Eukaryotic community distribution and its relationship to water physicochemical parameters in an extreme acidic environment, Río Tinto (Southwestern Spain). Appl Environ Microbiol 72: 5325-5330. · Amils R., González-Toril E., Fernández-Remolar D., Gómez F., Rodríguez N., Durán C. (2002). Interaction of the sulfur and iron cycles in the Tinto River ecosystem. Environmental Science & Bio/Technology 1: 299–309. · Klingelhöfer G., Morris R.V., Bernhardt B., Schroder C., Rodionov D.S., de Souza P.A., Yen A., Gellert R., Evlanov E.N., Zubkov B., Foh J., Bonnes U., Kankeleit E., Gutlich P., Ming D.W., Renz F., Wdowiak T., Squyres S.W., Arvidson R.E. (2004). Jarosite and hematite at Meridiani Planum from Opportunity’s Mossbauer spectrometer. Science 306: 1740–1745. · Ziegler S., Ackermann S., Gottlicher J., Majzlan J., Gescher J. (2009). Matrix composition and community structure analysis of a novel bacterial pyrite leaching community. Environ Microbiol 11: 2329-2338.

190 abstracts book EXTREMOphiles_2012P62 Extreme microbial sulfur isotope fractionation in a Mars analogue environment at Rio Tinto, SW Spain Esther Velascoa, Paul Masonb, Pieter Vroona, Wilfred Rölinga, Ricardo Amilsc,d and Gareth Daviesa

aFaculty of Earth and Life Sciences, Vrije University of Amsterdam, The Netherlands bDepartment of Earth Science, Utrecht University, The Netherlands cCentro de Astrobiologia, Madrid, Spain dCBM-SO, Universidad Autonoma of Madrid, Spain E-mail: [email protected]

Sulfur isotopes are likely to be a key tool for the detection of past or present life on Mars, where abundant sulfate minerals are present. To investigate the link between the activity of sulfate reducing microorganisms and sulfur isotope fractionation, we incubated sediment from a modern hyper-acidic, Fe-rich subaereal environment at Rio Tinto, SW Spain. This site has frequently been used as a geochemical analogue of Mars. Sediments were sampled from the upper part of the Rio Tinto (Marismilla) as well as the estuary (Moguer). Laboratory incubation where carried out at 30°C using an artificial input solution with sulfate in excess and following techniques developed by Stam (2012). The experiments were performed with an input solution at pH & and pH 3 and electron donors were provided by the natural substrate. Duplicate reactors were incubated for a total of 10 weeks. Initial data indicate moderate sulfate reduction rates of between 5 and 90 nmol cm-3 h-1 in Marismilla and between 5 and 45 nmol cm-3 h-1 in Moguer, independently of the inflow solution pH. Outflow solutions showed pH close to 7, regardless of inflow pH of 3 or 7, suggesting buffering within sediments. Sulfur isotope fractionation was extreme in the Moguer estuary, extending beyond the maximum of 47‰ as predicted by the standard Rees model (Ress 1973) of microbial sulfur isotope fractionation, suggesting that additional fractionation is possible (Brunner 2005) or indicating multiple cycles of reduction and oxidation of sulfate within the reactors. These data indicate that sulfur isotopes may have a potential to be sensitive indicators of biotic activity on Martian sulfate minerals.

References · Stam (2010) Chemical Geology 278: 23-30. · Rees (1973) GCA 37:1141-1161. · Brunner (2005) GCA 69: 4759-4771.

abstracts book 191 P63EXTREMOphiles_2012 Microbialites from hyper alkaline Lake Van: insights into astrobiological application Yasemin Gulecala and Yildirim Dilekb

aDepartment of Freshwater Biology, Faculty of Fisheries, Istanbul University,34130, Istanbul, Turkey bDepartment of Geology, Environmental Earth Science, Miami University, Oxford, OH, USA E-mail: [email protected]

Microbialites resemble coral reefs but can also be found in freshwater systems. Composed of sediments built up over many hundreds of years and multiple interactions between microorganisms such as diatoms and cyanobacteria and minerals, they can sequester carbon through a process called biologically-mediated carbonate precipitation, though just how this is done is still poorly understood. Some researchers think microbialites resemble structures that may have existed on Earth 450 million years ago, which could help them recognize signs of life on other planets. Lake Van harbors the largest known microbialites on Earth. The surface of these huge carbonate pinnacles is covered by coccoid cyanobacteria whereas their central axis is occupied by a channel through which neutral, relatively Ca-enriched, groundwater flows into highly alkaline (pH ~9.7) By sequencing a handful of samples taken from several surface layers of an active, microbialite in Lake Van, our aimed to learn more about the microbial communities that are associated with these structures. Determining which geochemical and biologic parameters in disparate microbialite ecosystems are critical to the biomineralization process will have relevant application to astrobiological studies. Primary productivity, biogeochemical cycling, lake-water geochemistry, and preliminary microbial community analyses are reported here to demonstrate the influences of community and geochemical parameters on mineral precipitation. The applicability Lake Van is assessed for astrobiological studies.

192 abstracts book EXTREMOphiles_2012P64 Osmoadaptative accumulation of moderately halophilic bacteria in subsurface Quaternary sediments of the Qaidam basin Kai Jiang, Yanfen Xue, Xiaowei Wang and Yanhe Ma

National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China E-mail: [email protected]

The Qaidam basin is the largest Cenozoic continental basin in china and preserved completely the Quaternary sedimentary records. We investigated osmoadaptative accumulation of approximately 400 of our own isolates from a total of 13 sediment samples along a vertical depth profile (from 3.2 to 1006 m) in this area using HPLC and 13C-NMR spectroscopy. These isolates were heterotrophic moderately halophilic bacteria (MHB) and affiliated withGammaproteobacteria , Actinobacteria and Firmicutes. Phylogenetic analysis indicated that most of the MHB seem to originate primitively from marine environments and relate to the genera Salinicoccus, Marinobacter, Halomonas, Idiomarina, Bacillus, Halobacillus, Halolactibacillus, Virgibacillus and Brevibacteriumde (Wang, 2010). MHB mainly take ‘the organic-osmolyte strategy’, that enable organisms to adapt to the hostile conditions by accumulating small organic molecules which were generally designated as compatible solute (Severin, 1992; Galinski, 1995). Based on the extensive screening we were able to show that most of these strains accumulated ectoine and betaine as the predominant compatible solutes. Trehalose, sucrose, proline, hydroxyectoine and other yet unidentified compounds were also occurred in some strains as the osmoprotectant. It is worth mentioned that a derivative of lysine, Nα-acetyl-L-lysine, was identified inSalinicoccus sp. H3B36 tested when these were grown under the salt stress. In this strain, Nα-acetyl-L-lysine reached high concentrations and served as the dominant compatible solute, especially at the moderate salinity (9% NaCl). Strain H3B36 was able to grow at salinities of 0-25% (w/v) NaCl (optimally at 9%, w/v), at pH5.5-9 (optimally at pH 7.5) and at 10-40°C (optimally at 30°C).

References · X. W. Wang (2010). Diversity of moderately halophilic bacteria in subsurface the Quaternary sediments of the Qaidam basin [D]. Beijing: Institute of Microbiology Chinese Academy of Sciences. · J. Severin, A. Wohlfarth and E. A. Galinski (1992). The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J Gen Microbiol 138:1629-1638. · E. A. Galinski (1995). Osmoadaptation in bacteria. Adv Microb Physiol 37: 272-328.

abstracts book 193 P65EXTREMOphiles_2012 Raman spectroscopy in halophile research: an additional tool for direct and fast identification of compatible solutes and pigments Jan Jehlickaa, Aharon Orenb, Howell G.M. Edwardsc and Petr Viteka

aInstitute of Geochemistry, Mineralogy and Mineral Resources, Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic bInstitute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel cCentre for Astrobiology and Extremophiles Research, University of Bradford, Bradford, UK, BD71DP E-mail: [email protected]

Hypersaline and evaporitic environments, recent as well as ancient, are of primary interest for two areas– geobiology and astrobiology. They contain much information related to the energy sources, survival conditions of halophiles. Identification of traces of extinct or extant organisms in rocks is important in the geobiological context.

Laboratory investigations of pure cultures of halophiles are presented to demonstrate the possibilities for the detection of the major biomarkers compatible solutes and pigments (carotenoids). We have earlier established a database of Raman spectra of a selection of the most commonly encountered compatible solutes (Jehlicka et al., 2012a). Studies with Halomonas elongata and Ectothiorhodospira marismortui confirmed that Raman spectroscopy can be used to detect such solutes in colonies of extremophilic microorganisms. Examples of pigments identified in halophiles cultures using Raman spectroscopy are given as well (Jehlickaet al., 2012b). Direct measurements on cultures provide fast and reliable identification of pigments. Specific carotenoids are suggested as tracers for halophiles, different from other carotenoids commonly found in algae. The results of studies of stratified microbial communities within evaporite gypsum crusts in hypersaline saltern evaporation ponds in Eilat (Israel) are presented. Here, the crusts contain vertical layers of orange unicellular cyanobacteria, green filamentous cyanobacteria, and purple sulfur bacteria. Carotenoids and compatible solutes can be detected in the frame of these mats. Additional adaptation of analytical protocols would improve fluorescence background elimination important for successful application of Raman spectroscopy and lower the detection limits for biomarkers to be identified in mats and sediments.

References · J. Jehlicka, A. Oren, P. Vitek. (2012a). Use of Raman spectroscopy for identification of compatible solutes in halophilic bacteria. Extremophiles 16: 507-514. · J. Jehlicka, A. Oren, H.G.M. Edwards (2012b). Raman spectra of osmotic solutes of halophiles. J. Raman Spec., in press (DOI 10.1002/jrs.3136).

194 abstracts book EXTREMOphiles_2012P66 Halotolerant and halophilic fungi isolated from an athalassohaline environment Irma Marín, Ana Morato and José P. Abad

Department of Molecular Biology, Faculty of Science, Universidad Autónoma de Madrid, 28049 Madrid, Spain E-mail: [email protected]

Life at high salt concentrations is assumed to be energetically expensive. Probably this is one of the reasons why the study of microorganisms that inhabit hypersaline environments has not been addressed until relatively recent times. The, also assumed, higher adaptative capabilities of prokaryotes versus eukaryotes, has made that the survival and proliferation of eukaryotes in those environments had been regarded as improbable. Thus, these extreme systems have been screened for bacteria and archaea, not appearing the first report showing that fungi inhabit solar salterns until the year 2000 (Gunde-Cimermanet al., 2000). Since then many new yeast and fungi species have been found in hypersaline environments around the World (Gunde-Cimerman et al., 2009), specially in thalassohaline environments. The fungal diversity of athalassohaline hypersaline waters has been studied more scarcely mainly in the Great Salt Lake and in the Dead Sea, during the winter when the salinity is considerably lower (Butinaret al., 2005; Oren and Gunde-Cimerman, 2012). We have screened for culturable fungi two athalassohaline lagoons located in the Southern subplateau of central Spain’s La Mancha region. The salted-medium isolated fungi have been assigned by ITS sequence analysis to species included in the genera Aspergillus, Alternaria, Penicillium, Eurotium, Cladosporium, Hypocrea, Davidiella, Neosartorya, Phoma, Fusarium, Ustilago and Phaeosphaeria, being the most abundant Aspergillus and Alternaria. The presence of the different species in the two studied lagoons, the tolerance to different salts concentrations and the need for them have been studied and the results will be shown and discussed in relation to other reports on fungi in hypersaline environments.

References · N. Gunde-Cimerman (2009). Halotolerant and halophilic fungi. Mycological Research 113: 1231-1241. · N. Gunde-Cimerman, P. Zalar, S. de Hoog, A. Plemenitas (2000). Hypersaline waters in salterns-Natural ecological niches for halophilic black yeast. FEMS Microbiol Ecol 32: 235-240. · L. Butinar, S. Santos, I. Spencer-Martins, A. Oren, N. Gunde-Cimerman (2005). Yeast diversity in hypersaline habitats. FEMS Microbiol Lett 244: 229-234. · A. Oren, N. Gunde-Cimerman (2012). Fungal life in the Dead Sea. Prog Mol Subcell Biol 53: 115-132.

abstracts book 195 P67EXTREMOphiles_2012 A clue to longevity-interplay of cell entombment, potassium supply, water activity and membrane potential inHalobacterium salinarum Dorthe Kixmüller and Jörg-Christian Greie

Department for Biology/Chemistry, University of Osnabrück, 49069 Osnabrück, Germany E-mail: [email protected]

Many extremely halophilic archaea belonging to the Halobacteriales have remarkable longevity. Viable cells have even been found in 250-million-year-old ancient Permo-Triassic subterranean halite deposits. Furthermore, life in hypersaline environments generally challenges organisms with a low water activity. However, the key systems responsible for this remarkable ability and the underlying physiological mechanisms have not yet been deciphered. Our studies reveal that the ATP-dependent K+ uptake system KdpFABC of Halobacterium salinarum is essential for survival under desiccation and salt crystal inclusion and, thus, can be identified as at least one of these systems in this organism. The presence of the kdp genes promotes survival of H. salinarum entombed in halite, compared with cells in which these genes were deleted. Expression of the kdp operon is found to be induced already under desiccating conditions without halite entombment. Furthermore, the morphology of cells included in halite resembles that of cells grown under potassium limitation. Therefore, a steady potassium supply, even under unfavorable energetic conditions, plays an important role in long-term survival and desiccation tolerance.

References · D. Kixmüller and J.-C. Greie (2012). An ATP-driven potassium pump promotes long-term survival of Halobacterium salinarum within salt crystals. Environ Microbiol Rep 4: 234-241. · D. Kixmüller, H. Strahl, A. Wende and J.-C. Greie (2011). Archaeal transcriptional regulation of the prokaryotic KdpFABC complex mediating K+ uptake in H. salinarum. Extremophiles 15: 643-652.

196 abstracts book EXTREMOphiles_2012P68 Octaheme nitrite reductases from haloalkaliphilic bacteria of the genus Thioalkalivibrio: adaptation to extreme environment Tamara Tikhonova, Alexey Tikhonov and Vladimir Popov

A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow 119071, Russia E-mail: [email protected]

Adaptation of microorganisms to extreme environment can be realized at the level of microbial community, cell processes and at the molecular biochemical level. Study of molecular adaptation mechanisms of the key proteins of haloalkaliphilic chemolithoautotrophic bacteria living in soda lakes under the dual effect of the alkaline pH and high salinity is a subject of particular interest. A new family of octaheme cytochrome nitrite reductases was found in genomes of different classes of proteobacteria (b, g, d) belonging to haloalkaliphilic bacteria (genus Thioalkalivibrio, Desulfurivibrio alkaliphilus, Desulfobulbaceae bacterium MLMS1) or to neutrophilic non-halophilic bacteria (genus Geobacter, Pelobacter propionicus, genus Burkholderia). Three proteins of this family were detected in the genomes of haloalkaliphilic sulfur-oxidizing bacteria of the genus Thioalkalivibrio, which dominates in the bacterial communities of the soda lakes with Na+ concentration up to 4.0-4.5 M and pH up to 10.65. Two of these proteins were isolated, catalytic properties were characterized and 3D structures were determined. It was shown that both proteins are constitutive, and their content is about 5-9 % of the soluble proteins of the cell homogenate. It is indicative of the significant role of this family of proteins in cell metabolism of the genus Thioalkalivibrio. The comparative analysis of the amino acid sequences of homologous octaheme nitrite reductases from haloalkaliphilic and neutrophilic non-halophilic bacteria showed that the sequences of five octaheme nitrite reductases from haloalkaliphiles are characterized by a high acidic amino acid (Asp+Glu) content and a more than a twofold decrease in the lysine content compared to proteins from non-haloalkaliphilic bacteria, resulting in the decrease in pI by three units. Simultaneously, the proteins from haloalkaliphilic bacteria are characterized by the high Arg and Glu content. All the above attests to structural adaptation of these proteins to high pH values and salt concentrations. Adaptation at the level of 3D structures will be discussed.

abstracts book 197 P69EXTREMOphiles_2012 From sequencing to gene discovery to function: desiccation tolerance in an Antarctic metagenome Dominique Andersona, Mark Taylora, Marla Tuffina, Don Cowanb and Craig Caryc

aInstitute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Bellville, South Africa bDepartment of Genetics, University of Pretoria, Pretoria 0002, South Africa cUniversity of Waikato, Hamilton, New Zealand E-mail: [email protected]

Analysis of the genomes of uncultured microorganisms not only provides a better understanding of microbial ecology, but may enable the discovery of novel biocatalysts and biomolecules (Schmeisser et al., 2007). The primary focus of many metagenomic research endeavours is the bioprospecting for novel products and as such, metagenomics has become a powerful tool for the discovery of unknown and improved gene products (Cowan et al., 2005). Although microbial metagenomics has the potential to generate large amounts of sequence data, the processing of this data is by no means a trivial task. Challenges which may be encountered during the downstream processing of data includes the assembly of the sequence reads, accuracy of ORF identification and functional annotation of sequence fragments and as such, the identification of novel genes is greatly influenced by bioinformatics and functional genomics (Chistoserdova, 2010). The suitability of metagenomic methods for gene mining in extreme environments which harbor very high levels of uncultured microorganisms has been demonstrated. Metagenomic DNA extracted from Antarctic mineral soil samples was used to construct a fosmid library representing 75 bacterial genomes. Functional screening of the fosmid library resulted in the identification of several clones with tributyrin hydrolysing activity, which were sequenced using Illumina (SOLEXA) technology. The short-read (36 bp) sequences obtained were used to construct the full genetic blue print of four cloned inserts which allowed for further investigation of the Antarctic metagenome. A putative bacterial desiccation tolerance gene encoding a Water Hypersensitivity response protein was identified. This study represents the first report of a prokaryotic WHy protein from an Antarctic Dry Valley metagenome, as well as the first attempt to characterise this bacterial protein in vivo and in vitro.

References · C. Schmeisser et al. (2007). Metagenomics, biotechnology with nonculturable microbes. Appl Micro Biotech 75: 955-962. · D. A. Cowan et al. (2005). Metagenomic gene discovery: Past, present and future. Trends Biotech 23: 321-329. · L. Chistoserdova (2010). Recent progress and new challenges in metagenomics for biotechnology. Biotech Lett 32: 1351-1359.

198 abstracts book EXTREMOphiles_2012P70 Mannosylglycerate protects staphylococcal nuclease with restriction of backbone and β-sheet motions Tiago M. Paisa, Pedro Lamosaa,b, Manolis Matzapetakisa David L. Turnera and Helena Santosa

aInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal bCentro de Ressonância Magnética António Xavier, Oeiras, Portugal E-mail: [email protected]

Compatible solutes are small organic compounds that accumulate in many organisms in response to stressful environmental conditions, such as those associated with osmotic and heat stress. Generally, solutes favoured by (hyper) thermophilic organisms are better protectors of proteins against thermal denaturation, which is a valuable trait for biotechnological applications.

The final goal of this work is to understand the molecular mechanisms underlying protein stabilization by ionic compatible solutes as those typically accumulated by (hyper)thermophiles. In particular, we want to study the effect of stabilizers on the internal dynamics of proteins and look for specific solute/protein interactions. Backbone and side chain sub-nanosecond motions were monitored, respectively, by NMR measurements of 15N and 13C spin relaxation rates. The results showed that backbone motions, but not side chain motions, were progressively restricted by increasing concentrations of mannosylglycerate. Conversely, the presence of urea resulted in an increase of the backbone sub-nanosecond motions. In fact there was a strong correlation between the restriction of fast backbone dynamics and changes in the melting temperature of the protein induced by increasing concentrations of mannosylglycerate. Slower motions (>ms time scale), were studied by using native state hydrogen exchange NMR experiments in the absence and presence of mannosylglycerate. By measuring exchange rates in the presence of mannosylglycerate as a function of urea concentration, it was possible to distinguish the effect of the solute on the local motions and on the global opening motions of the protein. Global opening motions appeared to be restricted by mannosylglycerate independently of protein location, suggesting a simple cancellation of the denaturant effect. On the other hand, local motions were specifically constrained in the β-sheets (Pais et al., 2012). This work reveals a link between solute-induced stabilization and restriction of protein motions at different time scales, and also shows that mannosylglycerate preferentially affects specific structural elements of the protein. This finding could be a basis to explain the observed variability of the degree of protection exerted by a given solute upon different proteins.

Reference · TM Pais, P. Lamosa, DL Turner, M Matzapetakis and H Santos (2012). Mannosylglycerate stabilizes staphylococcal nuclease with restriction of slowβ -sheet motions, Protein Science, DOI:10.1002/pro.2100.

abstracts book 199 P71EXTREMOphiles_2012 Microorganisms isolated from Antarctica Wakao Fukudaa, Kozo Yamadaa, Tomomi Kimuraa, Shigeo Arakia, Yohzo Chinoa, Yuki Miyoshib, Yuka Kondob, Hirokazu Okunoa, Tamotsu Kanaib, Haruyuki Atomib and Tadayuki Imanakaa

aDepartment of Biotechnology, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan bDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan E-mail: [email protected]

Environmental samples from Antarctica were collected by the summer party ofthe 46th Japanese Antarctic Research Expedition in 2004-2005, and interesting microorganisms were isolated from the environmental samples. A white rock sample containing a cryptoendolithic community was collected at the Skallen region, and more than ten different bacterial strains were obtained from the rock sample. One of them was strain 262-8, whose cells often occurred in pairs or chains. Comparative analyses of 16S rRNA gene sequences and physiological characteristics indicated that strain 262-8 should be classified in a new genus in the classAlphaproteobacteria , order Rhodospirillales, family Rhodospirillaceae. Therefore, a new genus and species with the name, Constrictibacter antarcticus gen. nov., sp. nov., was proposed (Yamada, 2011). In addition, strain 262-7, which showed an unprecedented range of morphological diversity in response to growth conditions, was isolated. On the other hand, freshwater samples containing microbial mats were collected at the bottom of lakes in Skarvsnes, and an oligotrophic bacterium forming red colonies (strain 120-1) was isolated from the freshwater sample. Comparative analyses of 16S rRNA gene sequences and physiological characteristics of strain 120-1 indicated that strain 120-1 should represent a new genus in the class Alphaproteobacteria, order Rhizobiales, family Rhodobiaceae. Therefore, a new genus and species with the name, Rhodoligotrophos appendicifer gen. nov., sp. nov., was proposed (Fukuda, 2011). In addition,Lysobacter sp. strain 107-E2, which can grow at -5 °C, was isolated from other fresh water sample.

References · K. Yamada, W. Fukuda, Y. Kondo, Y. Miyoshi, H. Atomi and T. Imanaka (2011). Constrictibacter antarcticus gen. nov., sp. nov., a cryptoendolithic microorganism in antarctic white rock.Int J Syst Evol Microbiol 61: 1973-1980. · W. Fukuda, K. Yamada, Y. Miyoshi, H. Okuno. H. Atomi and T. Imanaka (2011). Rhodoligotrophos appendicifer gen. nov., sp. nov., a bacterium with projections isolated from a lake in Skarvsnes, Antarctica. Int J Syst Evol Microbiol.

200 abstracts book EXTREMOphiles_2012P72 Genome-wide functional screening to identify nickel- resistance determinants in the extreme nickel- resistant Acidiphilium sp. PM Patxi San Martin-Uriza, Salvador Miretea, Manuel J. Gómeza, Pedro J. Alcoleab, Ricardo Amilsa,c and Eduardo Gonzalez-Pastora

aCentro de Astrobiología (INTA-CSIC) 28850 Torrejón de Ardoz, Spain bCentro de Investigaciones Biológicas (CSIC), 28040 Madrid, Spain cCentro de Biología Molecular Severo Ochoa. (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain E-mail: [email protected]

Heavy metals are double-edged swords for life. While nanomolar concentrations afford organisms catalytic versatility and enable electron transfer reactions, metal concentrations in the micromolar to millimolar range pose a threat to most life forms. Because the solubility of heavy metals increases with acidity, some of the most metal-resistant organisms are found in acidic environments. An example of such a resistant organism is Acidiphilium sp. PM, an alpha-proteobacterium isolated from the acidic, heavy metal-laden waters of Rio Tinto (Spain) [1]. Metal resistance studies performed with Acidiphilium sp. PM have revealed it possesses a remarkable ability to tolerate nickel (Ni) concentrations up to 1 M. To uncover the genes behind such extreme resistance, a shotgun genomic library was constructed in E. coli and then screened for Ni-resistant (NiR) clones. Five NiR clones were obtained, their inserts sequenced, and their ORFs identified using the annotation Acidiphiliumof sp. PM. In clones where several ORFs were present, the determinant(s) responsible for Ni-resistance were identified by in vitro transposon mutagenesis and/or subcloning. Several determinants were found to confer E. coli a NiR phenotype: operon hslUV encoding a heat-shock protein, a non-specific protein tyrosine kinase and a glycosyl transferase both of which are involved in polysaccharide biosynthesis, a dihydroxy-acid dehydratase involved in the synthesis of branched-chain amino acids and a TonB-dependent receptor. Given the diverse molecular functions and cellular localizations of these determinants, it is likely that Acidiphilium sp. PM relies on several different mechanisms to cope with Ni toxicity. For instance, heat-shock protein HslUV (also known as ClpQY) likely degrades and recycles mis-folded (hence non-functional) proteins that result from Ni toxicity. Proteins related to the biosynthesis of exopolysaccharide could cause changes in the composition and density of the external envelope, which could help maintain low cytoplasmic Ni concentrations. Finally, the fact that dihydroxy-acid dehydratase is rapidly inhibited by superoxide oxyanions (Brown & Yein, 1978), could make it a good scavenger of toxic free radicals generated by Ni.

References · San Martin-Uriz, P., et al., Draft genome sequence of the electricigen Acidiphilium sp. strain PM (DSM 24941). J Bacteriol 193: 5585-5586. · Brown, O.R. and F. Yein (1978). Dihydroxyacid dehydratase: the site of hyperbaric oxygen poisoning in branch-chain amino acid biosynthesis. Biochem Biophys Res Commun 85: 1219-1224.

abstracts book 201 P73EXTREMOphiles_2012 Niche specific oxidative stress response in extreme acidophiles Juan Pablo Cárdenasa,b,c, Mauricio Arenasa,c,d, Raquel Quatrinib,c and David S. Holmesa,c

aCenter for Bioinformatics and Genome Biology, Fundación Ciencia y Vida, Santiago, Chile bLaboratory of Microbial Ecophysiology, Fundación Ciencia y Vida, Santiago, Chile cDepto. Ciencias Biologicas, Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chile dCentro de Bioinformática y Simulación Molecular, Universidad de Talca, Talca, Chile E-mail: [email protected]

Microorganisms living in extremely acidic environments (1200 genomes). Here we report the finding that the highly conserved heme-associated catalases used by neutrophiles for hydrogen peroxide (H2O2) scavenging are rarely found in acidophiles and appear to have been replaced functionally by the non-heme iron containing protein rubrerythrin (Rbr). In addition, phylogenetic analysis suggests that the use of Rbr can be a niche specific property of acidophiles, as a result of lateral gene transfer between them. The reason for this niche specific preference for Rbr is unknown. However, clues are emerging based on phylogenetic analysis and other bioinformatic inferences. For example, in an extensive phylogenetic analysis of Rbr, there is a major clade that contains Rbr orthologs from hyperthermophiles of deep branching Bacteria and Archaea, suggesting that Rbr may be an ancient mechanism for ROS scavenging that retained a selective advantage when the early atmosphere changed from anaerobic to aerobic conditions. As oxygen levels in the early atmosphere increased, soluble iron would have been precipitated out diminishing the contribution of this major causative agent of stress, except in acidophilic environments. In these environments, soluble iron levels would have (and still do) remained high, requiring the protective action of Rbr that had evolved earlier in the anaerobic but high iron environments of primitive earth. On the other hand anaerobic neutrophilic descendants of the ancestral anaerobes generally (with notable exceptions) are exposed to significantly less stress from both soluble iron and oxygen and have evolved haem- containing catalyses that are fine tuned to sense and respond to fluctuations in oxygen levels. This property is not essential for aerobic acidophiles.

Acknowledgements: Fondecyt 1090451 and 1100887. CONICYT Grant for PhD studies, UNAB DI-116-12/I.

202 abstracts book EXTREMOphiles_2012P74 High hydrostatic pressure altersThermococcus barophilus metabolism Anaïs Cario, Florence Lormières and Philippe Oger

Laboratoire de Géologie de Lyon, CNRS UMR 5276, Ecole Normale Supérieure de Lyon, Université Lyon 1, 46 Allée d’Italie, 69364 Lyon cedex 07 (France) E-mail: [email protected]

Most of Earth’s biotopes are located in the deep-sea and the deep subsurface under elevated hydrostatic pressures (HHP) inhibitory to surface organisms, but which are required for optimal activity in deep-environment adapted microbes. Several HHP-adapted microbes, or piezophiles, have been isolated from such settings, but our understanding of the mechanisms for HHP adaptation remains limited to date. Experimental evidence clearly show that the adaptive strategies in bacterial piezophiles involve a set of specific genes, the fine tuning of gene expression, and/or structural adaptations of the proteome. Adaptive strategies in the domain archaea remain to be elucidated. Metabolic genes and transporters have been identified as one of the main genomic compartment influenced by variations in hydrostatic pressure in bacteria. We have explored the pressure-dependence of metabolism in T. barophilus strain MP, a piezophilic archaeon, which genome has been recently determined, to identify possible adaptive traits to HHP in this archaeon. Strain MP displays optimal pressure, temperature and salinity at 40 MPa, 85°C and 3%, respectively. Previous characterization of organic solute accumulation demonstrated that this strain possesses a piezophilic proteome. We have monitored the influence of pressure on pathways of the central metabolism of strain MP including lipid synthesis and membrane composition, energy metabolism and metabolic requirements. Strain MP was fed a series organic molecules ranging from simple C5 or C6 sugar or amino acids to large organic polymers, including but not restricted to starch, cellulose, chitin and peptone based on the basis of the presence in the genome of putative catabolic pathways. Experiments were performed at least in triplicates in a specifically-designed mineral base medium, and monitored over 72h to estimate growth rates and growth yields. We found that strain MP could be grown at best in mineral medium supplemented with acetate as a carbon and energy source and amino acids to ca. 20% of the growth rates and yields obtained in organic rich medium. 14 amino acids were found to be essential for growth under optimal pressure conditions. This number exceeds the number of amino acid synthesis pathways potentially lacking from the genome of strain MP. These amino acids were shown to be specifically depleted from the medium, indicating their transport and utilization inside the cell. Surprisingly, only 3 amino acids were required for growth at atmospheric pressure. All substrates proved to be excellent growth substrates at low and optimal pressure to the exception of 1) starch which could only support very limited growth under atmospheric pressure conditions and 2) glucose and maltose which only supported growth under atmospheric pressure. Repeated cultivation and starvation cycles in our defined medium allowed us to isolate a pressure-sensitive mutant of strain MP. Primary data show that this mutant is totally unable of grow at 40 MPa, the optimal pressure conditions for growth of the parent strain. Hydrostatic pressure has a strong impact on the metabolic properties of the piezophilic archaeon Thermococcus barophilus. The pressure-dependent divergence in metabolic requirements of strain MP reported here for the first time may influence the outcome of experiments performed under different pressure conditions. Although we have not found a rationale for this discrepancy, similar pressure-dependent amino acid utilization profiles were observed for another thermophilic and obligate piezophilic archaeon : strain CH1 of Pyrococcus yayanosii. A possible link with an adaptive strategy to HHP remains to be elucidated.

abstracts book 203 P75EXTREMOphiles_2012 Accumulation of compatible solutes in a member of the deep-branching phylum Planctomycetes - Rhodopirellula baltica

Filipa d’Avóa, Sofia Cunhaa, Ana Mingoteb, Pedro Lamosac, Milton S. da Costaa,d and Joana Costaa,d

aCentro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal bInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal cCentro de Ressonância Magnética António Xavier, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal dDepartment of Life Sciences, University of Coimbra, Apartado 3046, 3001-401 Coimbra, Portugal E-mail: [email protected]

Most organisms are skilled in adapting to changes that occur in their natural habitat by synthesis and accumulating of specific solutes. These solutes are referred to as compatible solutes because they can be accumulated to high levels by de novo synthesis or taken up from the environment without any interference with cellular metabolism (Brown, 1976). The intracellular accumulation of these osmolytes in response to stressful conditions was investigated in a member of the phylum Planctomycetes - Rhodopirellula baltica. This organism accumulates sucrose, trehalose, mannosylglucosylglycerate (MGG), glutamate and succinic acid depending on the tested growth conditions. In fact, the pool of compatible solutes accumulated by this organism was rather unique, since sucrose has only been found in cyanobacteria (Lunn, 2002), while MGG has only been identified in a unrelated thermophile of the genus Petrotoga (Jorge et al., 2007, Fernandes et al., 2010). In R. baltica, under optimal osmotic conditions, trehalose and glutamate were the major osmolytes. With rising levels of osmotic stresses, glutamate increased notably, while trehalose was replaced by sucrose and MGG. Several other stress conditions were tested allowing us to understand the role of each compatible solute in the stress adaptation of this representative of the monophyletic phylum Planctomycetes. References · A. D. Brown (1976). Microbial water stress. Bacteriol Rev 40:803-846. · J. E. Lunn (2002). Evolution of sucrose synthesis. Plant Physiol 128:1490-1500. · C. D. Jorge, P. Lamosa, and H. Santos (2007). á-D-mannopyranosyl-(1’!2)-á-D-glucopyranosyl-(1’!2)-glycerate in the thermophilic bacterium Petrotoga miotherma—structure, cellular content and function. FEBS J 274: 3120–3127. · C. Fernandes, V. Mendes, J. Costa, N. Empadinhas, C. Jorge, P. Lamosa, H. Santos and M.S. da Costa (2010). Two alternative pathways for the synthesis of the rare compatible solute mannosylglucosylglycerate inPetrotoga mobilis. J Bacteriol 192: 1624-1633.

204 abstracts book EXTREMOphiles_2012OL1

session 3 physiology and metabolism

abstracts book 205 OL1EXTREMOphiles_2012

206 abstracts book EXTREMOphiles_2012P76 Genome-wide dissection of the key enzymes and distinct pathways for PHBV production in Haloferax mediterranei Jing Hana, Jing Houa, Fan Zhanga,b, Shuangfeng Caia, Bo Fenga, Qiuhe Lua, Songnian Hub and Hua Xianga

aInstitute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China bBeijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China E-mail: [email protected]

Haloferax mediterranei has long been thought to be a promising producer of the desired bioplastic poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources. Here, we report the complete genome (3,904,707 bp) of H. mediterranei, the key enzymes (PhaA1/PhaA2, PhaB1/PhaB2, PhaE and PhaC) that are responsible for the PHBV production from acetyl-CoA and propionyl-CoA, the PhaP responsible for the PHA granule formation, and the most attractive propionate-independent pathways for generation of propionyl-CoA in PHBV biosynthesis in haloarchaea. Significantly, bioinformatic analysis indicated that this strain uses at least four pathways for propionyl-CoA biosynthesis, including the pyruvate/2-oxobutanoate, oxaloacetate/2-oxobutanoate, methylmalonyl-CoA, and acetyl-CoA carboxylation pathways. Introducing pathway intermediates and inactivating pathway-specific genes supported that these four pathways were involved in the biosynthesis of 3HV monomer. To further investigate the involvement of the acetyl-CoA carboxylation pathway in PHBV biosynthesis, 13C positional enrichment in 3HV monomer was analyzed, and the result was consistent with a role for the acetyl-CoA carboxylation pathway in PHBV biosynthesis. Notably, 13C metabolic flux analysis showed that the pyruvate/2-oxobutanoate pathway (53.0% flux) and the acetyl-CoA carboxylation pathway (30.6% flux) were the two main generators of propionyl- CoA from glucose. Microarray analysis of two H. mediterranei mutants, ΔphaEC and ΔphaA1, provided further evidence for these propionyl-CoA synthetic pathwaysin vivo and yielded insights into the relationship between central metabolism and PHBV biosynthesis. Taken together, this study has established the complete pathways for the PHBV biopolymer production in H. mediterranei, identified a new carboxylation pathway in haloarchaea, which would significantly enhance our understanding of carbon cycling in hypersaline environments, and facilitate the bioengineering of haloarchaea for bioplastic production in the future.

abstracts book 207 P77EXTREMOphiles_2012 The physiological intricacies in Caldicellulosiruptor saccharolyticus Sudhanshu Pawar and Ed WJ van Niel

Division of Applied Microbiology, Lunds Institute of Technology, Lund University, SE-22241, Lund, Sweden E-mail: [email protected]

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, obligate anaerobe, possessing superior characteristics to produce hydrogen from wide range of poly-, oligo- and monosaccharides. With possible exploitation of this organism in a view, its physiology is studied in depth to optimize hydrogen yields and productivities. A study on the fermentability of wheat straw hydrolysates by the organism showed reasonable tolerance towards the inhibitors present. In a growth medium designed for C. saccharolyticus, cysteine is present as a reducing agent as well as a source of sulfur. Our investigations have led to the conclusion that C. saccharolyticus is able to assimilate inorganic sulfur, replacing thus relatively expensive cysteine, which is also a source of H2S in the flue gas. Currently, a kinetic model of the central carbon metabolic pathway of C. saccharolyticusis in development to simulate its regulation at metabolic level as a function of the environmental changes. It is clear that the metabolic fluxes at the pyruvate node are regulated by a combination of redox cofactors and energy carriers. Insights in the regulation mechanisms, using the model as a tool, will help to perform genetic modifications in the organism to increase hydrogen productivity as well as to minimize the production of undesired by-products.

208 abstracts book EXTREMOphiles_2012P78 Thermodynamic challenges of the formate-oxidizing metabolism and implications for hydrogen production Hyun Sook Leea,b, Jae Kyu Lima,b, Seung Seob Baea,b, Tae Wan Kima, Jung-Hyun Leea,b and Sung Gyun Kanga,b

aKorea Ocean Research and Development Institute, Ansan, Korea bDepartment of Marine Biotechnology, University of Science and Technology, Daejeon, Korea E-mail: [email protected]

Formate could be considered as a potential energy source for microbial metabolism in hydrothermal - - ecosystems. The formate oxidation involving proton reduction to hydrogen (HCOO + H2O g HCO3 + H2) has been reported for two groups of microbes, hyperthermophilic Thermococcus strains and syntrophic consortia of a fermentative bacteria and a methanogen (Kim et al., 2010; Dolfing et al., 2008). Unlike to the syntrophic consortia, a hyperthermophilic archaeon Thermococcus onnurineus NA1 yielded hydrogen accumulation to a partial pressure of 1-7 bar until the values of Gibbs free energy change (ΔG) reached near to thermodynamic equilibrium. The bioenergetic requirement for the metabolism to conserve energy was demonstrated by the ΔG values as small as -10 kJ mol-1, which are less than the biological minimal quantum of energy, -20 kJ mol-1 (Jackson & McInerney, 2002), from submillimolar concentration of formate. Considering formate as a possible hydrogen storage material, the hydrogen production potential of the strain was assessed. The volumetric hydrogen production rate increased linearly with increasing cell density and resulted in the high specific hydrogen productivities of the highest one compared with any other hydrogen- producing microbial strain studied to date. Our results demonstrate that Thermococcus strains would be efficient formate utilizers, which could thrive on low levels of formate produced by biotic and abiotic processes in hydrothermal systems (Lang et al., 2010), and could be also good hydrogen producers.

References

· Kim YJ, et al. (2010) Formate-driven growth coupled with H2 production.Nature 467: 352-355. · Dolfing J, Jiang B, Henstra AM, Stams AJ, Plugge CM (2008) Syntrophic growth on formate: a new microbial niche in anoxic environments. Appl Environ Microbiol 74: 6126-6131. · Jackson BE, McInerney MJ (2002) Anaerobic microbial metabolism can proceed close to thermodynamic limits. Nature 415: 454-456. · Lang SQ, Butterfield DA, Schulte M, Kelley DS, Lilley MD (2010) Elevated concentrations of formate, acetate and dissolved organic carbon found at the Lost City hydrothermal field.Geochim Cosmochim Acta 74: 941-952.

abstracts book 209 P79EXTREMOphiles_2012 Analysis of the DNA polymerase B disruptant of hyperthermophilic archaeon Thermococcus kodakarensis Takashi Kushidaa, Issay Narumib, Shinsuke Fujiwarac, Tadayuki Imanakad and Hiroki Higashibataa,e

aGraduate school of Life Sciences, Toyo University, Ora-gun, Gunma, 374-0193, Japan bQuantum Beam Science Directorate, JAEA, Takasaki, Gunma, 370-1292, Japan cSchool of Science and Technology, Kwansei-Gakuin University, Sanda, Hyogo, 669-1337, Japan dCollege of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan eBio-Nano Electronics Research Centre, Toyo University, Kawagoe, Saitama, 350-8585, Japan E-mail: [email protected]

Archaea, the third domain of life, have eukaryal-like DNA replication machinery. Almost all genome- sequenced archaea possess one or more family-B DNA polymerases (Pol B) similar to eukaryotic replicative DNA polymerases α, δ, and ε. However, Euryarchaeota and Crenarchaeota, subdomains of Archaea, differ in their DNA replication systems. The gene encoding a family-D DNA polymerase (Pol D) has been found in the euryarchaeal genomes but not in the crenarchaeal ones. Pol D has a 3’-5’ proofreading exonuclease activity as well as the Pol B, and consequently, is able to synthesize DNA with high accuracy. Thermococcus kodakarensis, belonging to Euryarchaeota, possesses Pol B and Pol D. It is not known whether both DNA polymerases are essential for viability. At first, we focused on the gene encoding Pol B inT. kodakarensis, and the gene disruption experiment was carried out. Disruption ofpolB gene was not lethal in T. kodakarensis. The ΔpolB strain showed high sensitivity to DNA damage caused by UV, MMS, MMC, andγ -ray, respectively. PolB probably plays an important role in DNA repair rather than DNA replication.

210 abstracts book EXTREMOphiles_2012P80 Metabolic versatility of «Melioribacter roseus» gives insights into the evolution and deep subsurface origin of a novel phylum «Ignavibacteriae» Sergey Gavrilova, Olga Podosokorskayaa, Ilya Kublanova, Alexander Merkela, Vitaly Kadnikovb, Andrey Mardanovb, Nikolay Ravinb, Yulia Frankc, Olga Karnachukc and Elizaveta Bonch-Osmolovskayaa

a Winogradsky Institute of Microbiology, Prospekt 60-Letiya Oktyabrya 7, 117312 Moscow, Russia bBioengineering Center, Russian Academy of Sciences, Prospekt 60-Letiya Oktyabrya 7, 117312 Moscow, Russia cDepartment of Plant Physiology and Biotechnology, Tomsk State University, Prospekt Lenina 36, 634050 Tomsk, Russia E-mail: [email protected]

Moderately thermophilic bacterium «Melioribacter roseus», representing a novel phylum Ignavibacteriae»« (see Kublanov et al., this volume), was isolated from a microbial mat proliferating on wooden surface of a chute covered with hot water outflow of a 2775 meters-deep artesian oil explorating borehole (Podosokorskaya et al., 2012). Molecular analysis revealed the presence of ignavibacterial phylotypes both in the borehole outflow and the associated microbial mat. The majority of «M. roseus»–related blast hits appeared to belong to 16S rRNA genes retrieved from deep-sea sediments and deep subsurface habitats. «M. roseus» possesses high metabolic versatility being able to ferment various carbohydrates and utilize acetate as an electron donor for oxygen, Fe(III) oxide, arsenate, and nitrite respiration. Genome analysis revealed the presence of complete aerobic and Fe(III) respiratory chains as well as putative dissimilatory arsenate and nitrite reductases. Surprisingly, terminal oxygen reductases of «M. roseus» appeared to share strong similarity with oxygen detoxifying enzymes found previously only in strict anaerobes Desulfovibriales and Desulfuromonadales (Lamrabet et al., 2011). This allows predicting the emergence of «M. roseus» respiratory oxygen reductases from ancestral detoxifying enzymes as an evolutionary response to oxygenation of the organisms habitat. The electron acceptors utilized by «M. roseus» in anaerobic conditions are scarce in the modern habitat of the organism but are typically abundant in deep hot subsurface (Gold, 1992, Huber et al., 2000). This fact, together with the relation of our isolate to subsurface clones implies that the organism is a newcomer in its present habitat. Such an assumption is supported by the observed tolerance of «M. roseus» to high salinity, which is an inherent physiological feature of organisms inhabiting subsurface strata of marine genesis, such as that disclosed by the investigated borehole.

References · Podosokorskaya et al. (2012). Environ. Microbiol. submitted. · Lamrabet et al. (2011). Microbiology 157: 2720-2732. · Gold (1992). Proc. Natl. Acad. Sci. USA 89: 6045-6049. · Huber et al. (2000). Syst. Appl. Microbiol. 23: 305-314.

abstracts book 211 P81EXTREMOphiles_2012 Mutants of Pyrococcus furiosus as a means to study the roles of mannosylglycerate and di-myo-inositol phosphate in stress adaptation Ana M. Estevesa, Sanjeev K. Chandrayanb, Patrick M. McTernanb, Michael W. Adamsb, Nuno Borgesa and Helena Santosa

aInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal bDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA E-mail: [email protected]

Many marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supra-optimal temperature or salinity.Pyrococcus furiosus is a marine hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di- myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively (Martins & Santos 1995). The ability of these ionic organic solutes to stabilize proteins in vitro has been extensively demonstrated (Faria et al., 2008). Therefore, it was assumed that MG and DIP could protect cellular components against heat damage in vivo. However, a definite proof of the role of these solutes in stress adaptation of hyperthermophiles is still missing, largely due to the lack of genetic tools to produce suitable mutant strains. In a previous work, the gene for the synthesis of DIP was deleted in Thermococcus kodakarensis and the resulting mutant examined with respect to its growth properties under heat stress conditions (Borgeset al., 2012). Unfortunately, the DIP- deficient strain accumulated aspartate instead of DIP and the growth profile was unaltered, thereby preventing a clear conclusion on DIP´s role. In the meantime, tools for the genetic manipulation of P. furiosus became available (Bridger et al., 2011), making this organism a promising target for our purpose. Unlike T. kodakarensis, which takes up glutamate and aspartate in response to osmotic stress, P. furiosus does not use amino acids or other components of the external medium for osmo- or thermo-adaptation. In this work, genes PF0591 and PF1058 coding for key enzymes in the synthesis of MG and DIP, respectively, were deleted by double- crossover recombination (Bridgeret al., 2011) and a mutant deficient in both genes was also constructed. The resulting mutant strains (DPF0591, DPF1058, and DPF0591DPF1058) did not accumulate MG or DIP. Data on the growth parameters of the mutants and the parental strains under osmotic and heat stress as well as the solute accumulation profiles will be presented.

References · Lo Martins and H Santos (1995). Accumulation of mannosylglycerate and di-myo-inositol-phosphate by Pyrococcus furiosus in response to salinity and temperature. Appl Environ Microbiol 61: 3299-3303. · TQ Faria, A Mingote, F Siopa, R Ventura, C Maycock and H Santos (2008). Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms. Carbohydr Res 343: 3025-3033. · N Borges, R Matsumi, T Imanaka, H Atomi, H Santos (2010). Thermococcus kodakarensis mutants deficient in di-myo-inositol phosphate use aspartate to cope with heat stress. J Bacteriol 192:191-197. · Bridger, S. L., Clarkson, S. M., Stirrett, K., DeBarry, M. D., Lipscomb, G. L., Schut, G. J., Westpheling, J., Scott, R. A., Adams, M. W. W. (2011) Deletion strains reveal metabolic roles for key elemental sulfur responsive proteins in Pyrococcus furiosus. J. Bacteriol. 193: 6498-6504.

212 abstracts book EXTREMOphiles_2012P82 The biosynthesis of glycero-phospho-inositol: do Archaea «like» to be different? Marta V. Rodrigues, Rita Ventura, Christopher Maycock, Nuno Borges and Helena Santos

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal E-mail: [email protected]

The accumulation of low-molecular mass organic compounds (compatible solutes), is a common biochemical strategy to counterbalance changes in the external osmolarity, thereby preserving cell viability. Interestingly, marine hyperthermophiles accumulate compatible solutes to cope not only with fluctuations in the salinity of the external medium but also with temperature variations. Glycero-phospho-inositol (GPI) is a compatible solute thus far found only in the archaeon Archaeoglobus fulgidus and in hyperthermophilic bacteria of the genus Aquifex (Lamosa et al., 2006). We reported that cell extracts of A. fulgidus catalyse the synthesis of GPI from CDP-glycerol and L-myo-inositol-1P (Borges et al., 2006). Curiously, «GPI» accumulated in A. fulgidus had a 31P-NMR spectrum different from that of «GPI» synthesized by the bacteriumAquifex pyrophilus, suggesting distinct stereochemical configurations of the inositol or glycerol groups. Additionally, GPI obtained from beef liver phospholipids was identical to the solute extracted from A. pyrophilus. In summary, the stereochemistry of the archaeal GPI is distinct from that of bacteria and eukarya. By using specifically carbon-13 labelled myoL- - inositol-1P we demonstrated that the inositol moiety has the same configuration regardless of the bacterial or archaeal origin of the enzymes (Rodrigues et al., 2007). Therefore, the difference between the GPIs must arise from the stereochemical configuration of the glycerol group, which is provided by CDP-glycerol. This metabolite is produced by the enzyme glycerol-3-phosphate cytidylyltransferase. Therefore, we set out to establish the stereochemical configuration of CDP-glycerol produced by A. fulgidus and Aquifex aeolicus. To this end, we studied the specificity of the respective glycerol-3P cytidylyltransferases towards the two enantiomers of glycerol phosphate. sn-Glycerol-1-phosphate was synthesised by chemical means since it is not commercially available. The genes encoding putative glycerol-3P cytidylyltransferases of A. fulgidus and A. aeolicus were cloned and expressed in E. coli. These data provided the basis to explain the distinct stereochemical configuration of the glycerol moiety in GPI from archaea and bacteria.

References · P. Lamosa, L. G. Goncalves, M. V. Rodrigues, L. O. Martins, N. D. Raven, H. Santos (2006). Occurrence of 1-glyceryl-1-myo-inosityl phosphate in hyperthermophiles. Appl Environ Microbiol 72: 6169-6173. · N. Borges, L. G. Goncalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, H. Santos (2006) Biosynthetic pathways of inositol and glycerol phosphodiesters used by the hyperthermophileArchaeoglobus fulgidus in stress adaptation.J Bacteriol 188: 8128-8135. · M. V. Rodrigues, N. Borges, M. Henriques, P. Lamosa, R. Ventura, C. Fernandes, N. Empadinhas, C. Maycock, M. S. da Costa, H. Santos (2007). Bifunctional CTP:inositol 1-phosphate cytidylyltransferase/CDP-inositol:inositol- 1-phosphate transferase, the key enzyme for di-myo-inositol phosphate synthesis in several (hyper)thermophiles. J Bacteriol 189: 5405-5412.

abstracts book 213 P83EXTREMOphiles_2012 Caloramator boliviensis, a thermoanaerobe with interesting metabolic properties Carla Crespoa, Anselm Moshib and Bo Mattiassona

aDepartment of Biotechnology, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden b Food and Biotechnology Division, Tanzania Industrial Research and Development Organization, P.O.Box 23235, Dar-es Salaam, Tanzania E-mail: [email protected]

Caloramator boliviensis 45BT, a thermophilic anaerobic bacterium occurring naturally in an extreme habitat in Potosi – Bolivia, was isolated based on its capacity to grow and degrade lignocellulosic material and produce ethanol as main product (Crespo et al. 2011). Production of biologically derived compounds such as ethanol, hydrogen, butyrate, succinate, etc., from renewable substrates by saccharolytic, thermoanaerobes has been emphasized for potential and economical application due to several advantages e.g., broad spectrum of substrates, including pentoses, polymers and low cost substrates, easier product recovery, limited contamination risk, reduced operational costs (Canganella and Wiegel 1993; Cook & Morgan, 1994; Wiegel & Ljungdahl, 1986). Caloramator boliviensis (bo.li.vi.en’sis. L. masc. adj. boliviensis pertaining to Bolivia) a slightly curved rod, variable to Gram stain occurring singly or in chains, depending on the physiological state of the cell, grows on xylose, cellobiose, glucose, arabinose, sucrose, lactose, fructose, galactose, mannose, glycerol, xylan, carboxy methylcellulose and starch (Crespo et al. 2011). The first metabolic investigation C.of boliviensis done in low cellobiose concentration (5g/L), batch mode in closed vials, without shaking or pH regulation yielded ethanol as the main product (0.45 g/g cellobiose consumed) at high fermentation rate. However, at higher concentrations of cellobiose either in batch or fed batch mode under controlled pH (7.0 ± 0.5) a metabolic shift caused redistribution of products where lactate was detected as important product (0.25 – 0.50 g/g cellobiose consumed). Interestingly, NH4OH used as pH adjusting solution and extra supplement of nitrogen source re- routes faster the metabolism to lactate if compared to when NaOH was used. In batch mode under controlled pH, lactate was produced (0.25 gram of lactate per gram cellobiose consumed) which was then fermented leading to butyrate formation (Crespo and Mattiasson 2012). The versatility to: 1) produce value-added compounds e.g. ethanol, butyrate, succinate, propandiol, 2) shift metabolism, 3) operate at elevated temperatures make C. boliviensis an interesting thermoanaerobe. Studies at genome and transcriptome level are necessary to understand and manipulate the microorganism with a specific aim. References · G. Cook, and H. Morgan (1994). Hyperbolic growth of Thermoanaerobacter thermohydrosulfuricus (Clostridium thermohydrosulfuricum) increases ethanol production in pH-controlled culture. Appl Microbiol Biotechnol 41: 84-89. · F. Canganella and J Wiegel (1993). The potential of thermophilic clostridia. In: Woods D R, editor. The clostridia in biotechnology. Stoneham, Mass: Butterworths Publications. pp. 393-429. · C. Crespo, B. Mattiasson (2012). Butyrate production in Caloramator boliviensis and its relationship to lactate production and utilization. Submitted · C. Crespo, T. Pozzo, E. Nordberg-Karlsson, M.T. Alvarez, B. Mattiasson (2011) Caloramator boliviensis sp. nov., a novel thermophilic, ethanol-producing bacterium isolated from a hot spring in Bolivia. Int J Syst Evol Microbiol. In press. · J. Wiegel and L. Ljungdahl (1986). The importance of thermophilic bacteria in biotechnology. Crit Rev Biotechnol 3: 39-107.

214 abstracts book EXTREMOphiles_2012P84 Anoxybacillus – thermophilic facultative anaerobic bacteria with potential biorefining properties Emanuel Ron, Carla Crespo, Bo Mattiasson and Eva Nordberg Karlsson

Department of Biotechnology, Faculty of Chemistry, Lund University, 22100 Lund, Sweden E-mail: [email protected]

As the fossil fuels continues to increase in cost there is a renewed interest of environmentally friendly and efficient source of energy. The abundant polysaccharides in biomass resources have different monosaccharide composition and natural resistance to biodegradation which complicates its usage as a substrate. Starch, pectin, hemicellulose and cellulose are all potential targets as resources for microbial substrates for producing intermediate molecules or biofuels. The pre-treatment of biomass often include acidic environments and/or high temperatures which directs the attention to extremophiles which might tolerate these conditions. The Anoxybacillus genus contain Gram-positive thermophilic bacteria reported to degrade several polysaccharides including xylan, starch and cellulose making representatives from the genus interesting as candidates for biorefining purposes. Some Anoxybacillus species are facultative anaerobic which can be advantageous due to high growth rates when grown aerobically and the ability to switch to an anaerobic production phase, potentially increasing over-all production efficiency of certain metabolites. Anoxybacillus flavithermus was originally categorized as Bacillus flavithermus but was isolated as a novel genus and renamed Anoxybacillus together with Anoxybacillus pushkinoensis. After the division 15 more species have been found to belong to the genus of Anoxybacillus as of today. The two original species were reported as strictly anaerobic but were later revised to facultative anaerobes. The aim of this study is to characterize the following Anoxybacillus species: A. yunnanensis, A. flavithermus WK1, A. flavithermus 2641 and A. rupiensis. The project focuses on use of polysaccharide based sources of substrate such as starch, xylan, cellulose but also their mono- and di-saccharide derivatives. The project will also focus on analysis of product formation, in order to pinpoint metabolites of interest. Results from this screening will be displayed, along with initial results on the competence of the Anoxybacillus species for transformation.

abstracts book 215 P85EXTREMOphiles_2012 Sugar degradation in the haloarchaeon Haloferax volcanii Ulrike Johnsen, Moritz Sutter, Andreas Pickl and Peter Schönheit

Institut für Allgemeine Mikrobiologie, University of Kiel, 24118 Kiel, Germany E-mail: [email protected]

H. volcanii utilizes various hexoses, e. g. fructose, and the pentoses D-xylose and L-arabinose as a sole carbon and energy source. (1) The uptake of fructose and its degradation to triose phosphates and (2) the pathways of D-xylose and L-arabinose degradation and their transcriptional regulation were analysed. The studies include genomic and transcriptional analyses, growth experiments with knock-out mutants of candidate genes, and overexpression and characterization of enzymes involved. (1) Fructose uptake is mediated by a bacterial like phosphoenolpyruvate-dependent phosphotransferase system (PTS), generating fructose-1-phosphate, which is further phosphorylated to fructose-1,6-bisphosphate (FBP) by fructose-1-phosphate kinase (1PFK). FBP is cleaved to triose phosphates by a bacterial like class II fructose-1,6-bisphosphate aldolase (FBA). This is the first report of the functional involvement of a bacterial type PTS in sugar metabolism of archaea (Pickl et al., 2012). (2) Both, D-xylose and L-arabinose in H. volcanii are degraded in an oxidative pathway to α-ketoglutarate (αKG) as an intermediate. D-Xylose is oxidized by inducible xylose specific dehydrogenase (XDH) to D-xylonate followed by two dehydratase reactions (xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase) to yield α-KG semialdehyde that is oxidized to α-KG by α-KG dehydrogenase (Johnsen et al., 2009). The first step of L-arabinose degradation is catalysed by an inducible L-arabinose dehydrogenase (AraDH), which differs from XDH by kinetic properties and phylogenetic affiliation. Deletion mutants experiments indicate that L-arabinoate conversion to α-KG involves the same set of enzymes as reported for D-xylonate degradation toα -KG. Further, a transcriptional activator of both, D-xylose and L-arabinose degradation pathways was identified.

References · A. Pickl, U. Johnsen and P. Schönheit (2012). Fructose degradation in the haloarchaeonHaloferax volcanii involves a bacterial type phosphoenolpyruvate-dependent phosphotransferase system, fructose-1-phosphate kinase, and class II fructose-1,6-bisphosphate aldolase. J Bacteriol 194: 3088-97. · U. Johnsen, M. Dambeck, H. Zeiss, T. Fuhrer, J. Soppa, U. Sauer and P. Schönheit (2009). D-Xylose degradation pathway in the halophilic archaeon Haloferax volcanii. JBC 284: 27290-27303.

216 abstracts book EXTREMOphiles_2012P86 Identification of a global regulator of glucose metabolism in Sulfolobus solfataricus Olha V. Rohulyaa, Sonja-Verena Albersb and Arnold J.M. Driessena

aDepartment of Molecular Microbiology Groningen Biomolecular Sciences and Biotechnology Institute, and the Kluyver Centre for the Genomics of Industrial Microorganisms, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands bMolecular Biology of Archaea, Max Planck Institute for terrestrial Microbiology, Karl-von -Frisch-Strasse 10, D-35043 Marburg, Germany E-mail: [email protected]

Sulfolobus solfataricus belongs to the group of hyperthermoacidophilic archaea that grow optimally aerobically at high temperature (62-92 °C) and acidic pH (pH 2-4). It belongs to Crenarchaeota phylum and in recent years a full set of genetic tools has become available. The central metabolic pathway in S. solfataricus contains many unique features compared to the classical pathways described for bacteria and eukaryotes. S. solfataricus uses a novel semiphosphorylated Entner- Doudoroff pathway for sugar metabolism [1,2], and this raises the question how it is regulated. Here, we report the discovery of a gene that appears to act as global regulator of carbon metabolism. Deletion of the gene resulted in a strain that shows an improved growth on glucose medium, which can be attributed to an increased flux through the semiphosphorylated ED pathway because of the upregulation of a number of its key enzymes.

References · Ahmed H, Ettema T.J., Tjaden B., Geerling A.C., van der Oost, J. and Siebers, B. (2005) The semi- phosphorylative Entner-Doudoroff pathway in hyperthermophilic archaea: a re-evaluation. Biochem J 390: 529-540. · Snijders A.P.L., Walther J., Peter S., Kinnman I., de Vos, Marjon G. J., van de Werken, H. J. G., et al. (2006) Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis. Proteomics 6: 1518-1529.

abstracts book 217 P87EXTREMOphiles_2012 Dissimilatory sulfate reduction in the crenarchaeote «Vulcanisaeta moutnovskia» Nikolai A. Chernyh, Margarita L. Miroshnichenko, Olga L.Kovaleva, Maria I. Prokofeva, Nikolai V. Pimenov, Alexander V. Lebedinsky and Elizaveta A. Bonch-Osmolovskaya

Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow 117312, Russia E-mail: [email protected]

«Vulcanisaeta moutnovskia» is an obligately anaerobic acidophilic crenarchaeote with a pH range of growth from 3.5 to 6.5 and a temperature range from 60 to 98°C. It is a metabolically versatile archaeon capable of fermenting proteinaceous substrates and some sugars. Elemental sulfur and thiosulfate are reduced to hydrogen sulfide if added (Prokofeva et al., 2005). In the genome announcement of «Vulcanisaeta moutnovskia» (Gumerov et at., 2011), presence of genes of the sulfate reduction pathway was mentioned. However, no detailed analysis was presented. The present study demonstrated the presence in the «Vulcanisaeta moutnovskia» genome of all genes currently recognized to be involved in dissimilatory sulfate reduction, and provided evidence of the occurrence of the process. In the genomes of Caldivirga maquilingensis and Thermoproteus tenax, crenarchaeotes earlier briefly mentioned to be capable of sulfate reduction (Itoh et al.,1999; Siebers et al., 2011), we failed to find some of the presumably indispensable genes.

References · V. M. Gumerov, A. V. Mardanov, A. V. Beletsky, M. I. Prokofeva, E. A. Bonch-Osmolovskaya, N.i V. Ravin, and K. G. Skryabin (2011). Complete Genome Sequence of «Vulcanisaeta moutnovskia» Strain 768-28, a Novel Member of the Hyperthermophilic Crenarchaeal Genus Vulcanisaeta. J Bacteriol 193: 2355-6. · T. Itoh, K. Suzuki, P. C. Sanchez, and T. Nakase. 1999. Caldivirga maquilingensis gen. nov., sp. nov., a new genus of rod-shaped crenarchaeote isolated from a hot spring in the Philippines. Int J Syst Bacteriol 49: 1157-1163 · M.I. Prokofeva,, I. V. Kublanov, O. Nercessian, T. P. Tourova, T. V. Kolganova, A. V. Lebedinsky, E. A. Bonch- Osmolovskaya, S. Spring, C. Jeanthon (2005). Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats. Extremophiles 9: 437-448. · B. Siebers, M. Zaparty, G. Raddatz, B. Tjaden, S.V. Albers, S.D. Bell, F. Blombach, A. Kletzin, N. Kyrpides, C. Lanz, A. Plagens, M. Rampp, A. Rosinus, M. von Jan, K.S. Makarova, H.P. Klenk, S.C. Schuster, R. Hensel. 2011, The complete genome sequence of Thermoproteus tenax: a physiologically versatile member of the Crenar- chaeota. PLoS One.6: e24222.

218 abstracts book EXTREMOphiles_2012P88 Physiological characterization of Halomonas pacifica and Halomonas campisalis isolated from saline alkaline lakes in the Libyan Sahara Abdolkader A. Mohmmeda and D. James Gilmourb

aFaculty of Science, Sebha University, Sebha - Libya bDepartment of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK Email: [email protected]

Alkaline saline lakes are widespread as the result of complex interactions of the geological, climatic, and biogeochemical conditions and are formed in closed drainage basins. Gaber – oun and Um – Alma lakes are alkaline saline lakes in the south of the Libyan Sahara. There were only a few reports (Ajali et al., 1984) on their microbial diversity before the current work was undertaken. Five Gram-negative bacterial strains belonging to the family of Halomonadaceae were isolated from the lakes by subjecting the isolates to high salinity medium. Two of the Halomonas species isolated (H. pacifica and H. campisalis) were chosen for further study on the basis of novelty (the little studied H. pacifica [Wang et al., 20089]) and on dual stress tolerance (high pH and high salinity) shown by H. campisalis (Mormile et al., 1999). The effects of NaCl and pH on growth were studied, both species showed optimum growth at 0.5 M NaCl, but H. campisalis alone was able to grow in the absence of NaCl. H. pacifica grew better than H. campisalis at high salinities in excess of 1 M NaCl and was clearly moderately halophilic. H. pacifica showed optimum growth at pH 7 to 8, but in contrast H. campisalis could grow well at pH values up to 10 13C-NMR spectroscopy was used to determine and identify the compatible solutes accumulated by the two species in rich and minimal media at different concentrations of NaCl. H. pacifica accumulated betaine in rich (LB) medium with ectoine only appearing at the highest salinity tested (2.5 M NaCl). In contrast, in M9 minimal medium, no betaine was detected and ectoine and hydroxyectoine were accumulated. Exactly the same pattern of compatible solute accumulation was seen for H. campisalis.

References · A. Ajali, J. E. Furet and J. Tanti (1984). Diatoms of a hypersaline lake in the Libyan Sahara. 8th Diatom Symposium pages 645-653. · Y. Wang et al. (2008). Halomonas sulifodinae sp. nov., a halophilic bacterium isolated from a salt mine in China. Int. J Sys Evol Microbiol 58: 2855-2858. · M. R. Mormile et al. (1999). Halomonas campisalis sp. nov., a denitrifying moderately haloalkaliphilic bacterium. Syst Appl Microbiol 22: 551-558.

abstracts book 219 P89EXTREMOphiles_2012 C14-AHL signalling molecules enhances biofilm formation on metal sulfides by the extremophilic bacterium Acidithiobacillus ferrooxidans Alex Gonzáleza,d, Sören Bellenbergb,d, Sigde Mamania,c, Lina Ruiza, Alex Echeverríad, Laurent Soulèree, Alain Doutheaue, Cecilia Demergassod, Wolfgang Sandb, Yves Queneaue, Mario Verab and Nicolas Guiliania

aDepartment of Biology, Faculty of Sciences, University of Chile, Santiago, Chile bBiofilm Centre Aquatische Biotechnologie, Universität Duisburg – Essen, Duisburg, Germany cCNRS, Institut de Microbiologie de la Méditerranée, Laboratoire de Chimie Bactérienne et Université de la Mediterranée, Faculté des Sciences de Luminy, Marseille, France dBiotechnology Center, Universidad Católica del Norte, Antofogasta, Chile eICBMS, Chimie Organique et Bioorganique, UMR 5246, CNRS, Université Lyon 1, INSA, Lyon, France E-mail: [email protected]

Research on bioleaching must consider as goals the enhancement of bioleaching rates to improve the metallurgical performance in industrial processes activity and their inhibition in order to mitigate AMD/ARD. Comprehension of the physiology and the adaptive molecular mechanisms of bioleaching microorganisms is thus an extremely important issue. Bacterial attachment and biofilm formation on minerals increases leaching activities due to the formation of a reaction space between the sulfide ore surface and the cell. Biofilm development is a complex process that is regulated by cell-to-cell communication mechanisms named Quorum Sensing (QS) in many Gram-negative bacteria. Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautrophic Gram-negative bacterium that has an important role in metal leaching and AMD/ARD production. It belongs to the consortia of microorganisms related with bioleaching processes, and possesses a functional type AI-1 QS system that involves acyl-homoserine lactone (AHLs) as autoinducer molecules. The At. ferrooxidans QS system (afeI/afeR) has been proposed that could be involved in the production of EPS and consequently in the regulation of biofilm formation. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacteriumAcidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using QS signalling molecules. Our results revealed that AHL-signalling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of At. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous At. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances (EPS). These results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/ rock drainage.

220 abstracts book EXTREMOphiles_2012P90 Ferritin distribution in organisms developing in an extreme acidic environment, Río Tinto Ricardo Amilsa,b, Lourdes Rufoc, Nuria Rodrígueza and Vicenta de la Fuentec

aCentro de Astrobiología (CSIC-INTA), 28055 Torrejón de Ardoz, Spain bCentro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco. 28049 Madrid, Spain cDepartmento de Biología, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain E-mail: [email protected]

Ferritin is a universal protein that plays an important role in storage and management of iron and to prevent cell damage produced by oxidative stress. Phylogenetically ferritin appears to be a very ancient protein because it has been found in all three domains of life. Although there is fear amount of information of ferritin localization in plant and animals, its distribution in other organisms is rather scarce. Plant ferritin is associated with plastids which contain electron dense granules in the form of phytoferritin, and in animal cells can be found distributed in several organelles and in the cytoplasm. We have previously reported the detection of high concentration of iron in rhizomes and leaves of Imperata cylindrica, a perennial grass isolated from the banks of Río Tinto, an extreme acidic environment. The use of Mösbauer spectroscopy and XRD indicated that iron accumulate in this plant as jarosite and ferritin (Rodríguez et al., 2005). The use of immunocytochemistry allowed the localization and subcellular distribution of ferritin I.in cylindrica (de la Fuente et al., 2012). The highest density of immunolabeling in shoots was observed in the intracellular space of cell tissues, near the cell wall, and in the cytoplasm. In leaves, ferritin was detected in the proximity of the middle lamella of cell walls, following a similar path to that observed in shoots. These results further expanded the localization of ferritin in cell components other than chloroplast and mitochondria in plants. To clarify if the observed localization of ferritin inI. cylindrica was peculiar of this plant or corresponded to a more general pattern existing in organisms exposed to the extreme conditions of pH and heavy metal content of Río Tinto, ferritin was identified immunocytochemically in different organisms isolated from the Tinto basin:Acidiphilium sp. (bacteria), Penicillium janthinellum (fungi), Dunaliella sp. (algae), Enteromorpha compressa (Linnaeus) Greville (plant), and compared with I. cylindrical.

References · N. Rodríguez, N. Menéndez, J. Tornero, R. Amils and V. de la Fuente (2005) Internal iron biomineralization in Imperata cilíndrica, a perennial grass: chemical composition, speciation and plant localization. New Phytol 165: 781-789. · V. de la Fuente, N. Rodríguez and R. Amils (2012) Immunocytochemical analysis of the subcellular distribution of ferritin inImperata cylindrica (L.) Raeuschel, an iron hyperaccumulator plant. Acta Histochem 114: 232-236.

abstracts book 221 P91EXTREMOphiles_2012 Molecular dynamics response of Halobacterium salinarum to environmental stress V. Marty, E. Fabiani, F. Gabel, G. Zaccai, and B. Franzetti

Institut de Biologie Structurale, Extremophiles and Large Molecular Assemblies Laboratory, CNRS UMR 5075, Grenoble, France E-mail: [email protected]

In vivo molecular dynamics of the Halobacterium salinarum proteome and cytosol under various stress conditions was measured by neutron scattering experiments coupled with microbiological characterization. The effects of hyposaline and thermal stress, respectively, were assessed by measuring intracellular salt concentrations and the accumulation of the chaperonin and protease protein quality control machineries. It revealed that the PAN-Proteasome system contributes importantly to the low salt stress response.

Under stress conditions,in vivo, the neutron scattering experiments detected molecular dynamics alterations reflecting a softening of protein structures consistent with unfolding. The results revealed extreme halophilic behaviour at the level of the proteome, which is more severe than has been suggested by in vitro studies on individual proteins. They revealed that, in halophiles, a moderate drop in the environmental salt concentration has a similar effect on cell physiology as thermal stress. The experiments showed that the neutron scattering method provides a promising tool to study molecular dynamics modifications in the proteome of living microorganisms induced by factors altering protein folds.

References · H. Chamieh, V. Marty, D. Guetta, A. Perollier, B. Franzetti (2012) Stress regulation of the PAN-proteasome system in the extreme halophilic archaeon Halobacterium. Extremophiles, in press.

222 abstracts book EXTREMOphiles_2012P92

session 4 proteins and enzymes

abstracts book 223 OL1EXTREMOphiles_2012

224 abstracts book EXTREMOphiles_2012P92 Functional and structural characterization of Dye-linked L-proline dehydrogenase from hyperthermophiles Takenori Satomuraa, Ryushi Kawakamib, Shin-ichiro Suyea, Haruhiko Sakurabac and Ohshima Toshihisad

aDepartment of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui, Japan bAnalytical Research Center for Experimental Sciences, Saga University, 1 Honjo-machi, Saga, Japan, cDepartment of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa, Japan dMicrobial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Japan E-mail: [email protected]

Dye-linked l-proline dehydrogenase (Dye-L-proDH) catalyzes the oxidation of l-proline to «∆-pyrroline-5- carboxylate in the presence of artificial electron acceptors. Two types of Dye-L-proDH have been identified in hyperthermophilic archaea so far reported (Kawakami et al., 2012). The first type is a heterotetrameric αβγd-type Dye-L-proDH which exhibits not only Dye-L-proDH activity but also NADH dehydrogenase activity, and has two electron transfer proteins (Kawakami et al., 2012). The second one is a heterooctameric αβγd-type Dye-L-proDH which has FAD, FMN, ATP and Fe as the cofactors in its molecule and creates a new electron transfer pathway (Kawakami et al., 2012). The third one is a homodimeric Dye-L-proDH which has been recently found and exhibits the highest thermostability among these archaeal Dye-L-proDHs (Kawakami et al., 2012; Satomura et al., 2012). In addition, we succeeded in crystallization and X-ray analysis of the third type Dye-L-proDH fromAeropyrum pernix (Sakuraba et al., 2012). Here we report about the structural characterization of the third type A. pernix Dye-L-proDH.

References · R. Kawakami, T. Satomura, H. Sakuraba, and T. Ohshima (2012). L-proline dehydrogenases in hyperthermophilic archaea: distribution, function, structure, and application. Appl Microbiol Biotechnol 93: 83-93. · T. Satomura, Y. Hara, S. Suye, H. Sakuraba, and T. Ohshima (2012). Gene expression and characterization of a third type of dye-linked L-proline dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix. Biosci Biotechnol Biochem 76: 589-593. · H. Sakuraba, T. Satomura, R. Kawakami, K. Kim, Y. Hara, K Yoneda, and T. Ohshima (2012). Crystal structure of a novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix. J Biol Chem, in press.

abstracts book 225 P93EXTREMOphiles_2012 Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii Diya Alsafadi and Francesca Paradisi

Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland E-mail: [email protected], [email protected]

The efficiency of biocatalytic redox reactions catalyzed by alcohol dehydrogenases (ADHs) has been investigated in recent years as it offers a facile route to enantiopure alcohols (1). Microorganisms living under extreme conditions (extremophiles) have been an important source of enzymes with unique structural features and properties (2). Several extremophilic ADHs have been identified and proposed as promising biocatalysts, with major focus on their ability to work at high temperatures (thermophilic ADHs) (3,4). Halophilic ADHs, on the other hand, have been relatively less explored (5). Halophilic enzymes function under extremely high salt concentration and are stable under «dry condition» (low water concentration). Mesophilic enzymes in organic solvents are generally denatured and their catalytic activity disappears. Stability and activity in media other than acqueous remains a significant challenge. Because salt has the effect of reducing water activity, halophilic enzymes may become the enzymes of choice for biocatalysis in other low water activity environments like aqueous/organic and non-aqueous media. We recently reported on the identification and the biochemical characterization of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii. HvADH2 displayed much greater tolerance to organic solvents than HvADH1 (5). Hence, it is of great importance to evaluate the potential application of HvADH2 in chemical synthesis, and we further examined the effect of water-miscible organic solvents [dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile (ACN) and methanol (METH)] in aqueous solution on the activity and conformational stability of ADH2. The alterations to the tertiary structure of the enzyme were followed by means of ûuorescence spectroscopic measurement.

References · J.M. Woodley, (2008),Trends Biotechnol 26, 321-327. · R. Karan, M.D. Capes, S. Dassarma (2012), Aquat Biosyst 8: 1-15. · J.E. Guy, M.N. Isupov, J.A. Littlechild (2003), Aeropyrum pernix, J Mol Biol 331: 1041-1051. · H. Radianingtyas, P.C. Wright, (2003) FEMS Microbiol Rev 27: 593-616. · L.M. Timpson, A.K. Liliensiek, D. Alsafadi, J. Cassidy, M.A. Sharkey, S. Liddell, T. Allers, F. Paradisi (2012) Appl Microbiol Biotechnol, DOI: 10.1007/s00253-012-4074-4.

226 abstracts book EXTREMOphiles_2012P94 Slow unfolding pathway of a hyperthermophilic protein, Tk-RNase H2, examined by pulse proteolysis using a super-stable protease, Tk-subtilisin Kazufumi Takanoa, Jun Okadab, Yuichi Kogab and Shigenori Kanayab

a Kyoto Prefectural University, Kyoto 606-8522, Japan bOsaka University, Suita 565-0871, Japan Email: [email protected]

The unfolding speed for some hyperthermophilic proteins is dramatically slower than that for their mesostable homologs. Ribonuclease H2 from a hyperthermophilic archaeon, Thermococcus kodakarensis, (Tk-RNase H2) is stabilized by its remarkably slow unfolding rate (Mukaiyama et al., 2004). In this work, we examined the slow unfolding pathway of Tk-RNase H2 by pulse proteolysis using a super-stable protease, subtilisin-like serine protease fromThermococcus kodakarensis (Tk-subtilisin) (Tanakaet al., 2007). Tk-subtilisin has its enzymatic activity in highly concentrated GdnHCl where Tk-RNase H2 unfolds slowly. The native state of Tk-RNase H2 is completely resistant to Tk-subtilisin, whereas the unfolded state of Tk- RNase H2 (induced by 4 M GdnHCl) can be degraded by Tk-subtilisin. In pulse proteolysis, some degradation products of Tk-RNase H2 were detected by Tricine-SDS-PAGE. We identified the cleavage sites in Tk-RNase H2 by N-terminal sequencing and MALDI-TOF mass spectrometry, and constructed mimics of Tk-RNase H2 unfolding intermediate by protein engineering. The mimics were biophysically characterized. Through these experiments, we found that Tk-RNase H2 includes two different forms with N-state and N’- state in the native state. The N-state has the large hydrophobic surface in the C-terminal region. In the early stage of unfolding, the N-state changes to an intermediate state (A-state) which is digested by Tk-subtilisin at the C-terminal region. In contrast, the N’-state is a Tk-subtilisin resistant form. In the slow unfolding pathway, the A-state shifts to the N’-state which gradually unfolds.

References · Mukaiyama et al. (2004) Biochemistry 43, 13859. · Tanaka et al. (2007) J. Biol. Chem. 282, 8246.

abstracts book 227 P95EXTREMOphiles_2012 Acylpeptide hydrolases in Sulfolobus solfataricus: an enzyme system with «surprising» features M. Gogliettino, M. Balestrieri, E. Cocca, M. Rossi and G. Palmieri

Institute of Protein Biochemistry, National Research Council (CNR-IBP), Naples, Italy E-mail: [email protected]

Acylpeptide hydrolase (APEH) is a member of the prolyl oligopeptidase (POP) family of serine peptidases, which catalyzes the removal of N-terminally blocked amino acid residues from peptides, producing an acetyl amino acid and a peptide with a free N terminus shortened by one amino acid residue. So far, these enzymes have been studied in a number of eukaryal and archaeal organisms, and recently in a psychrophilic bacterium. The role of this enzyme in the physiology of the mammalian cells is not yet clear; but, it has been recently suggested that it may function cooperatively with proteasome in the protein-turnover processes (Perrieret al., 2005) and it could be involved in the secondary antioxidant defense system (Palmieri et al., 2011; Shimizu et al., 2004). In fact the prevention of accumulation of oxidized cellular proteins is one of the major functions of the degradative machinery, which rapidly eliminates damaged proteins whose increase would interfere with normal cell function and viability. Therefore APEH could represent a promising therapeutic target relevant for a wide array of diseases caused by rising of misfolded proteins, associated, for example, with tumor onset and development (Palmieri et al., 2011; Shimizu et al., 2004). On this background, what about archaeal microorganisms, which, unlike the mammalian counterparts, show a multiplicity of apeh genes whose function, is still unknown? In this study, for the first time, we explored the possiblerole ofAPEH in a hyperthermophilic microorganism, providing a good starting point for understanding the biological role of this class of enzymes in Archaea. To this aim, we carried out a comparative analysis of two APEH (APEHSs and APEH-3Ss), which we isolated from S. solfataricus, investigating— the structural/functional properties and the regulation of gene expression. Interestingly, our results revealed that the different APEHs could be identified as stress-regulated proteins possibly playing a complementary function in allowing the cell life under diverse conditions. Specifically, the two APEH gene expression patterns were modulated differently in response to stress conditions, indicating that the dynamical environmental changes of the «extreme» native habitat could be responsible in selecting from time to time the enzyme suitable to the needs of the cells.Our analyses evidenced that, while APEHSs displayed the typical properties of the archaeal APEH family, APEH-3Ss showed intermediate features between those from archaeal and mammalian counterparts. Moreover, the phylogenetic investigations showed that

APEHSs was widely distributed in several archaeal species and the putativeAPEH -3Ss-homologs were found only in Crenarchaeota phylum, which is described to be more related to the Eukarya Kingdom. Therefore, APEH-3Ss could be the founder of a new class of acylpeptide hydrolases.

References: · Perrier J, Durand A, Giardina T, Puigserver A (2005). Biochimie 87: 673-685. · Palmieri G, Bergamo P, Luini A, Ruvo M, Gogliettino M, et al. (2011). PLoS ONE 6: e25888. · Shimizu K, Kiuchi Y, Ando K, Hayakawa M, Kikugawa K (2004). Biochem Biophys Res Com 324: 140-146.

228 abstracts book EXTREMOphiles_2012P96 Construction of bifunctional biomass-degrading enzymes from thermophiles Skander Elleuche, Mazen Rizk and Garabed Antranikian

Institute of Technical Microbiology, Hamburg University of Technology, D-21073 Hamburg, Germany E-mail: [email protected]

Lignocellulosic biomass degrading enzymes are of high importance in the recycling of photosynthetically fixed carbon. Cellulose and hemicellulose are the major components of plant cell walls. These polysaccharidic compounds are tightly intertwined and compactly packed and their hydrolysis is a difficult task. For this reason, the improvement of cellulose and hemicellulose degrading enzymes has considerable importance for versatile applications in industry. Since enzymatic hydrolysis of cellulose and hemicellulose compounds requires the action of multiple glycoside hydrolases, various strategies were developed in the course of evolution. Few bacteria are able to form multi-component enzyme complexes, known as cellulosomes, while others have separate enzymes or even isozymes working in synergy. These separate bacterial enzymes work in tandem to efficiently degrade polysaccharides. Moreover, a number of enzymes has evolved a modular organization consisting of bi- or multifunctional catalytic sites. Nature’s invention to form such complexes can be mimicked in laboratories, owing to a number of different molecular and genetic techniques. Gene fusion methods, which are extensively used to purify recombinant proteins or monitor gene expression, can be also used for the generation of artificial bifunctional polysaccharide digesting hydrolases. In this study, ligation-based end-to-end fusion approaches were used to generate heat-stable xylanase-cellulase, cellulase-α-glucosidase and chimeric double cellulase enzymes. Detailed characterization of enzymatic properties and synergetic effects will be presented.

abstracts book 229 P97EXTREMOphiles_2012 Structural insight into the evolution and broad substrate specificity of the fourth type of tRNA splicing endonuclease Akira Hirataa, Kosuke Fujishimab,c, Ryota Yamagamia, Takuya Kawamuraa, Jillian F. Banfieldd,e, Akio Kanaib and Hiroyuki Horia,g

aDepartment of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan bInstitute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan cNASA Ames Research Center, Moffett Field, CA 94035, USA dDepartment of Earth and Planetary Science, University of California, Berkeley, CA 94270, USA eEnvironmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA gVenture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan E-mail: [email protected]

In the past, archaeal tRNA splicing endonucleases (EndA), which remove introns from precursor tRNA, were classified into the three types, homodimer α( 2), homotetramer (α4) and heterotetramer (α2b2). Extensive structural and biochemical studies revealed a mechanism underlying the RNA cleavage and recognition by the three types of archaeal EndAs. Recently, we found the fourth type of archaeal EndA from an ultrasmall archaeon Candidatus Micrarchaeum acidiphilum (Fujishima et al., 2011), referred to as ARMAN (Archaeal Richmond Mine Acidphilic Nanoorganism)-2, which was discovered in an acid mine drainage site at Iron Mountain in Northern California. Our bioinformatics and biochemical analyses revealed that the ARMAN-2 EndA has a novel three- unit (e) architecture that consists of two duplicated catalytic á units and one structural b unit encoded on a single gene. Furthermore, we found that the e2 homodimer structure is essential for enzymatic activity and broad substrate specificity. However, the precise architecture of ARMAN-2 EndA remains unknown, raising the intriguing two questions: first, how are the three units arranged and assembled to form the e2 homodimer?; second, what of structural properties confer the broad substrate specificity? To address these questions, we determined the X-ray crystal structure of ARMAN-2 EndA. The structure could be solved as a e2 homodimer. The e protomer was separated into three units, aN,a, and bC. The aN-a and a-bC units were fused by two linker loops. The overall structure of ARMAN-2 EndA is very similar to those of three types of archaeal EndAs, suggesting the common evolutionary origin of them. In this meeting, further structure-guided mutational study will be present.

References · K. Fujishima, J. Sugahara, C. S. Miller, B. J. Baker, M. D. Giulio, K. Takesue, A. Sato, M. Tomita, J. F. Banfield and A. Kanai (2011). A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity. Nucleic Acids Res 39: 9695–9704.

230 abstracts book EXTREMOphiles_2012P98 Transfer RNA recognition mechanism of Thermus thermophilus folate/FAD-dependent tRNA methyltransferase (TrmFO) Ryota Yamagamia, Koki Yamashitaa, Hiroshi Nishimasub, Chie Tomikawaa, Anna Ochia, Chikako Iwashitaa, Akira Hirataa, Ryuichiro Ishitanib, Osamu Nurekib, and Hiroyuki Horia, c aDept. of Materials Sciience and Biotechnology, Graduate School of Science and Engineering, Ehime University, Japan bDept. of Basic Biophysics.and.Biochemistry.,Graduate School of Science, University of Tokyo, Japan cVBL, Ehime Univ, Japan E-mail: [email protected]

Various modifications (more than 100) have been found in tRNA. The 5-methyluridine at position 54 (m5U54) is a universally conserved modified nucleoside in eubacterial and eukaryotic tRNA in addition to some archaeal tRNA. This modification plays a significant role in stabilization of tRNA structure. The m5U54 modification in Gram-positive and some Gram-negative bacteria including Thermus thermophilus is generated by folate/ FAD-dependent tRNA (m5U54) methyltransferase [TrmFO] (Urbonavicius et al., 2005; Nishimasu et al., 2009). TrmFO requires 5, 10-methylenetetrahydrofolate (MTHF) as a 1-carbon donor. However, MTHF is unstable and radioisotope labeled compound is not commercially available. We previously reported the semi-quantitative TrmFO assay system. Our assay system was coupled with the MTHF production by serine hydroxymethyltransferase [SHMT] (Nishimasu et al., 2009). Here we improved the TrmFO assay system and focused on the substrate tRNA recognition mechanism of TrmFO. We designed 47 mutant tRNA variants and measured their methyl group acceptance activities. The results indicated that TrmFO essentially recognizes the conserved G53-C61 base pair and U54U55C56 sequence in the T-arm. Furthermore, the A58 of tRNA is an important positive determinant for TrmFO. Moreover, we found that A38 prevents the incorrect methylation of U32 in the anticodon-loop. In addition, we found that the existence of m1A58 modification accelerates the TrmFO reaction. This result suggests that there is the synergistic effect of5 m U54, m1A58 and s2U54 modifications on m5s2U54 formation in livingThermus thermophilus cells.

References · J, Urbonavicius, S. Skouloubris, H. Myllykallio and H. Grosjean (2005). Nucleic Acids Res 33: 3955-3964. · H. Nishimasu, R. Ishitani, K. Yamashita, C. Iwashita, A. Hirata, H. Hori, and O. Nureki, (2009). Proc Natl Acad Sci USA 106: 8180-8185.

abstracts book 231 P99EXTREMOphiles_2012 Transfer RNA modifications in thermophilic bacteria Takuya Kawamuraa, Chie Tomikawaa, Takayuki Ohirab, Yasushi Inouec, Akihiko Yamagishic, Tsutomu Suzukib and Hiroyuki Horia, d

aDepartment of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan bDepartment of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan cDepartment of Molecular Biology, School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji 192-0392, Japan dVenture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan E-mail: [email protected]

Numerous modified nucleosides have been identified in tRNA, however there is little knowledge concerning with modified nucleosides in archaeal tRNA.Thermoplasma acidophilum is a thermophilic archaeon, which grows at 55°C in pH1.8 medium under anaerobic condition. To clarify the modified nucleosides inT. acidophilum, we purified tRNALeu from total tRNA by the solid DNA probe affinity column chromatography. The RNA sequence of this tRNA was determined by Kuchino’s post-labeling method and liquid chromatography-mass spectrometric analysis. To our surprise, T. acidophilum tRNALeu has unique modifications 4(s U9, archaeosine13 and m7G49) 4 2 1 1 in addition to the common modifications (s U8, archaeosine15, m 2G26, m G37, 39, 55, Cm56, and m A58) of archaeal tRNA. The s4U9 have not been reported in archaeal tRNA: the s4U9 has been reported only in eubacterial and eukaryotic tRNAs. The archaeosine13 and m7G49 are the modifications at novel positions in all tRNAs reported thus far. Thus, these tRNA modifications suggest thatT. acidophilum has considerably different RNA modification enzymes from the other organisms reported.

232 abstracts book EXTREMOphiles_2012P100 Prediction of novel phosphor-sugar metabolic pathway by functional genomics of acidothermophilic archaeon, Sulfolobus tokodaii Yutaka Kawarabayasi

Laboratory for Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Fukuoka, 8128581 Fukuoka, Japan E-mail: [email protected]

Already over 1,300 kinds of entire genomic sequences of microorganisms were determined. Within them, over 100 entire genomic sequences of archaea were included. The information predicted from the genomic sequence of these microorganisms provided useful information on genes or gene product. Also some genes were assigned on the general metabolic pathways. However, only limited numbers of genes for carbohydrate metabolic pathways were detected on the genomic data of Sulfolobus tokodaii. It means that the genomic data is not sufficient for recognition of entire feature of microorganisms. Then, functional analysis of each gene was performed. Seven sugar-1-phosphate nucleotidylyltransferases homologues were detected within the genome of Sulfolobus tokodaii. Among them, the ST0452 protein was detected as the protein with unusual long C-terminus. Thus, this protein was chosen as a target for detailed analyses of activities and functions. The GlcNAc-1-phosphate uridyltransferase activity and glucosamine-1-phosphate acetyltransferase activities were detected on the ST0452 protein. This result indicated that the ST0452 protein could catalyse the last two reactions within the UDP-GlcNAc biosynthesis pathway. It was also shown that the ST0452 possessed GalNAc- 1-phosphate uridyltransferase and galactosamine-1-phosphate acetyltransferase activities. This observation revealed that the ST0452 protein could be involved in the novel metabolic pathway for construction of UDP- GalNAc from galactosamine-1-phosphate. It can be concluded that to understand the actual activity or function of each protein, expression of gene and following experimental analyses are necessary. Therefore, it can be thought that the unimaginable activities or functions are hidden within the genomic data of archaea.

References · Z. Zhang et al. (2005). Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon,Sulfolobus tokodaii strain 7. J Biol Chem 280: 9698-9705. · Z. Zhang et al. (2007). Increasing in Archaeal GlcNAc-1-P uridyltransferase activity by targeted mutagenesis while retaining its extreme thermostability. J Biochem 141: 553-562. · Z. Zhang, et al. (2010). Identification of novel acetyltransferase activity on the thermostable protein ST0452 from Sulfolobus tokosaii strain 7. J Bacteriol 192: 3287-3293.

abstracts book 233 P101EXTREMOphiles_2012 Creation of a thermostable NADP-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase by site-directed mutagenesis Hironaga Akitaa, Katsumi Doib, Yutaka KawarabayashiC and Toshihisa Ohshimab

aBioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581 Japan bMicrobial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Japan cFunctional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Japan E-mail: [email protected]

Meso-diaminopimelate dehydrogenase (DAPDH, EC 1.4.1.16) catalyzes the NAD(P)- dependent oxidative deamination of meso-diaminopimelate and functions in the lysine metabolism. We screened various thermophiles to find stable DAPDH and found it in a thermophile isolated from compost. The thermophile was identified as Ureibacillus thermosphaericus strain A1, which grew well aerobically at around 55°C. We purified the enzyme and characterized. The DAPDH showed highly specific for meso-diamino- pimelate and NADP, and much more thermostable (no loss of activity at 60°C) than those from mesophiles so far reported. We succeeded gene cloning in Escherichia coli and sequenced determination of the enzyme (326 amino acid residues, a homodimer of 40 kDa). As the next step, we tried creation of thermostable NADP- dependent D-amino acid dehydrogenase (DAADH) from DAPDH by site-directed mutagenesis. We prepared the mutated enzyme of Gln154Leu, Asp158Gly, Thr173Ile, Arg199Met and His249Asn as described previously (Vedha-Peters K. et al., 2006), and purified the mutant enzyme expressed in E. coli to homogeneity. The mutant enzyme showed no loss of activity at 65°C, indicating the mutant enzyme was more thermostable than the parental DAPDH. In the presence of NADP, the mutant enzyme catalyzed the oxidative deamination of several D-amino acids including D-cyclohexylalanine, D-isoleucine and D-2-aminooctanoate but not meso-diaminopimelate, confirming the creation of an NADP- dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. This indicates that the DAADH may be applicable to the synthesis and sensing of D-amino acids.

References · Akita H, Fujino Y, Doi K, Ohshima T (2011). AMB Express 1: 43. · Akita H, Doi K, Kawarabayasi Y, Ohshima T (2012). Biotechnology Letters, in press.

234 abstracts book EXTREMOphiles_2012P102 Study of novel recombinant proline dipeptidase from anaerobic organotrophic archaeon Thermococcus sibiricus Elvira Slutskayaa, Ekaterina Bezsudnovaa, Anton Trofimovb, Dmitry A. Korzhenevskyc, Vadim Gumerovd and Vladimir Popova,c

aA.N.Bach Institute of Biochemistry Russian Academy of Sciences, Leninsky pr. 33, Moscow, 119071, Russia bEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str.32, 119991 Moscow, Russia cRussian National Research Centre «Kurchatov Institute», Akademika Kurchatova sq. 1, Moscow 123182, Russia dCentre «Bioengineering» Russian Academy of Sciences, Prosp. 60-let Oktyabrya, bld. 7-1, Moscow, 117312, Russia E-mail: [email protected]

Proline dipeptidase (prolidase) is a metallopeptidase that specifically hydrolyzes dipeptides with a prolyl residue in the carboxy terminus (NH2–X–/–Pro–COOH). Novel prolidase (TsProl) was isolated from cell extracts of the hyperthermophilic anaerobic organotrophic archaeon Thermococcus sibiricus. The gene was cloned and expressed in E. coli. The recombinant TsProl fused to a His-tag was purified to apparent homogeneity by heat treatment followed by Ni-NTA affinity chromatography and gel filtration. The catalytic activity is increased for a 60% by the addition of Mn2+ ions. The TsProl exhibited narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Val, Ala Phe) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 8.5 and a temperature of 90°C. The affinity of TsProl for the substrates Met-Pro, Phe-Pro, Val-Pro and Ala-Pro is 4.8 mM, 6.1 mM, 6.9 mM and 3.3 mM, respectively. The crystals of the TsProl were grown by the vapor-diffusion hanging-drop technique at 40C.The crystal structure has been solved at 2.6 A resolution. An analysis of intersubunit contacts showed that TsProl has a dimeric structure in the crystals. The predominant form of the enzyme in solution as estimated by gel filtration is also a dimer with a molecular mass of 82 kDa. The monomer has a two-domain structure with characteristic dinuclear metal cluster in C-domain typical of other dipeptidases. The comparison of the structure of TsProl with the structures of two homologous thermostable dipeptidases from P. horikoshii (Ph) and P. furiosus (Pf) revealed large structural similarity between the counterparts: Z-score was 19.5 and 15.4 and RMSD (Cα) was 1.19 and 1.92 A, respectively. A comparison of the amino acid composition of TsProl with Ph and Pf dipeptidases shows no difference in the contents of charged amino acids. This fact is in good agreement with the observed high thermostability of the counterparts. Remarkable differences are observed among hydrophobic amino acids content: the total amount of phenylalanine + tyrosine is the highest for TsProl. Such high content of large hydrophobic residues could results in the maximum density of molecular packings for TsProl. Moreover, most of phenylalanine residues are located in metal cluster containing C-domain that might explain the substrate preferences of the enzyme. The study was supported by the Ministry of Education and Science of the Russian Federation (state contract no. 16.512.11.2175) References · R. Kitchener, A. Grunden (2012). Prolidase function in proline metabolism and its medical and biotechnological applications. J Appl Microbiol PMID: 22512465. · C. Theriot, S. Tove, A. Grunden (2010). Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii. Appl Microbiol Biotechnol 86:177-188.

abstracts book 235 P103EXTREMOphiles_2012 Characterization of recombinant thermostable L-asparaginase from Thermococcus kodakaraensis KOD1 Sung-Jun Hong, Yun-Ha Lee, Jae-Ho Shin

School of Applied Bioscience, Kyungpook National University, Daegu, 702-701 South Korea E-mail: [email protected]

L-Asparaginase(E.C.3.5.1.1) catalyzes hydrolysis of L-asparagine into L-aspartate and ammonia. This enzyme is present in various living organisms including animals, plants and bacteria. For food industrial purpose, L-asparaginase is suggested to use for reducing acrylamide content of starch baked foods. A gene encoding putative L-asparaginase,tk 1656, was cloned from a thermophilic archaean, Thermococcus kodakaraensis KOD1 (GenBank Accession No. NC_006624). The PCR amplified L-asparaginase gene with 6xHis- tag was sub-cloned into pET21a(+) and subsequently transformed to Escherichia coli BLR(DE3). The expressed recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimum temperature and pH of the recombinant L-asparaginase were 90ºC and pH 8.0, respectively. Up to 50% of the enzyme activity was remained after treatment of 90ºC for 16 h. Moreover, after pre-incubation in pH 3.5 to 11.5 for 24 h at 4ºC, at least 80% of the enzyme activity was remained.

Both heat and pH stability is indicating that the enzyme is industrial useful. Calculated Km, Vmax and Kcat values by Lineweaver-Burk plot were 4.3 mM, 1,558 U/mg and 933 s-1 respectively. Ni2+ and Mg2+ increased the recombinant enzyme activity. The acrylamide level in wheat starch baked bread could be lowered by treating the recombinant L-asparaginase after making dough.

236 abstracts book EXTREMOphiles_2012P104 The natural transformation machinery in Thermus thermophilus HB27: A pilus-independent DNA transporter comprising unique motor ATPase and secretin complexes R. Salzera, J. Burkhardta, J. Vonckb and B. Averhoffa

aMolecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Goethe University, Frankfurt/ Main, Germany bDepartment of Structural Biology, Max-Planck Institute of Biophysics, Frankfurt/Main, Germany E-mail: [email protected]

To get insights into the structure and function of DNA translocators we chose the thermophileT. thermophilus HB27 as model organism since it exhibits the highest natural transformation frequencies known to date. A genome-wide genetic screen followed by mutant studies led to the identification of 16 distinct competence proteins (Averhoff 2009), several of them were found to play a dual role in transformation and piliation. But the question whether the pilus structures itself are essential for DNA uptake was still unanswered. Here we report on structural and functional analyses of the AAA-ATPase PilF, a unique motor component and the secretin PilQ. Both were found to be essential for natural transformation and piliation. PilF carries a unique N-terminal triplicated GSPII domain and a C-terminal tetracysteinmotif involved in zinc binding (Roseet al., 2011). Mutant studies revealed that two of the cysteines are essential for Zn2+ binding, piliation, twitching motility and adhesion, but not for natural transformation. Recently, we reported on the novel structure of a PilQ complex, comprising a stable cone and cup structure and six ring structures (Burkhardt et al., 2011). Structural analyses of a set of PilQ deletion derivates in T. thermophilus HB27 identified 136 N-terminal residues, encoding an unusual ααβααββα fold as a ring building domain. Deletion of this domain had a dramatic effect on piliation but did not abolish natural transformation. Taken together, these findings provide clear evidence that the pilus structures are not essential for natural transformation.

References · Averhoff B. (2009) FEMS Microbiol Rev 33:611-626. · Rose I., Biukoviæ G., Aderhold P., Müller V., Grüber G., Averhoff B. (2011) Extremophiles 15: 191-202. · Burkhardt J., Vonck J., Averhoff B. (2011) J Biol Chem 286: 9977-9984.

abstracts book 237 P105EXTREMOphiles_2012 Purification and characterization of a thermophilic feather-degrading enzyme from a moderate thermophile Bacillus sp. FN60 Mio Kojima and Akira Inoue

Bio Nano Electronics Research Centre, Toyo University, 2100 Kujirai, Kawagoe Saitama 350-8585, Japan E-mail: [email protected]

We isolated a feather-degrading moderate thermophile Bacillus sp. FN60 from the soil sample of poultry farm. The isolate completely degraded feather pieces, wools and dog hairs after liquid culture at 55ºC (pH 7.0) for 2-3 days. Strain FN60 is a Gram-positive, spore-forming, rod-shaped bacterium and was identified with B. thermoruber based on 16S rDNA analysis. The keratinase enzyme produced by strain FN60 was refined using ammonium sulfate precipitation, negative-ion DEAE Toyopearl exchange chromatography, and hydroxyapatite chromatography. The molecular weight of this enzyme was 35 kDa. The enzyme exhibited activity at pH 5.0-11.0 and 30-80 °C with azokeratin as a substrate, although the optimum pH and temperature for keratinase activity were pH 7.0 and 60 °C, respectively. Moreover this enzyme also maintained residual activity after incubation at 90 °C for 30min and thus had thermostability.

This enzyme is one of the serine-type proteases. Thermophilic WF146 protease and this enzyme had 93% homology in the amino acid sequence. Since this enzyme differed from WF146 protease in molecular weight, heat resistance, they are suggested to be different enzymes.

References · J. Wu, Y. Bian, B. Tang, X. Chen, P. Shen, Z. Peng (2004). Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops. FEMS Microbiology Letters 230: 251-258.

238 abstracts book EXTREMOphiles_2012P106 Molecular basis for polysaccharides degradation capabilities of Desulfurococcus fermentans, a hyperthermophilic crenarchaeon Anna Perevalovaa,c, Ida Steenb, Runar Stokkeb, Dwi Susantic, Elizaveta Bonch-Osmolovskayaa and Biswarup Mukhopadhyayc,d

aWinogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia bCentre for Geobiology, University of Bergen, Bergen, Norway cVirginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA dDepartments of Biochemistry and Biological Sciences, Virginia Tech, Blacksburg, VA, USA E-mail: [email protected]

Desulfurococcus fermentans is the first reported cellulolytic archaeon (Perevalova et al., 2005). This hyperthermophilic anaerobic crenarchaeon produces hydrogen from fermentative degradation of cellulose, various other carbohydrates, and peptides at temperatures of 80-82oC. To better understand the molecular basis for these activities, complete genome ofD. fermentans was sequenced at the US Department of Energy’s Joint Genome Institute (Susantiet al., manuscript in preparation). TheD. fermentans genome reveals the presence of membrane-bound hydrogenases and is notable for the absence of typical cellulases, suggesting that novel glycohydrolases likely facilitate efficient hydrogen production from cellulose. To understand the mechanisms of cellulose degradation inD. fermentans a comparative proteomic analysis was performed. Here to identify the differences in abundances of proteins during growth on cellulose and starch, whole cell lysate proteins were subjected to the gel-electrophoresis-LC-MS/MS analysis (Aebersold and Mann, 2003). A total of 110 validated proteins were identified as specific to the protein pool of cellulose-grown cells and 156 proteins were associated with growth on starch. Some of these were candidate glycohydrolases and were chosen for enzymology work. Here we report the results from studies on one of the glycohydrolase homologs that according to bioinformatics data and RT-PCR analysis seems to form a membrane-bound complex. We have expressed the catalytic subunit of this complex in E. coli. Purified recombinant protein exhibited glycohydrolase activity on three carbohydrate substrates, xylan, mannan, galactomannan, but not cellulose. Whether other components of the membrane complex are necessary for this glycohydrolase to exhibit cellulose activity is a focus in our future investigations.

References · Perevalova et al., 2005. Desulfurococcus fermentans sp. nov., a new hyperthermophilic archaeon from a Kamchatka hot spring, with an emended description of the genus Desulfurococcus. Int J Syst Evol Microbiol 55: 995-999. · Susantiet al. Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia. (manuscript in preparation). · R. Aebersold and M. Mann 2003. Mass spectrometry-based proteomics. Nature 422: 198-207.

abstracts book 239 P107EXTREMOphiles_2012 A new chemo-enzymatic tool for the GOS synthesis using a mutant of the thermophilic b-galactosidase from Alicyclobacillus acidocaldarius Andrea Strazzullia, Beatrice Cobucci-Ponzanoa, Perugino Giuseppea, Maria Michela Corsarob, Emiliano Bedinib, Mosè Rossia and Marco Moraccia

aInstitute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy bDipartimento di Chimica Organica e Biochimica, Università di Napoli «Federico II», Complesso Universitario di Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy E-mail: [email protected]

The galacto-oligosaccharides (GOS) belong, for their indigestible nature, to the group of prebiotics, which beneficially affect the host by stimulating the growth and/or activity of colon bacteria. GOS naturally occur in human milk, and commercially available products are broadly used in infant formula, biscuits, food for critical illnesses1. In addition, several studies on both infants and adults have shown that consumption of GOS resulted in a significant increase in Bifidobacteria2. The synthesis of GOS is commonly performed by mesophilic β-galactosidases via transglycosylation starting from lactose. The drawback of this application is the limited final yields due to the intrinsic hydrolytic activity of the enzyme and the subsequent formation of free glucose that inhibits the synthesis of products3. The large demand for these important nutraceuticals requires the development of new efficient and economically sustainable methods for their synthesis. To expand the chemo-enzymatic tools for the GOS synthesis we have characterized, in detail, a mutant of a non catalytic residue of the GH42 b-galactosidase (Aab-Gal)4 from the thermophilic bacterium A. acidocaldarius, named Glu361Gly. We demonstrate here that this is able to promote GOS synthesis, showing a strongly reduced hydrolytic activity and catalyzing transgalactosylation reactions with high efficiency on different acceptors.

References · Coulier, L., Timmermans, J., Bas, R., Van Den Dool, R., Haaksman, I., Klarenbeek, B., Slaghek, T., and Van Dongen, W.(2009) Journal of Agricultural and Food Chemistry, 57: 8488-8495. · Piirainen, L., Kekkonen, R. A., Kajander, K., Ahlroos, T., Tynkkynen, S., Nevala, R., and Korpela, R. (2008) Annals of Nutrition & Metabolism 52: 204-208. · Park, A.R. and Oh, D.K.(2010) Applied microbiology and biotechnology, 85: 1279-1286. · Di Lauro, B., Strazzulli, A., Perugino, G., La Cara, F., Bedini, E., Corsaro, M. M., Rossi, M., and Moracci, M. (2008) Biochimica et Biophysica Acta 1784:292-301.

240 abstracts book EXTREMOphiles_2012P108 New groups of potentially novel enzyme specificities in the extremophilic glycoside hydrolase family GH57 Karol Blesak and Stefan Janecek

Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, SK-84551 Bratislava, Slovakia E-mail: [email protected]

The glycoside hydrolase (GH) family 57 (Janecek, 2012) consists of more than ~700 proteins from Archaea (roughly 1/4) and Bacteria (roughly 3/4) mostly from thermophiles. Less than 20 GH57 members have already been biochemically characterized as real (almost exclusively) amylolytic enzymes (Cantarel et al., 2009). Five enzyme specificities are well established in the family: α-amylase, amylopullulanase, branching enzyme, 4-α-glucanotransferase and α-galactosidase (Blesak and Janecek, 2012) with one large group of the so-called α-amylase-like homologues probably without the enzyme activity (Janecek and Blesak, 2011). An incomplete TIM-barrel as a catalytic domain (Imamuraet al., 2003) and five conserved sequence regions (Zonaet al., 2004) are the main characteristics of the family GH57. The main goal of the present bioinformatics study was to describe two novel groups within the family GH57 that could represent á-amylases (124 sequences) and maltogenic amylases (12 sequences). Both groups may define new GH57 subfamilies distinguishable from other subfamilies recognized previously by their specific features especially in their conserved sequence regions. Acknowledgments This work was supported by the Slovak Research and Development Agency under the contract No. LPP- 0417-09 and by the VEGA grant No. 2/0148/11.

References · K. Blesak and S. Janecek (2012). Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57. Extremophiles 16: 497-506. · B. L. Cantarel, P. M. Coutinho, C. Rancurel,et al. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37 (Database issue): D233-D238. · H. Imamura, S. Fushinobu, M. Yamamoto, et al. (2003). Crystal structures of 4-α-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor. J Biol Chem 278: 19378-19386. · S. Janecek (2012). Glycoside hydrolase family 57. In: CAZypedia, URL: http://www.cazypedia.org/. · S. Janecek, K. Blesak (2011). Sequence-structural features and evolutionary relationships of family GH57 α-amylases and their putative α-amylase-like homologues. Protein J 30: 429-435. · R. Zona, F. Chang-Pi-Hin, M. J. O’Donohue and S. Janecek (2004). Bioinformatics of the family 57 glycoside hydrolases and identification of catalytic residues in amylopullulanase fromThermococcus hydrothermalis. Eur J Biochem 271: 2863-2872.

abstracts book 241 P109EXTREMOphiles_2012 A relatedness between the CAZy alpha-amylase families GH57 and GH119 - a novel clan? Stefan Janecek, Karol Blesak and Andrea Kuchtova

Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, SK-84551 Bratislava, Slovakia E-mail: [email protected]

The glycoside hydrolase (GH) family 119 was established in the CAZy server (Cantarel et al., 2009) in 2006 based on a sequence of a novel α-amylase IgtZ from Bacillus circulans (Watanabe et al., 2006). At present there are only 5 additional hypothetical proteins in the GH119. Except for a short remark in the CAZy on a distant relatedness of GH119 to GH57 (Cantarel et al., 2009), nothing has been known about the GH119 catalytic residues and catalytic-domain fold. The family GH57 belongs, on the other hand, to well-described CAZy GH families with more than ~700 members mostly from thermophiles (Cantarel et al., 2009). Five confirmed GH57 specificities of α-amylase, amylopullulanase, branching enzyme, 4-α-glucanotransferase and β-galactosidase (Blesak and Janecek, 2012) adopt an incomplete TIM-barrel catalytic domain (Imamuraet al., 2003) and share five conserved sequence regions (Zonaet al., 2004). The main goal of the present bioinformatics study was to identify both catalytic residues within the predicted incomplete catalytic TIM-barrel for the family GH119 members. The relatedness between the families GH119 and GH57 was revealed based on a detailed in silico analysis involving sequence comparison in combination with BLAST searches and structure modelling. The families GH57 and GH119 may thus define a new CAZy clan. Acknowledgments This work was supported by the Slovak Research and Development Agency under the contract No. LPP- 0417-09 and by the VEGA grant No. 2/0148/11.

References · K. Blesak and S. Janecek (2012). Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57. Extremophiles 16: 497-506. · B. L. Cantarel, P. M. Coutinho, C. Rancurel,et al. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37 (Database issue): D233-D238. · H. Imamura, S. Fushinobu, M. Yamamoto, et al. (2003). Crystal structures of 4-α-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor. J Biol Chem 278: 19378-19386. · H. Watanabe, T. Nishimoto, M. Kubota, et al. (2006). Cloning, sequencing, and expression of the genes encoding an isocyclomaltooligosaccharide glucanotransferase and an α-amylase from a Bacillus circulans strain. Biosci Biotechnol Biochem 70: 2690-2702. · R. Zona, F. Chang-Pi-Hin, M. J. O’Donohue and S. Janecek (2004). Bioinformatics of the family 57 glycoside hydrolases and identification of catalytic residues in amylopullulanase fromThermococcus hydrothermalis. Eur J Biochem 271: 2863-2872.

242 abstracts book EXTREMOphiles_2012P110 Archaeal glycosylation: deciphering the machinery in Sulfolobus solfataricus Maria Carmina Ferraraa, Beatrice Cobucci-Ponzanoa, Andrea Carpentierib, Angela Amoresanob and Marco Moraccia

aInstitute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy bDepartment of Chemical Sciences, University of Naples «Federico II», Naples, Italy E-mail: [email protected]

Glycosylation is one of the most prevalent post-translational modifications that expand the diversity of the proteome by the addition of different glycan moieties. Once believed to be restricted to Eukarya, it is now clear that Bacteria and Archaea are also capable of performing N-glycosylation. The first example of prokaryotic N-glycosylated protein was reported 30 years ago in the haloarchaeon Halobacterium salinarum (Mescher et al., 1976). Moreover the prevalence and the structural variety of the N-glycosylated proteins are more prominent in Archaea than in Bacteria. However, while the eukaryal and bacterial N-glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Archaea combines particular aspects of the bacterial and eukaryal pathways, such as monomeric oligosyltransferases and dolichol phosphate carrier respectively, along with traits unique to this life form (Calo et al., 2010). Within the last few years, the efforts of several groups have served to elucidate different aspects of N-glycosylation in euryarchaeal model species (Abu-Qarn and Eichler 2010). Preliminary results indicate that glycosylation in Crenarchaea, a phylum evolutionarily distant from Euryarchaea, is indispensable for cell survival (Lindås et al., 2008) and even more widespread than in Euryarchaea (Koerdt et al., 2010). Nevertheless, the study of the steps and the components of the machinery involved in the crenarchaeal glycosylation and of the nature of the glycosylated proteins is still in its infancy. Here, we report the identification and biochemical characterization of a novel β-glucosidase/β-N- acetylglucosaminidase in Sulfolobus solfataricus P2. This enzymatic activity, encoded by the ORF SSO3039, is classified in the family GH116 according the database CAZy (http://www.cazy.org) and represents the first member of this family able to hydrolyze N-acetyl-β-D-glucosaminides. Moreover, by using a glycoproteomic approach we analyzed the sugar composition and structure of the glycoproteins from S. solfataricus. A better understanding of crenarchaeal glycosylation will provide new insights into this post-translational modification across evolution as well as protein processing under extreme conditions.

References · Mescher MF and Strominger JL (1976) Journal of Biological Chemistry 251: 2005-2014. · Calo D, Kaminski L and Eichler J, (2010) Glycobiology 20: 1065-1076. · Abu-Qarn M and Eichler J (2006). Molecular Microbiology 61:511-25. · Lindås AC, Karlsson EA, Lindgren MT, Ettema TJ, Bernander R (2008)Proc Natl Acad Sci USA 105: 18942-6. · Koerdt A, Gödeke J, Berger J, Thormann KM, Albers SV (2010) PLoS One 5, e14104.

abstracts book 243 P111EXTREMOphiles_2012 Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway Riku Aonoa, Takaaki Satoad, Ayumu Yanoa, Shosuke Yoshidaa, Yuichi Nishitanib, Kunio Mikibd, Tadayuki Imanakacd and Haruyuki Atomiad

aDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan bDepartment of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan cDepartment of Biotechnology, College of Life Sciences, Ritsumeikan University, Noji-Higashi, Kusatsu, Shiga 525-8577, Japan dJST, CREST, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan E-mail: [email protected]

In this study, we performed enzymatic analysis of two novel enzymes, AMP phosphorylase (AMPpase) and ribose-1,5-bisphosphate isomerase (R15Pi), from the hyperthermophilic archaeon Thermococcus kodakarensis. The enzymes are presumed to be members of a novel metabolic pathway in the Archaea involved in AMP metabolism (Sato et al. 2007). In terms of R15Pi, we observed that enzyme activity was extremely low when using chemically synthesized ribose 1,5-bisphosphate (R15P), compared to levels using substrate enzymatically prepared with AMPpase. By evaluating the effects of compounds in AMPpase reaction mixture, we found that R15Pi was dramatically activated by AMP. In addition, investigating the effects of various compounds similar to AMP revealed that ADP also activated R15Pi. The enzyme was specific for R15P and did not recognize other sugar compounds. As the substrate specificity of AMPpase was previously examined using R15Pi as a coupling enzyme, the lack of activity towards NMPs other than AMP may have been due to the low levels of R15Pi in the absence of AMP. Using AMP-activated R15Pi, we re-evaluated the substrate specificity of AMPpase. We found that AMPpase exhibited phosphorylase activity towards CMP and UMP in addition to AMP. The [S]-v plot of the enzyme towards AMP was sigmoidal with an increase in activity observed at AMP concentrations higher than 3 mM. Our results suggest that this pathway is involved in the breakdown of various nucleoside 5’-monophosphates in response to accumulation in the intracellular concentration of AMP or ADP.

References · T. Sato, H. Atomi, and T. Imanaka (2007). Archaeal type III RuBisCOs function in a pathway for AMP metabolism. Science 315: 1003-1006.

244 abstracts book EXTREMOphiles_2012P112 Dynamic, ligand-dependent conformational change

triggers the reaction of ribose-1,5-bisphosphate isomerase from Thermococcus kodakarensis

Takaaki Satoad, Akira Nakamurab, Riku Aonoa, Masahiro Fujihashibd, Yosuke Nishibab, Shosuke Yoshidaa, Ayumu Yanoa, Tadayuki Imanakacd, Kunio Mikibd and Haruyuki Atomiad

aDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan bDepartment of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan cDepartment of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan dJST, CREST, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan E-mail: [email protected]

We are interested in the unique metabolism of hexoses and pentoses in Archaea and have previously discovered a novel pathway that converts the ribose moiety of AMP into 3-phosphoglycerate, an intermediate of central carbon metabolism (Sato et al. 2007). Ribose-1,5-bisphosphate isomerase (R15Pi) is a novel enzyme identified as a member of this AMP metabolic pathway. The enzyme catalyzes the isomerization of d-ribose 1,5-bisphosphate (R15P) into ribulose 1,5-bisphosphate (RuBP), the substrate for archaeal ribulose-1,5- bisphosphate carboxylase/oxygenases. Here we report the crystal structures of R15Pi from Thermococcus kodakarensis KOD1 (Tk-R15Pi) with and without its substrate or product. Tk-R15Pi is a hexameric enzyme formed by the trimerization of dimer units. Biochemical analyses show that, among the anomers of R15P, Tk-R15Pi only accepts the a-anomer. Mutation of Cys133 and Asp202 led to mutant proteins with undetectable levels of activity, indicating that these residues are essential for RuBP production. The crystal structure suggested that Arg227 is involved in subunit interaction. The mutant protein R227E exhibited a lower molecular size and a decrease in activity, suggesting that this Arg plays a key role in hexamerization, and that the hexameric assembly is important for R15Pi activity. Comparison of the determined structures reveals that the unliganded and product-binding structures are in an open form, whereas the substrate-binding structure adopts a closed form, indicating domain movement upon substrate binding. The conformational change to the closed form optimizes active-site configuration and also isolates the active site from the solvent, which may allow deprotonation of Cys133 and protonation of Asp202 to occur. The structural features of the substrate-binding form and biochemical evidence lead us to propose that the isomerase reaction proceeds via a cis-phosphoenolate intermediate (Nakamura et al. in press).

References · T. Sato, H. Atomi, and T. Imanaka (2007). Archaeal Type III RuBisCOs Function in a Pathway for AMP Metabolism. Science 315: 1003-1006. · A. Nakamura , M. Fujihashi, R. Aono, T. Sato, Y. Nishiba, S. Yoshida, A. Yano, H. Atomi, T. Imanaka, and K. Miki (in press) Dynamic, ligand-dependent conformational change triggers the reaction of ribose-1,5-bisphosphate isomerase from Thermococcus kodakarensis KOD1. J. Biol. Chem.

abstracts book 245 P113EXTREMOphiles_2012 Single molecule force spectroscopy study of cold shock protein from the hyperthermophile Thermotoga maritima Katarzyna Tycha,b, Toni Hoffmanna,b, David J. Brockwellb and Lorna Dougana,b

aMolecular and Nanoscale Physics Group, School of Physics and Astronomy, University of Leeds, LS2 9JT, UK bAstbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, LS2 9JT, UK E-mail: [email protected]

The Cold shock protein (Csp) family are small proteins with a highly conserved beta-barrel structure. While their expression is up-regulated by a sudden decrease in temperature, as part of the cold stress response of many bacteria, Csps are also present under normal conditions and may have a role in the response to other forms of stress. Their function is to stabilize single-stranded nucleic acids via their nucleic-acid binding motifs. The details of their cold-stress protective action are, however, not yet fully understood. Csp domains are found in many other proteins from bacteria and even vertebrates, such as the Y-box proteins, which have significance in the tumor growth of human cancers. We are using an atomic force microscopy (AFM) force spectroscopy instrument with variable temperature capabilities to examine the conformational dynamics of extremophilic proteins at the single molecule level. In this experimental technique, a cantilever with a known spring constant is used to apply a constant stretching force or a constant stretching velocity along the end-to-end length of the protein, driving the protein into a fully extended unfolded state. By examining molecules one at a time, the individual dynamics of protein sub-populations can be measured, in the context of their environment. We use this approach to measure the mechanical stability and the underlying energy landscape of single protein domains in a range of different temperature environments. Previous work has shown that a high thermal stability does not necessarily correspond to a high mechanical stability, therefore studying proteins in this way provides an additional tool for their characterisation in a physiologically interesting context. Csp from Thermotoga maritima (Tm) lends itself well as a model protein for AFM studies as it is small (66 residues), has a known structure and known thermodynamic and kinetic stabilities. To study this protein by AFM methods, poly-protein chains were expressed and purified that consisted of threeTm Csp domains interdigitated with four «fingerprint» I27 domains (the 27th immunoglobulin-like domain of titin). As I27 has previously been extensively studied by AFM force spectroscopy, the fingerprint domains serve as both a mechanical force standard, and a fingerprint enabling those force-extension traces where polyproteins are extended by force, to be identified. Force-extension traces of the polyprotein chains were collected at four different temperatures and five different pulling speeds, and the peak-to-peak distances and peak unfolding forces for each domain measured. We discuss our findings in the context of other thermophilic proteins studied and speculate as to the origin of mechanical and thermal stability in such proteins.

246 abstracts book EXTREMOphiles_2012P114 A novel alkaline xylanase from alkaliphilic Bacillus sp. SN5 Wenqin Bai, Yanfen Xue, Cheng Zhou, Yanhe Ma

National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China E-mail: [email protected]

A xylanase gene (xyn11A) was cloned from a genomic library of alkaliphilic Bacillus sp. SN5. The full-length gene consisted of 1,101 bp and encoded a polypeptide of 366 amino acids. The xylanase Xyn11A belonged to family 11 of glycoside hydrolases and comprised a catalytic domain and a xylan-binding domain joined by a short Gly/Pro-rich linker region. The intact xylanase Xyn11A and the CBM-linker-truncated xylanase Xyn11A- LC were expressed in Escherichia coli BL21 (DE3), purified to electrophoretic homogeneity and subsequently characterized. The optimum temperature of Xyn11A and Xyn11A-LC were both 55 ºC. The optimum pH of Xyn11A was 7.5, while Xyn11A-LC showed broad optimum pH from 5.5 to 8.0. Xyn11A and Xyn11A-LC showed high pH stability from 8.0 to 11.0. Xyn11A and Xyn11A-LC retained 18% and 64% of its initial activity after 30- min incubation at 60 ºC, respectively. For beechwood xylan, the specific activity of Xyn11A and Xyn11A-LC were 4539.6 and 6214.2 U/mg, respectively. CBM of Xyn11A affected enzyme stability, optimum pH, specific activity to soluble xylan and affinity to insoluble xylan. The CBM-linker-truncated xylanase Xyn11A-LC was a family 11 alkaline xylanase with the highest specific activity reported to date. Therefore, it was a promising candidate to use in pulp bleaching process. In order to study the relationship between structure and function, the recombinant enzyme Xyn11A-LC was crystallized. The three-dimensional structures of Xyn11A-LC and highly alkaliphilic xylanase XynJ (PDB accession number 2DCJ, Lu N et al.,2000) were compared. An increased number of salt-bridges were in the alkaliphilic xylanase XynJ. Salt-bridges were introduced in the structure of Xyn11A-LC by site-directed mutagenesis. Interestingly, Salt-bridge in the catalytic cleft didn’t improve the alkaliphilicity of Xyn11A-LC, but raised the optimum temperature from 55ºC to 60ºC. In order to improve the thermophilicity and thermostability of Xyn11A-LC, the N-terminus of Xyn11A-LC and the thermophilic xylanase TfxA (Irwin D, et al.1994) were compared. Hydrophobic interaction of aromatic amino acids was introduced in the structure of Xyn11A-LC by site-directed mutagenesis, which not only increased the optimum temperature by 5ºC but also improved the thermostability of Xyn11A-LC. Therefore, the salt bridge and hydrophobic interaction contributed to improvement of the thermophilicity and thermostability of Xyn11A-LC.

References · Lu N, Moriyama H, Nakamura S, Sato T, Tanaka N (2000). Crystallization and initial X-ray analysis of alkaline xylanase. Acta Crystallogr D Biol Crystallogr 56: 464-465. · Irwin D, Jung ED, Wilson DB (1994). Characterization and sequence of aThermomonospora fusca xylanase. Appl Environ Microbiol 60: 763-770.

abstracts book 247 P115EXTREMOphiles_2012 Polimeryzing laccase-like activity in antarctic psychrophiles and thermophiles R. Peraltaa,b, L. Blameya, J. Pereiraa and J. M. Blameya,b

aFundación Científica y Cultural Biociencia, Santiago of Chile bDepartment of Chemistry and Biology. Universidad de Santiago de Chile E-mail: [email protected]

Lignin is one of the three main constituents in wood and the second most abundant polymer on Earth. It is typically considered a recalcitrant material that is resistant to microbial decomposition; only specialized biota, predominantly fungi, are able to synthesize extracellular enzymes that break down this structure into biologically usable forms. It can be found in the cell wall in about 23%, forming an amorphous matrix together with hemicelluloses (Hammel et al. 1997) and it is basically composed of p-hydroxyphenyl, guaiacyl and syringyl units. Laccase, a versatile multi-copper oxidase, is one of the major redox enzymes involved in lignin modification catalyzing the oxidation of lignin components using molecular oxygen as the electron acceptor, which in turn is reduced to water. Advantage can be taken on the ability of laccase to catalyze the oxidation of ortho and p-diphenols, amino phenols, phenolic dimers of lignin, even of non-phenolic compounds in the presence of some intermediates (Bourbonnais & Paice 1992) to form reactive radicals for further polymerization in order to couple target molecules producing polymers with novel properties. For instance, laccases are being investigated for purposes such as functionalisation of lignocellulose by oxidatively incorporating antimicrobial agents as chitosan or catechin, for the development of «intelligent fabrics» (Silva et al., 2011). Little is known about extremophilic bacterial or archaeal laccases and nothing is known about any antarctic counterpart. It is important to consider that in contrast to fungal laccases, bacterial laccases are highly active and much more stable at high temperatures, high pH and high concentrations of chloride and copper ions (Rosconi et al., 2005). The stability presented by extremophilic laccases allows them to be functional under harsh industrial conditions and to prevent protein degradation during storage. In our laboratory, we have detected phenoloxidase activity in different antarctic psychrophiles isolates belonging to Bacillus, Pseudomonas and Shewanella genera that were grown in a media using sulfonated lignin as a sole carbon source. Besides, we have quantified laccase-like activity on an extracelullar protein concentrate from a Bacillus isolate, obtaining an activity of 47 U/mg protein. Antarctic thermophilic consortia also showed laccase-like activity 3 U/mg protein on crude extracts. Further work on characterization, is part of the ongoing work.

References · K. Hammel (1997). Fungal degradation of lignin. In Driven by Nature: Plant litter and decomposition. Eds G. Gadisch and K. E. Giller. Wallingford, UK: CAB International: pp 22-45. · R. Bourbonnais and M. Paice (1992). Demethylation and delignification of kraft pulpTrametes by versicolor laccase in the presence of 2,2’-azinobis-(3-ethylbenzthiazoline- 6-sulphonate). Applied Microbiology and Biotechnology 36: 823-827. · C. Silva et al. (2011). Antimicrobial and antioxidant linen via laccase-assisted grafting.Reactive & Functional Polymers 71: 713-720. · F. Rosconi (2005). Purification and characterization of a periplasmic laccase produced by Sinorhizobium meliloti. Enzyme and Microbial Technology 36: 800-807.

248 abstracts book EXTREMOphiles_2012P116 Tetrathionate hydrolase from the marine acidophilic sulfur-oxidizing bacterium Acidithiobacillus thiooxidans strain SH Tadayoshi Kanaoa, Yuki Hashimotob, Tatsuya Togeb, Hiroshi Kikukawab and Kazuo Kamimuraa

aDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, 700-8530 Okayama, Japan bFaculty of Agriculture, Okayama university, Okayama, Japan E-mail: [email protected]

Acidithiobacillus thiooxidans strain SH was isolated from seawater (Kamimura et al., 2003). This strain is acidophilic, chemolithoautotrophic bacterium which is capable of oxidation of various reduced inorganic sulfur compounds as energy sources. Acidophilic sulfur-oxidizing bacteria which are tolerant to NaCl might be useful in developing remedial technology for salt-containing environments contaminated with heavy metals. Therefore, it is important to study the sulfur oxidation metabolism of such a NaCl tolerant acidophile. The strain SH showed well growth on basal salt medium containing 2% NaCl and 5 mM tetrathionate at pH 4.0. Tetrathionate hydrolase (4THase) which catalyzes hydrolysis of tetrathionate is one of the unique enzymes in the dissimilatory sulfur metabolism of the genus Acidithiobacillus. 4THases have been reported from Acidithiobacillus species (A. ferrooxidans, A. thiooxidans and A. caldus), Acidiphiliun acidophilum and thermo- acidophilic archaeon Acidianus ambivalens. We have detected 4THase activity in the strain SH cells grown on tetrathionate. The gene encoding 4THase (SH-tth) was isolated by colony hybridization from genome library of the strain SH. The deduced amino acid sequence without putative signal peptide consists of 480 amino acid residues with a predicted molecular mass of 51.9 kDa. The primary structure showed high similarity with the enzyme from A. caldus and A. ferrooxidans (85% and 75%, respectively). SH-tth was cloned and expressed in Ecsherichia coli cells. The recombinant SH-Tth was synthesized in inclusion bodies and no 4THase activity could be detected. However, the recombinant protein was successfully activated by application of our refolding method (kanao et al., 2010). The recombinant SH-Tth showed the maximum activity at pH 3.0. The activity of SH-Tth was enhanced up to 180% in the presence of NaCl. In contrast to SH-Tth, 4THase from A. ferrooxidans (Af-Tth) was significantly inhibited (less than 20%) by 0.5M NaCl. This halophilic property of SH-4THase implies the strain SH is marine bacterium.

References · K. Kamimura et al. (2003) Marine acidophilic sulfur-oxidizing bacterium requiring salts for the oxidation of reduced inorganic sulfur compounds. Extremophiles 7: 95-99. · T. Kanao et al. (2010) Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding. FEMS Microbiol Lett309: 43-47.

abstracts book 249 P117EXTREMOphiles_2012 Characterization of the extremophile homologs of mitoNEET Emi Hagiudaa, Yoshiharu Miyajima-Nakanoa, Daijiro Ohmorib, Teruo Kusanoa, Shinichi Matsushitaa, Sergei A. Dikanovc, Takashi Kumasakad and Toshio Iwasakia aDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan bDepartment of Chemistry, Juntendo University, Inba, Chiba 270-1695, Japan cDepartment of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA dJapan Synchrotron Radiation Research Institute (SPring-8/JASRI), Sayo, Hyogo 679-5198, Japan E-mail: [email protected]

The mammalian mitochondrial outer membrane protein, named MitoNEET, is a potential pharmacological and clinical target of the insulin-sensitizer pioglitazone (for the treatment of type 2 diabetes). Deficiency of mitoNEET expression in mice resulted in compromise in the respiratory capacity of cardiac mitochondria. Recent crystallographic studies on the recombinant soluble domain of human mitoNEET have revealed that it is a homodimer with each subunit binding a unique [2Fe–2S] cluster in a three-cysteine plus one-histidine ligand environment. The mitoNEET superfamily is widely distributed across various organisms from extremophiles to mammals, although the in vivo function of this new iron-sulfur protein superfamily remains elusive. Toward this goal, we have overproduced in Escherichia coli and characterized recombinant prototypal mitoNEET homologs from selected extremophiles, for which the whole genomic DNA sequences are known and the genetic manipulation systems have been established: (i) Cm-mitoNEET from a unicellular extremophilic red alga Cyanidioschyzon merolae that inhabits sulphate-rich hot springs (pH 1.5, 45°C) and contains only one each of mitochondrion and plastid; and (ii) TthNEET0026 from an extreme thermophileThermus thermophilus HB8, for which we determined the crystal structure at 1.8 Å resolution. Biochemical, spectroscopic and structural comparisons of these extremophile mitoNEET homologs with mammalian mitoNEET will be presented.

References

· T. Iwasaki et al. (2009). Continuous-wave and pulsed EPR characterization of the [2Fe-2S](Cys)3(His)1 cluster in rat mitoNEET. J Am Chem Soc 131: 13659-13667. · A. Kounosu et al. (2008). Crystallization and preliminary X-ray diffraction studies of the prototypal homologue of mitoNEET (Tth-NEET0026) from the extremophile Thermus thermophilus HB8. Acta Crystallogr Sect F 64: 1146-1148. · M. Matsuzaki et al. (2004). Genome sequence of the ultra small unicellular red alga Cynanidioschyzon merolae 10D. Nature 428: 653-657.

250 abstracts book EXTREMOphiles_2012P118 Structural basis for metallosulfur recognition of archaeal Rieske protein scaffold Toshio Iwasakia and Takashi Kumasakab aDepartment of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan bJapan Synchrotron Radiation Research Institute (SPring-8/JASRI), Sayo, Hyogo 679-5198, Japan E-mail: [email protected]

Proteins containing Rieske-type [2Fe-2S](His)2(Cys)2 clusters are involved in a wide range of biological electron transfer reactions such as aerobic respiration, photosynthesis, and biodegradation of various aromatic compounds. In contrast to regular plant-type and vertebrate-type [2Fe–2S] ferredoxins with complete cysteinyl ligations, the Rieske-type cluster has an asymmetric iron–sulfur core, with the Sg atom of each of the two cysteine residues coordinated to one iron site and the Nd atom of each of the two histidine residues coordinated to the other iron site. Our previous ligand-substitution studies on hyperthermophilic archaeal Rieske proteins as tractable models indicate the importance of the types and the spacing of ligands in the in vivo cluster-recognition/ insertion/assembly in metallosulfur protein scaffolds. A deeper understanding of the design of the metal-binding site and evolutionary divergency from the same protein template to promote biological functionalities requires structural information at atomic resolution. In this work we will present high-resolution X-ray crystal structures of the archaeal Rieske protein from Sulfolobus tokodaii named sulredoxin (SDX) and its site-directed variant: (i) native SDX (isolated from the archaeon) at 1.15-Å resolution, (ii) recombinant wild-type SDX (overproduced in Escherichia coli) at 1.7-2.5-Å resolution, and (iii) the SDX-triple variant with an engineered rubredoxin-like mononuclear Fe(Cys)4 site (by introduction of three-residue substitutions into theS. tokodaii SDX sequence) at 1.63-Å resolution. Notably, the recombinant wild-type SDX heterologously overproduced in E. coli was obtained as iron- and zinc-bound forms (unlike the native protein), and each representative structure was successfully determined. These structural data will be discussed with respect to the 3D recognition and assembly of a biological [2Fe-2S] cofactor into a Rieske iron-sulfur protein scaffold.

References · T. Iwasaki et al. (2005). Rational design of a mononuclear metal site into the archaeal Rieske-type ferredoxin scaffold. J Biol Chem 280: 9129-9134. · T. Iwasaki et al. (2006). Crystallization and preliminary X-ray diffraction studies of a hyperthermophilic archaeal Rieske protein variant (SDX-triple) with an engineeredrubredoxin-like mononuclear iron site. Acta Crystallogr. Sect. F 62: 993-995.

abstracts book 251 P119EXTREMOphiles_2012 Selective isotope labelling of extremophile metalloproteins Risako Fukazawaa, Myat T. Linb, Yoshiharu Miyajima-Nakanoa, Amgalanbaatar Baldansurenc, Shinichi Matsushitaa, Sylvia K. Choid, Sergei A. Dikanovc, Robert B. Gennisb,d and Toshio Iwasakia aDepartment of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan bDepartment of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA cDepartment of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA dCenter for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA E-mail: [email protected]

Amino acid-selective isotope labelling is an extremely powerful method to elucidate specific contributions of particular residues in the reaction mechanisms and/or folding of a target protein by vibrational (e.g., resonance Raman and FTIR) and magnetic resonance (e.g., NMR and pulsed EPR) spectroscopies. Such spectroscopic data can provide a deeper understanding of the dynamic aspect of a protein at work, often aided by the X-ray crystal structure which provides an important static overview in the lattice. Several methods have been developed for selective isotope labelling of proteins. One of the most convenient and cost-effective procedures is to employ amino acid auxotrophic bacteria as the host strains for the overproduction of target proteins. However, no suitable auxotrophic strains are commercially available for high-level expression of the foreign genes coding for metalloenzymes from extremophilic bacteria and archaea, because (i) their high-level expression, e.g., in Escherichia coli, often requires tRNA genes for the cognate rare codons and (ii) specific growth conditions must be set for effective overproduction of holoproteins in a form suitable for biophysical studies. To overcome these problems, we have reported the construction of a set of cost-effective, high-yield auxotrophs in commonly used E. coli expression strains C43(DE3) and BL21(DE3). In this work we adapted some of these strains by incorporation of tRNA genes for the E. coli rare codons and developed heterologous expression procedures suitable for amino acid-selective isotope labelling of simple iron-sulfur proteins from extremely thermophilic bacteria and archaea. It is demonstrated that the new auxotrophs can be used for high- level, heterologous expression of selectively15 N/13C-labelled, exteremophile metalloproteins. These strains will be made available to the scientific community through a public strain bank.

Reference · M. T. Lin et al. (2011). A rapid and robust method for selective isotope labeling of proteins. Methods 55: 370-378.

252 abstracts book EXTREMOphiles_2012P120 Active site structure of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) Kazuya Hasegawaa, Shinichi Matsushitab, Asako Konosub, Amgalanbaatar Baldansurenc, Risako Fukazawab, Sergei A. Dikanovc, Toshio Iwasakib and Takashi Kumasakaa

aJapan Synchrotron Radiation Research Institute (SPring-8/JASRI), Sayo, Hyogo 679-5198, Japan bDepartment of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan cDepartment of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61801, U.S.A. E-mail: [email protected]

Proteins containing Rieske-type [2Fe-2S](His)2(Cys)2 clusters are involved in a wide range of biological electron transfer reactions such as aerobic respiration, photosynthesis, and biodegradation of various aromatic compounds. The available crystal structures and spectroscopic analyses of Rieske-type proteins indicate that a lower potential Rieske-type [2Fe-2S] cluster tends to have less extensive hydrogen bonding network around the cluster. In our previous studies, we have established the heterologous overexpression system in Escherichia coli of an archaeal low-potential Rieske-type ferredoxin (ARF) fromSulfolobus solfataricus strain P1 with homology to bacterial oxygenase-associated Rieske-type ferredoxin. S. solfataricus ARF has served as a tractable model for ligand-substitution studies on this protein family. In this work, we will present the in-depth study of the active site structure ofS. solfataricus ARF probed by X-ray diffraction analysis at 1.85-Å resolution and by pulsed electron spin resonance technique in conjunction with site-specific stable amino acid isotope labelling. We will also report the effect of synchrotron high-flux

X-ray irradiation on the oxidation state of the archaeal Rieske-type [2Fe-2S](His)2(Cys)2 cluster reduction in ARF crystals. The active site structure of ARF will be compared with those of other mesophile Rieske-type ferredoxins reported so far.

References · T. Iwasaki et al. (2005). Rational design of a mononuclear metal site into the archaeal Rieske-type ferredoxin scaffold. J Biol Chem 280: 9129-9134. · T. Iwasaki et al. (2009). Two-dimensional pulsed electron spin resonance characterization of 15N-labeled archaeal Rieske-type ferredoxin. FEBS Lett 583: 3467-3472. · A. Konosu et al. (2010). Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1. Acta Crystallogr Sect F 66: 842-845.

abstracts book 253 P121EXTREMOphiles_2012 Engineering the enzyme LipBL from Marinobacter lipolyticus to identify the residues involved in the hydrolytic activity Dolores Péreza, Filip Kovacicb, Susanne Wilhelmb, Karl-Erich Jaegerb, María Teresa Garcíaa, Antonio Ventosaa and Encarnación Melladoa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain bInstitute for Molecular Enzyme Technology (IMET), Heinrich-Heine University, Düsseldorf, Forschungszentrum Jülich, Germany E-mail: [email protected]

The halophilic bacterium Marinobacter lipolyticus produces a lipolytic enzyme from the family VIII named LipBL (Pérez et al., 2011). Family VIII of lipolytic enzymes represents a group of enzymes with tempting properties. LipBL as most lipolytic enzymes from this family possesses two possible nucleophilic serines located in the S-X- X-K b-lactamase motif and the G-X-S-X-G lipase motif. Recent studies revealed serine in the S-X-X-K motif as the catalytic residue but the role of serine within the common lipase consensus sequence G-X-S-X-G was not yet systematically studied.

The heterologous co-expression of His6-tagged LipBL with the cytoplasmic molecular chaperones GroEL/ GroES and subsequent purification in presence of ATP were necessary to obtain the pure and catalytically active LipBL. Site directed mutagenesis studies performed to map the active site of LipBL revealed that the mutation of serine and lysine in b-lactamase motif (S72-M-T-K75) to alanine abolished enzymatic activity of LipBL in contrast to the mutation of serine in the lipase consensus motif (G-X-S321-X-G). Furthermore, mutagenesis studies were performed to understand the role of the G-X-S-X-G motif and other, among family VIII esterases, conserved amino acids. Mutations in conserved G-X-S-X-G motif altered the biochemical properties and the substrate specificity of LipBL. Molecular modeling results indicate the position of G-X-S321-X-G motif in a loop close to the catalytic center of LipBL, presumably representing a substrate binding site of LipBL.

Reference · D. Pérez, S. Martín, G. Fernández-Lorente, M. Filice, J.M. Guisán, Ventosa, A., García, M.T. & Mellado, E. (2011). A novel halophilic lipase, LipBL, with applications in synthesis of Eicosapentaenoic acid (EPA). PlosOne 6, e23325.

254 abstracts book EXTREMOphiles_2012P122 Optimization of tellurite reducing enzyme from moderately halophilic bacterium, Salinicoccus iraniensis Sana Alavia, Mohammad Ali Amoozegara and Khosro Khajehb a Extremophiles Laboratory, Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran bDepartment of Biochemistry, Faculty of Science, Tarbiat Modares University, Tehran, Iran E-mail: [email protected]

Salinicoccus iraniensis, a novel moderately halophilic, Gram-positive bacterium, was isolated from textile industry wastewater in Qom, Iran. The Strain was strictly aerobic, non-motile, non-sporulating and oxidase- and catalase-positive.Salinicoccus has the ability to tolerate high concentration of the toxic tellurium, tellurite 2- (TeO3 ). The MIC of this bacterium has been determined 12mM, which is high enough for bacteria to tolerate (Amoozegar et al., 2008). It was not only the high tolerance but also the ability of the strain to reduce tellurite into elemental form (black precipitation) that attracted us to understand the mechanism(s) by which organism do so. Therefore, it is going to be the first report on investigation of enzyme(s) responsible for tellurite reduction ability of a halophilic bacterial strain. In this report, the influence of different tellurite concentration on the bacterial growth and it‘s removal were measured under aerobic condition by colorimetric method using DDTC as described by Turneret al., 1992. The enzyme activity was measured spectrophotometrically at 340nm by following the oxidation of NADH. Tellurite reductase mixture consisted of 500µl Tris–HCl buffer (pH 8) contained 6mM potassium tellurite hydrate, 25µl of cell extract and 6µl NADH (10mM) as described by Etezad et al with some modification. Optimization of enzyme production by the strain was carried out with one factor at a time method (OFAT). Enzyme activity and bacterial growth were assayed. At the same time tellurite removal in that condition also has been determined. The factors which have been optimised were time (6-48h), primary inoculum concentration (1-10%), primary tellurite concentration (0.1-1mM), pH (6.5-10), temperature (5-45ºC), agitation (100-300rpm), different carbon and nitrogen sources, various salts concentration including NaCl, KCl, MgCl2, NaNO3, Na2SO4,

Na2SeO3, NaCH3COOH, Na2HPO4. The following are results arising from these experiments: Time 30h, Primary inoculum 3%, Tellurite concentration 0.1, pH 7.5, Different concentrations of salts; NaCl(15%), KCl(10%),

Na2SO4(10%), NaCH3COOH(15%), MgCl2(15%), NaNO3(5%). References · M. A. Amoozegar, P. Schumann, M. Hajighasemi, M. Ashengroph and M. R. Razavi (2008). Salinicoccus iranensis sp. nov., a novel moderate halophile. Int J Sys Evol Microbiol, 10.1099/ijs.0.65221-0. · S. M. Etezad, K. Khajeh, M. Soudi, P. Tajer Mohammad Ghazvini, B. Dabirmanesh (2009). Evidence on the presence of two distinct enzymes responsible for the reduction of selenate and tellurite inBacillus sp. STG-83. J Enz Mic Tec 2009.04.004. · M. Kabiri, M. A. Amoozegar, M.Tabebordbar, K. Gilany, G. H. Salekdeh (2009). Effects of selenite and tellurite on growth, physiology, and proteome of a moderately halophilic bacterium. J Proteome Res 8: 3098-3108. · R. Turner.; J. Weiner, D. E. Taylor, (1992). Use of diethylditiocarbamate for quantitative determination of tellurite uptake by bacteria. Anal Biochem 204: 292–295.

abstracts book 255 P123EXTREMOphiles_2012 The intriguing inositol-1-phosphate cytidylyltransferase activity of Rhodothermus marinus Carla D. Jorge, Nuno Borges and Helena Santos

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal E-mail: [email protected]

CDP-inositol is an intermediate in the synthesis of di-myo-inositol-1,3’-phosphate (DIP) and, thus far, no other function has been ascribed to this metabolite. DIP and derivatives accumulate in response to heat stress in organisms adapted to hot environments, especially in hyperthermophiles. The synthesis of DIP involves the consecutive actions of inositol-1-phosphate cytidylyltransferase (IPCT) and di-myo-inositol phosphate phosphate synthase (DIPPS). The first enzyme catalyses the activation myoof -inositol-1-phosphate into CDP-inositol at the expense of CTP, while the second activity adds another molecule ofmyo -inositol-1-phosphate to yield DIPP, the phosphorylated form of DIP. Dephosphorylation of this intermediate into DIP is catalysed by a yet unknown phosphatase. The genes encoding IPCT and DIPPS from several (hyper) thermophiles have been expressed in E. coli, and their products characterized (Rodrigues et al., 2007). Additionally, the evolutionary history of the DIP biosynthetic enzymes was studied [Gonçalveset al., 2012]. All organisms known to accumulate DIP possess homologues of IPCT and DIPPS genes, either separated or fused in a single gene product. Curiously, Rhodothermus marinus, a thermophilic bacterium that does not accumulate DIP, has a gene encoding a putative IPCT (33% of identity with known IPCTs). This gene is present in an operon-like structure together with putative CDP- alcohol phosphatidyltransferase andmyo -inositol-1-phosphate synthase. To gain insight into the physiological role of the putative IPCT and CDP-alcohol phosphatidyltransferase fromR. marinus, the respective genes were expressed in E. coli. We found that recombinant IPCT synthesises CDP-inositol using myo-inositol-1-phosphate and several nucleotide donors (CTP, ATP and UTP). Surprisingly, the enzyme also recognized glucose-6 phosphate or glucose-1-phosphate as substrates, yielding CDP-glucose. The CDP-alcohol phosphatidyltransferase did not show DIPPS activity. The presence of a CDP-inositol synthesising enzyme in an organism apparently unable to synthesise DIP is an intriguing result and suggests either the involvement of CDP-inositol in a yet unknown pathway or that the role of the enzyme is unrelated with the activation of inositol.

References · M. V. Rodrigues, N. Borges, M. Henriques, P. Lamosa, R. Ventura, C. Fernandes, N. Empadinhas, C. Maycock, M. S. da Costa and H. Santos (2007) Bifunctional CTP:inositol-1-phosphate cytidylyltransferase/CDP- inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper) thermophiles. J Bacteriol 189:5405-5412. · L.G. Gonçalves, N. Borges, F. Serra, P. L. Fernandes, H. Dopazo and H. Santos (2012). Evolution of the biosynthesis of di-myo-inositol phosphate, a marker of adaptation to hot marine environments. Environ Microbiol 14: 691-701.

256 abstracts book EXTREMOphiles_2012P124 Bacterial subfamily II isocitrate dehydrogenase: Catalytic and structural adaptations to low and high temperatures Anita-Elin Fedøya, Hanna-Kirsti S Leirosb, Ida H Steena,c and Nils-Kåre Birkelanda,c

aDepartment of Biology, University of Bergen, 5020 Bergen, Norway bThe Norwegian Structural Biology Centre, University of Tromsø, 9037 Tromsø, Norway cCentre for Geobiology, University of Bergen, N-5020 Bergen, Norway E-mail: [email protected]

In this study, isocitrate dehydrogenase (IDH) from the psychrophilic Desulfotalea psycrophila (DpIDH), the mesophilic Desulfitobacterium hafniesis (DhIDH), the thermophilic Clostridium thermocellum (CtIDH) and the hyperthermophilic Thermotoga maritima (TmIDH) are presented, all with primary sequence identity >60%. IDH was chosen as the target enzyme since it is ubiquitous in nature, due to its central role in the Krebs’ cycle and as an á-ketoglutarate producer for glutamate biosynthesis. Since IDH is expressed in organisms living at both high and low temperatures, we are able to identify structural determinants and relate them to stability, flexibility and activity. We determined the hyper- and thermophilicTm IDH and CtIDH to have an activity optimumT ( opt) at

90°C and 70°C, respectively, and an apparent melting temperature (Tm) of 98.3°C and 67.9°C, respectively. The quantitative structural analysis revealed stabilization of the N and C termini by long-range ionic interactions, together with a high number of ion-pars, a hydrophobic interior and many hydrogen bonds per residue, but the multi-member (>3) ionic networks are only found inTm IDH. The psychrophilic DpIDH has an unusual high

Tm of 66.9°C, with a Topt of 35°C. The active site of DpIDH was temperature sensitive, and the Km values for isocitrate were high at low temperatures and increased dramatically at elevated temperatures, probably due to a negatively area close to the active site and the overall negative charged enzyme. The high Tm was explained by stabilizing ionic interaction in the N and C termini and enlarged aromatic clusters, balanced with a strategic placement of a large methionine cluster in the hinge region and destabilizing amino acids close to the active site promoting flexibility to perform the catalysis at low temperature. Here, we present six crystal structures, an open TmIDH, an open and a locked structure of CtIDH, two closed and one locked structure of DpIDH. Each structure shows a snapshot of the movements during catalysis, representing a specific enzymatic state, and taken together they provide us enough information to deduce a catalytic mechanism.

References · M. Karlstrom, I. H. Steen, D. Madern, A. E. Fedoy, N.K. Birkeland, and R. Ladenstein (2006). The crystal structure of a hyperthermostable subfamily II isocitrate dehydrogenase from Thermotoga maritima. FEBS Journal 273: 2851-2868. · A. E. Fedoy, N. Yang, A. Martinez, H.K. Leiros, and I. H. Steen (2007). Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability. J Mol Biol 372: 130-149.

abstracts book 257 P125EXTREMOphiles_2012 Mineral respiration under extreme acidic conditions: from a supramolecular organization to a copper helper protein Magali Roger, Cindy Castelle, Frédéric Biaso, Marie-Thérèse Giudici-Orticoni and Marianne Ilbert

Laboratoire de Bioénergétique et Ingénierie des Protéines (BIP), IMM-CNRS-AMU, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France E-mail: [email protected]

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic gram-negative bacterium that derives energy from the oxidation of ferrous iron, elemental sulfur and various sulfur compounds at pH 2 using oxygen as electron acceptor. The study of this bacterium presents economic and fundamental biological interests because of its use in the industrial extraction of copper and uranium from ores, oxidation of metal sulfide A.by ferrooxidans enables the solubilisation of these precious metals. For this reason, its respiratory chain has been analyzed in details in recent years. Studies showed the presence of a functional supercomplex that spans the outer and the inner membranes and allows a direct electron transfer from the extracellular Fe(II) ions to the inner membrane cytochrome c oxidase (CcO) minimizing energetic losses. All proteins involved in this complex are involved in the electron pathway at the exception of AcoP, which was found tightly bound to CcO. AcoP, is a novel multifunctional copper-binding protein belonging to the cupredoxin family, with at least two roles (i) as a chaperon-like protein involved in the stability and activity of CcO under acidic conditions and (ii) as a linker between the cytochrome c and the CcO to improve electron transfer. The role, organisation and reactivity of AcoP’s copper are investigated using site-directed mutagenesis combined with functional and spectroscopic approaches. Preliminary results illustrate that AcoP might represent a new subfamily of cupredoxin.

258 abstracts book EXTREMOphiles_2012P126

Extracellular α-amylase from an isolated extreme halophilic archaeon Halorubrum xinjiangense Mohammad Ali Faramarzi and Mahsa Moshfegh

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy & Biotechnology research Centre, Tehran University of Medical Sciences, P. O. Box 14155-6451, Tehran 14174, Iran E-mail: [email protected]

Halophilic microorganisms from the Archaea domain (Haloarchaea) are the main source of extreme halophilic enzymes. As proteins and enzymes in haloarchaea are adapted to react in very high salt concentrations (3”5 M NaCl) they could be used in many harsh processes such as biotransformations and biosensors which require stable and active enzymes. α-Amylases are endohydrolases that catalyze the hydrolysis of α-1-4-glucosidic linkages in starch, glycogen and some related carbohydrates, producing glucose and oligosaccharides. Halophilic amylases are also able to degrade starch and some other carbohydrates residues in polluted hypersaline waters. In the present study, an extracellular amylase producing halophilic archaeon was isolated from the salty Lake of Orumieh located in northwest of Iran and identified as Halorubrum xinjiangense on the basis of 16s rDNA. The isolate was cultivated in 2L-Erlenmeyer flasks containing 500 ml of growth medium (250 g/L total salts, 5 g/L peptone and 10 g/L starch; pH 7.2) and shaken at 150 rpm for 2 days at 40°C. The supernatant was precipitated by gradually adding the pre-chilled ethanol up to 80% (v/v) saturation and stirred for 2 h at -20°C. The precipitant was collected by centrifugation at 16,000 g for 15 min and dissolved in a minimum volume of 20 mM Tris HCl (pH 8.0) containing 4 M NaCl and 50 mM CaCl2. The concentrated enzyme was dialyzed against the same buffer overnight and purified to an electrophoretically homogenous state by the affinity chromatography. The molecular mass of the purified enzyme was estimated to be about 60 KDa and demonstrated optimal activity at NaCl 4 M, 50 mM CaCl2,pH 8.0 and 60°C. The enzyme was active up to 70°C and pH 9.0 with 85.6% and 90% of the optimal activity. The purified enzyme was highly stable in the present of various organic solvents. These properties show the enzyme potential applications in starch-processing industries with high salt concentration, organic solvent and high temperature.

References · B. Van den Berg (2003). Extremophiles as a source for novel enzymes. Curr Opin Microbiol 6: 213-218. · M. Shafiei, A. A. Ziaee, M. A. Amoozegar (2011). Purification and characterization of an organic-solvent- tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F. J Ind Microbiol Biotechnol 38: 275-281. · M. Vidyasagar, S. Prakashs, C. Litchfield and K. Sreeramulu (2006). Purification and characterization of thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101. Archaea 2: 51-57.

abstracts book 259 P127EXTREMOphiles_2012 A new thermophilic microorganism from Deception Island, Antarctica: Isolation and characterization of a microorganism with biotechnological potential. Patricio A. Flores M.a,b and Jenny M. Blamey A.a,b

aFundación Científica y Cultural Biociencia, Santiago, Chile bUniversidad de Santiago de Chile, Santiago, Chile E-mail: [email protected]

Due to its intrinsic characteristics, the Antarctic continent is a source of extreme microorganisms. Millions of years of isolation have produced unique adaptive responses to its extreme environment. One of the greatest richesness of Antarctica is the existent biodiversity and the mechanisms of adaptation of its organisms to extreme conditions. Although the Antarctic climate is mainly cold, the presence of several geothermal sites in Antarctica, including thermal springs, fumaroles, hot soils and hydrothermal vents provides ideal environments for the development of thermophilic and hyperthermophilic microorganisms. Enzymes produced by (Hyper)thermophiles, called thermoenzymes, are interesting for both, academic and industrial research purposes, mainly for their high thermostability and thermophilia. (Li et al., 2005; Shirialdi et al., 2002; Kujo et al., 1998). Glutamate dehydrogenase (GDH) is an enzyme that plays a key role in the metabolism of carbon and nitrogen (DiRuggiero et al. 1995). It belongs to the family of oxidoreductases and it is widely distributed. It reversibly catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate and ammonium (Miñambres et al., 2000; Kujo et al., 1998). In this work, a new thermophilic microorganism has been isolated (PID15) from samples collected in the Antarctic Scientific Expedition ECA 46, from Deception Island. This microorganism grows optimally at 50°C and pH 8.0. Electron microscopy shows rod cells of 2 to 8 μm of length. Phylogenetic analysis of 16S rRNA gene revealed that this microorganism is closely related to Bacillus gelatini. This is the first time this thermophile has been found in Antarctica. From this new microorganism, using FPLC chromatography, GDH enzyme has been purified. Characterization of this enzyme including optimum temperature and pH as well as other important biochemical properties such as thermostability will be presented in this work.

Acknowledgements This work was supported by INNOVA-CORFO 07CN13PXT-64 and Instituto Antártico Chileno INACH

References · J. Diruggiero and F. Robb (1995). Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. Applied and Environmental Microbiology 61: 159–164. · C. Kujo and T. Ohshima (1998). Enzimological characteristics of the hyperthermoestable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents. Applied and Environmental Microbiology 64: 2152-2157. · W. Li, X. Zhou and P. Lu (2005). Structural features of thermozymes. Biotechnology Advances 23: 271-281. · B. Miñambres, E. R. Olivera, R. A. Jensen and J. M. Luengo (2000). A new class of glutamate dehydrogenases (GDH). The Journal of Biological Chemistry 50: 39529-39542. · C. Schiraldi and M. De Rosa (2002). The production of biocatalysts and biomolecules from extremophiles. Trends in Biotechnology 20: 515-521.

260 abstracts book EXTREMOphiles_2012P128 A heterotrimeric PCNA from Metallosphaera sedula Fumiya Iwataa, Hidehiko Hirakawaa and Teruyuki Nagamunea,b

aDepartment of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Japan bDepartment of Biotengineering, School of Engineering, University of Tokyo, Japan E-mail: [email protected]

Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein that serves as a platform for DNA-related enzymes in eukaryotes and archaea. PCNAs form homotrimers in eukaryotes and the most of archaea. However, it has been found that some chrenarchaea possess three PCNA genes. Although three PCNAs from chrenarchaeon Sulfolobus solfataricus (SsoPCNA) form a single SsoPCNA1-2-3 heterotrimer (Dionne et al., 2003), PCNAs from Sulfolobus tokodaii and Aeropyrum pernix form homotrimer as well as heterotrimer. Hyperthermoacidophilic archaeon Metallosphaera sedula belongs to the Sulfolobaceae family which contains S. solfataricus and S. tokodaii. M. sedula has three distinct PCNA genes (msed051, msed1792 and msed2250) never examined before. In this study, we expressed the three M. sedula PCNAs using Escherichia coli and demonstrated that the three PCNAs form a heterotrimer. First, amino acid sequences of MsePCNAs were compared to those of SsoPCNAs. Msed051 had 36.5, 16.0 and 16.4 % of identities withSso PCNA1, SsoPCNA2 and SsoPCNA3, respectively. Msed1792 and Msed2250 had the highest identities with SsoPCNA2 and SsoPCNA3, respectively. Thus, Msed051, Msed1792 and Msed2250 were termed MsePCNA1, MsePCNA2 and MsePCNA3, respectively. Next, the recombinant MsePCNAs were expressed in E.coli and purified. The size exclusion chromatography analysis revealed that MsePCNA1, MsePCNA2 and MsePCNA3 existed as monomer by themselves. MsePCNA3 did not interact with MsePCNA1 or MsePCNA2, while MsePCNA1 and MsePCNA2 formed a heterodimer. When the equimolar mixture of three MsePCNAs was subjected to size exclusion chromatography, the three proteins were co-eluted. This result indicated that MsePCNA1, MsePCNA2 and MsePCNA3 formed a heterotrimer. Finally, the pull-down assay was carried out. MsePCNA1 and MsePCNA2 pulled down each other, while MsePCNA3 did not pull down MsePCNA1 or MsePCNA2. However, MsePCNA3 pulled down the heterodimer of MsePCNA1 and MsePCNA2. Therefore, the three PCNAs from M.sedula form a heterotrimer in a step-wise manner; MsePCNA1 and MsePCNA2 form a heterodimer and then MsePCNA3 binds to the heterodimer.

References · I. Dionne, R. K. Nookala, S. P. Jackson, A. J. Doherty and S. D. Bell (2003). Mol Cell 11: 257-282.

abstracts book 261 P129EXTREMOphiles_2012 Extremophilic arsenite-oxidizing and arsenate- reducing bacteria L. Blameya, F. Sarmientoa,b, J. Pereiraa, C. Gaetea,cand J.M. Blameya,d

aFundación Científica y Cultural Biociencia, Chile bUniversity of Georgia, USA cUniversidad Andres Bello, Chile dDepartment of Chemistry and Biology. Universidad de Santiago, Chile E-mail: [email protected]

Arsenic is a metalloid, toxic to living cells. Redox reactions involving arsenic are of common occurrence and biogeochemically found in various environments. The release of arsenic into the environment by natural processes includes volcanic eruptions, erosion of rocks and anthropogenic processes. Pentavalent and trivalent arsenic are toxic to cells, being As+3 the most common oxidation state present in the environment. Arsenate -3 (As04 ) itself is an anionic specie with similar structure to phosphate capable of inhibit the phosphorylation - process generating a highly unstable arsenylated product (Gretchen et al., 1992). Arsenite (As02 ) is a toxic specie to cells due to its ability to bind sulfhydryl groups of proteins and dithiols such as glutaredoxin (Gretchenet al, 1972, Knowlesand et al., 1983). The oxidation of As (III) has been identified in several genera of bacteria includingPseudomonas, Alcaligenes, Thiomonas, Herminiimonas, Thermus and Acinetobacter (Oren et al., 2006). The biological oxidation of Arsenite [As (III)] to Arsenate [As (V)] is considered in many cases primarily a detoxification mechanism since As (V) is much less toxic than the As (III).The aerobic oxidation is catalyzed by arsenite oxidase (Aro), a member of the DMSOR family of molybdoenzymes. Here we report the isolation of a microorganism H3 and a stable consortia named MF1 from the Ascotán salt lake in the Atacama Desert. The microorganism, denominated H3, was identified as a member of the genus Bacillus, grows aerobically with an optimum temperature and pH of 37°C and 6,5. Additionally, this last microorganism is a moderate halophile, with an optimum growth at 2,5 M of NaCl (Linet al., 2006). MF1, was cultured aerobically at 20 °C and has an optimum pH of 6,6. The microorganism and consortia have heterotrophic growth at room temperature and both were able to reduce and oxidize arsenate or arsenite in a range from 10 to 30 mM. Arsenite oxidase and arsenate reductase were detected in both cultures MF1 and H3, when As III and As V was present in the growth medium. The activity of the enzymes was confirmed throught a qualitative assay and a zymogram. An arsenite oxidizing enzyme from MF1 was purified by anion exchange and molecular exclusion liquid chromatography, using an FPLC system. Additionally, Aro gene from this microorganism was amplified by PCR using a specific set of primers. Molecular weight of the protein was determined by gel electrophoresis. Additionally partial characterization of H3 enzymes is currently being performed. MF1 and H3 are potential producers of these enzymes with potential application in bioremediation processes.

References · L.A. Gretchen, W. Jeffrey and H. Russ (1992). The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum containing hydrxylase. J Biol Chem 267: 23674-23682. · F. W. Oehme, (1972) Toxicology.5: 215. · F. C. Knowlesand and A. A. Benson (1983).Trends Biochem Sci 8: 178-180. · C. Lin, L. Guanghui, C. Rensing and G. Wang (2009) BMC Microbiology, 9: 4. · A. Oren (2006). Life at high salt concentrations.Prokaryotes 2: 263-282.

262 abstracts book EXTREMOphiles_2012P130 Properties of 3-Isopropylmalate dehydrogenase from the deep-sea and non deep-sea Shewanella strains Yuki Hamajimaa, Takayuki Nagaeb, Nobuhisa Watanabeb, c, Chiaki Katod, Yasuyuki Kato-Yamadaa and Takeo Imaia aDepartment of Life Science, College of Science, Rikkyo University, 3-34-1 Nisiikebukuro, Tosima, Tokyo, Japan bDepartment of Biotechnology, Graduate School of Engineering cSynchrotron Radiation Research Center, Nagoya University, Furo-cho, Chikusa, Nagoya, Japan dInstitute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa, Japan E-mail: [email protected]

Deep-sea bacteria have various adaptations to the high-pressure and low temperature environment. One of the important adaptations is that such bacterial enzymes have high-pressure tolerance. 3-isopropylmalate dehydrogenase (IPMDH) is a key enzyme for leucine biosynthesis, so we focus on this enzyme to study the pressure adaptation mechanisms. We performed the extraction and purification of IPMDHs from atmospheric pressure adapted bacteria Shewanella oneidensis MR-1 and the psychrophilic and piezophilic deep-sea species Shewanella benthica DB21MT-2. These IPMDHs were compared on the activities under the high-pressure conditions. The results indicated that the activity of the enzyme from deep-sea strain DB21MT-2 was higher than that of the enzyme from strain MR-1 at 200 MPa. The enzyme of deep-sea strain was more tolerant under high-pressure conditions. In the next step, we are interested in what kind of the structural differences affect on the enzyme activity under high-pressure conditions. The chimeric enzymes were constructed using the IPMDH N-terminal side, middle part and C-terminal side from strains MR-1 and DB21MT-2, and measured the enzyme activities under pressure conditions. The results indicated that the middle part of the enzyme could be essential to keep the pressure tolerance. Moreover, it was generally believed that the heat stability of the enzyme co-related with the pressure tolerance. But our results suggested that the more heat stable enzyme in the chimeric enzymes could be less active under the high-pressure condition. In conclusion, the center domein of the IPMDH enzymes from the piezophilic strain is essential to keep the activity under higher-pressure conditions. At the same time, this domain also causes the low heat stability in deep- sea strains. Our data indicated the reverse correlations between heat stability and pressure tolelanceÿFurther studies are now in progress.

abstracts book 263 P131EXTREMOphiles_2012 Analysis of lipolytic enzyme genes from the bio-plastic degrading piezophilic Moritella sp. strain JT01 isolated from the Japan Trench Chiaki Katoa, Takayoshi Sekiguchia,b, Chinatsu Zamaa,b, Mona Windmeissera,c, Yuki Hamajimaa,d, Makiko Enokib and Haruyuki Kanehirob,e

aInstitute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan bDepartment of Marine Science, Tokyo University of Marine Science and Technology, Tokyo, Japan cInstitute for Technical Microbioligy, Technical University Hamburg-Harburg, Germany dDepartment of Life Science, RIKKYO University, Tokyo, Japan eOtsuma Women’s University, Tokyo, Japan E-mail: [email protected]

Moritella sp. strain JT01 was isolated from the Japan Trench at a depth of 5,356 m, which was able to grow optimally at 30 MPa and 5-8°C conditions (Sekiguchiet al., 2010a, Sekiguchi, et al., 2010b). This strain was also able to degrade some of bio-plastic materials (aliphatic polyesters), ca. polye -caprolactone (PCL), by using the lipolytic enzymes. Purpose of this study is to identify the lipolytic enzyme genes and analysis of the molecular mechanisms of their pressure adaptation. Shotgun cloning procedure was performed using Escherichia coli host-vector system, and lipolytic activity positive clones were screened on LB agar plate with 1% glycerol tributyrate and suitable antibiotics. Several positive clones were obtained, and finally about six different genes were identified. From the sequencing data, some of the genes were closely related with hypothetical genes reported from the genome analyses. Thus, it could be possible that some of the obtained genes might be novel lipolytic enzymes. In conclusion, we have succeeded to clone several lipolytic enzyme genes from deep-sea piezophilicMoritella sp. strain JT01, which was able to degrade the PCL materials. We are interested to analyse those enzymatic activity under different pressure conditions, so further studies are now in progress.

References · T. Sekiguchi, et al. (2010a). Isolation and characterization of biodegradable plastic degrading bacteria from deep-sea environments. JAMSTEC Rep Res Dev 11: 33-41. · T. Sekiguchi, et al. (2010b). Procedure for isolation of the plastic degrading piezophilic bacteria from deep- sea environments. J Jpn Soc Extremophiles 9: 25-30.

264 abstracts book EXTREMOphiles_2012P132 Screening of yeast from the semi-arid region of Bahia, with sucrase activity in aiming to use them as adjuvant therapy in sucrose intolerance Patricia Morais Lopes Pereiraa, Sandra Aparecida de Assisb and Elinalva Maciel Pauloa aUniversidade Estadual de Feira de Santana Biological Sciences (UEFS) 44.036.900 - Feira de Santana, Bahia, Brazil bUniversidade Estadual de Feira de Santana Health Sciences – UEFS, Bahia, Brazil E-mail: [email protected]

The semi-arid region of Bahia is characterized as an extremophile environment due to low rainfall with periods of drought and high temperatures. In this region there is a wide diversity of micro-organisms that hasn’t been explored yet, but with great expectation of biotechnology prospection. Yeasts are located in this scenario for being producers of different kinds of metabolites such as enzymes, dyes, and biopolymers. (Jayaniet al., 2005). Yeasts normally synthesize the sucrase-isomaltase, an enzyme that catalyzes the hydrolysis and fermentation of α-1 ,6-glucosides such as sucrose and isomaltose, respectively (Teste, et al, 2010). Its deficiency in the body causes the sucrose-isomaltose intolerance which is a congenital anomaly, which represents the second most common primary deficiency of disaccharides in humans with a prevalence between 0.1 and 0.5%. (Luiz, et al., 2005). There is no cure for this disorder, but it can be controlled by introducing the sucrase enzyme into diet, which may be capable of exerting an effect on the sucrose hydrolysis in the intestine of these deficiency patients, avoiding the effects of mal absorption and diarrhea. This work aimed to select yeast isolated from semi-arid region of Bahia, with significant production and activation of sucrase. In order to achieve it, we tested five enzyme extract (coded as E300, E306, E322, E327 and E33D). They were obtained from five different yeast strains, with the following results of enzymatic activity and specific activity: 0.05 / 1.24, 0, 01/0, 35, 0.14 / 3.15, 0.05 / 0.81, 0.19 / 5.31 for the respective enzyme extract. The chosen extract, E322, was obtained from the strain of the yeast Kluyveromyces marxianus. It has been performed kinetics of cellular production and kinetics of enzymatic production of this yeast. According to the results, it could be observed that its generation time is 4.30 hours and the growth rate 10.8 min-1, it also presented an optimum enzymatic activity at 24 hours and at the end of incubation time (40h) the maximum output of total protein content of 83.13 mg / mL. Based on these results we can infer the use of this yeast as probiotic to control and / or to minimize the harmful effects caused by deficiency of sucrase in individuals with this anomaly.

References · Jayani, et al. (1994). Microbial pectinolytic enzymes: review.Process Biochemistry 66: 1003-1010. · M.A Teste, J.M; François, J.L Parrou. (2010). Characterization of a new multigene family encoding isomaltases in the yeast Saccharomyces cerevisiae, the IMA family. Journal of Biological Chemistry 285: 26815-26824. · V. F. C Luiz et al. (2005).Terapia Nutricional nas Intolerâncias e Alergias Alimentares. The Electronic Journal of Pediatric Gastroenterology, Nutrition and Liver Diseases.

abstracts book 265 P133EXTREMOphiles_2012 Molecular cloning and characterization of Anoxybacillus flavithermus T1 esterase/lipase Laura Chis, Monica Hriscua, Gergely Ronab, Beata G. Vértessyb and Florin Dan Irimiea

aDepartment of Biochemistry and Biochemical Engineering, Faculty of Chemistry and Chemical Engineering, Babeº-Bolyai University Cluj-Napoca, 400028 Cluj-Napoca, Romania bInstitute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1113, Budapest, Hungary E-mail: [email protected]

The previously identified thermophileAnoxybacillus flavithermus strain T1 (GenBank ID: JQ267733), isolated from a hot spring in Tãºnad, Romania, produces an extracellular esterase/lipase (Est/Lip), which despite its low activity displays a remarkable thermostability in partially purified preparations. The gene encoding for Est/Lip was cloned by PCR amplification from genomic DNA. The protein, with predicted molecular weight of 28.03 kDa, is encoded by an open reading frame of 741 bps. The primers used were designed for periplasmic expression, and a pET 20 b(+) vector was employed. Expression was achieved in E. coli BL DE 3. The main protein in the periplasmic fraction displayed lipolytic activity and was purified on Ni-agarose. SDS-PAGE-estimated molecular weight of the purified Est/Lip was 25 kDa. The thermophilic enzyme was most active at 60-65°C and in the low alkaline pH range (7.5-8.5) and displayed a half-life at 60°C of about 5 hours. Stability at higher temperatures, as well as resistance to different organic solvents and metal ions were also investigated. The enzyme showed highest activity with short chain fatty acid p-nitrophenyl esters (acetate, propionate, butyrate), but displayed a certain degree of activity with long chain fatty acid esters (palmitate, oleate), which justifies its classification as an esterase/lipase.

Acknowledgements LC and MH acknowledge the financial support received through the Sectoral Operational Programme for Human Resources Development 2007-2013, co-financed by the European Social Fund, under the project numbers POSDRU/88/1.5/S/60185 «Investing in people!» and POSDRU 89/1.5/S/60189 «Postdoctoral Programs for Sustainable Development in a Knowledge-Based Society», respectively.

266 abstracts book EXTREMOphiles_2012P134 Physiological role of eicosapentaenoic acid in the cold- adaptation mechanism of an Antarctic bacterium, Shewanella livingstonensis Ac10 Jun Kawamoto, Nobuyoshi Esaki and Tatsuo Kurihara

Institute for Chemical Research, Kyoto University, Japan E-mail: [email protected]

Polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid, are known to have various beneficial effects on human health. PUFAs existing as acyl components of membrane phospholipids alter the physicochemical properties of lipid bilayers, including permeability, curvature, fluidity, and thickness, and affect the function of membrane proteins. However, molecular mechanisms how PUFAs influence the functions of membrane proteins are not well understood. A cold-adapted microorganism, Shewanella livingstonensis Ac10 isolated from Antarctic seawater, produces EPA as an acyl chain of its membrane phospholipids at 4°C. When EPA-biosynthesis genes were disrupted, the EPA-lacking mutant showed the growth retardation and formed filamentous cells at 4°C, but not at 18°C, suggesting that EPA-containing phospholipids have an important role in the cold adaptation of this bacterium. We also found that, in the EPA-lacking mutant grown at 4°C, the abnormal folding of a cold-inducible outer membrane protein, Omp74, was observed. In order to elucidate the physiological role of EPA at low temperatures, we performed in vitro reconstitution of recombinant Omp74 with the liposomes containing or not containing EPA and analyzed the effect of the presence of EPA on the folding of Omp74. The larger amounts of folded Omp74 were observed in the liposomes containing EPA than those without EPA. Circular dichroism analysis indicated that Omp74 rapidly interacts with the membrane surface and forms ß-sheet structures in the EPA-containing liposome at 4ÚC. We analyzed the protease sensitivity of reconstituted Omp74 with the liposomes containing or not containing EPA to estimate whether EPA affects on the minor structure of Omp74. When Omp74 was reconstituted with palmitoleic acid containing phospholipids, not containing EPA, the digested peptides contain the C-terminal fragment of this protein, but not with EPA-containing phospholipids, indicating that Omp74 changes the conformation of its C-terminal region depending on the presence of EPA-containing phospholipids. These results suggest that EPA-containing phospholipids accelerate the membrane insertion and folding of Omp74 and play a role as a molecular chaperone of Omp74 at low temperatures.

abstracts book 267 P135EXTREMOphiles_2012 Improvement of thermostability of a bacteriophage and a bacterium by heat-shock proteins Yun Jae Kim, Hyun Sook Lee, Jung-Hyun Lee and Sung Gyun Kang

Marine Biotechnology Research Center, Korea Ocean Research & Development Institute, Ansan, Korea E-mail: [email protected]

In generally, bacteriophages (T4 and lambda) and a bacterial strain (Escherichia coli) exhibit heat sensitivity. Heat-shock proteins including chaperone and protease are expressed when cells are exposed to heat or other environmental stresses. According to a recent report, a chaperone from hyperthermophilic archaea showed increased thermostability of E. coli proteins in vitro and also enhanced survivability of E. coli at high temperature. Based on the analysis of genome databases, we selected several heat-shock proteins; a Lon protease and a proteasome-activating nucleotidase (PAN) from Thermococcus onnurineus NA1, a small heat-shock protein (sHSP) from Pyrococcus sp. NA2, and a chaperonin from Pyrococcus furiosus DSM3638. In this study, we tested the effect of these heat-shock proteins on the thermostability of T4 phage and E. coli. In vitro studies revealed that the viability of the phage was enhanced in the presence of purified Lon or sHSP proteins at 65°C. In addition, recombinantE. coli strain which expressed a PAN or a chaperonin, maintained the survivability (60%) for 16 hours at 50°C. Interestingly, the recombinantE. coli to express a chaperonin could grow up to 46°C. These results show that heat-shock proteins could be applied to industrial fields that require the thermostability of phages and bacteria, such as phage therapy, food fermentation, and biotechnology.

268 abstracts book EXTREMOphiles_2012P136 Characterization of the glutamine synthetase GlnA2 of Halobacillus halophilus Anna Shyan, Melanie Thompson, Daniela Hartmann, Michaela Tausendschön, Inga Hänelt and Volker Müller

Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Goethe University, Frankfurt/ Main, Germany E-mail: [email protected]

Glutamine and glutamate are the major compatible solutes in the moderate halophileHalobacillus halophilus under moderate salinities (Saumet al., 2008). One of the isogenes/-enzymes of glutamine synthetase (glnA2) was shown based on in vivo experiments to be the osmoregulated, chlorid-stimulated key player in salinity- dependent glutamine and glutamate biosynthesis (Saum at al., 2006). Here, we investigated the purified GlnA2 protein showing that the enzyme itself unites the osmo- and chlorid-sensor, the regulator and the catalyser for glutamine and glutamate synthesis, independent of any other cellular component. The enzymatic activity as well as substrate specificity and affinity of the protein in dependence of different salinities and chlorid concentrations is presented. Based on these results we will discuss a regulatory mechanism on a molecular level.

References · S.H. Saum and V. Müller (2008). Regulation of osmoadaption in the moderate halophile Halobacillus halophilus: chloride, glutamate and switching osmolyte stategies. Saline Systems 4: 4. · S.H. Saum, S.F. Sydow, P. Palm, F. Pfeiffer, D. Oesterhelt and V. Müller (2006). Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus. J Bacteriol 188: 6808-6815.

abstracts book 269 P137EXTREMOphiles_2012 Thermal stability of a [2Fe-2S] ferredoxin from Cyanidioschyzon merolae can be modified by a single amino acid substitution Yuko Uenoa, Ayumi Sandob, Hiroaki Tokiwab, Yukio Morimotoc, Yasuyuki Kato-Yamadaa and Takeo Imaia

aDepartment of Life Science, College of Science, Rikkyo (St. Paul’s) University, Toshima-ku, Tokyo, 171-8501, Japan bDepartment of Chemistry, College of Science, Rikkyo (St. Paul’s) University, Toshima-ku, Tokyo, 171-8501, Japan cDivision of Quantum Beam Material Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka, 590-0494, Japan E-mail: [email protected]

Cyanidioschyzon merolae (Cm) is a single-cell red algae, isolated from acidic and moderately hot spring, having unique characteristics that hold single nucleus, plastid, mitochondrion, which can provides a model system for studying the origin of eukaryotic cells (Matsuzakiet al., 2004). Ferredoxin (Fd) is a well known iron- sulfur protein, serves as an intermediate electron carrier in diverse metabolic pathways. Even thermophilic and mesophilic homologous proteins have nearly identical sequence and overall structures, its thermal stability has remarkable difference. This is said to be implemented by a combination of subtle differences and various factors, for example, increased number of ionic interactions, increased extent of hydrophobic-surface burial, etc. However, it is still a matter of debate which factors are the main determinants for increasing the thermal stability. Here, we found a critical point that greatly affects the thermal stability of CmFd. By combining structural analysis and theoretical calculations, we propose a novel molecular strategy for thermal stability. CmFd has approximately 20°C higher thermal stability compared to Spinach Fd (Yamaoka et al., 2011), due to its optimum growth temperature. Comparison of these two Fd’s tertiary structures with other thermophilic Fd revealed that a hydrogen bond cluster specifically exists in thermophilic Fd. By producing several point mutants of CmFd, we found that Val57 plays an important role for protein thermal stability. By changing Val57 to Ala, the protein’s stability as measured by the electron transfer activity decreased about 10°C compared to the wild type. Even a single amino acid mutation greatly affected CmFd’s thermal stability. We succeeded in crystallizing this V57A mutant CmFd and ran a fragment molecular orbital (FMO)-interfragment interaction energy (IFIE) analysis. Comparison of the crystal structure and the interaction energies between the amino acid residues calculated from FMO-IFIE could be a new process to reveal a proteins strategy to gain thermostability.

References · G. Vogt et al. (1997). Protein thermal stability: hydrogen bonds or internal packing? Fold 2: S40-S46. · A. Yamaoka, Y. Ozawa, Y. Ueno et al. (2011).Cyanidioschyzon merolae ferredoxin: A high resolution crystal structure analysis and its thermal stability. FEBS Letters 585: 1299-1302. · M. Matsuzaki et al. (2004). Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon melolae 10D. Nature 428: 653-657.

270 abstracts book EXTREMOphiles_2012P138 Cold-active and alkali-stable proteases from bacteria isolated from the polyextreme ikaite columns in Greenland Jeanette Eva Lylloff, Mikkel Glaring and Peter Stougaard

University of Copenhagen, Department of Plant and Environmental Sciences, DK 1871 Frederiksberg C, Denmark E-mail: [email protected]

The ikaite columns found in Ikka Fjord in South Western Greenland constitute a very rare environment. The interior of the columns is cold (4°C), alkaline (pH 10.4), and display low salinity (0.9%) and harbor a microbial community adapted to this polyextreme environment. The aim of this study is to identify novel cold-active proteolytic enzymes from cold-adapted bacteria from the ikaite columns. Cultured bacteria were isolated directly from ikaite material on different types of media and screened for production of proteolytic enzymes. The genomes of two bacterial isolates were sequenced and genes predicted to code for proteolytic enzymes were identified by bioinformatics. Currently, selected protease-encoding genes are being cloned and recombinantly expressed. Proteolytic enzymes will be characterised in relation to industrial applications, e.g. substrate specificity and pH-, salt-, and temperature-stability and -optimum. Application of the cold-active proteases may lead to low temperature proceses, for example laundry processes, hydrolysis of food proteins, and production of bioactive peptides.

abstracts book 271 P139EXTREMOphiles_2012 Purification of ribosome from extremely halophilic archaeon Haloarcula japonica and RFHR 2D analyses Kaoru Nakasone and Fuminori Aono

Department of Chemistry and Biotechnology, Faculty of Engineering, Kinki University, 739-2116 Hiroshima, Japan E-mail: [email protected]

Extremely halophilic triangular-shaped archaeon Haloarcula japonica isolated from saltern field in Japan grows optimally with 20% NaCl. The «Salt-in» strategy by the extreme halophile to uptake high-concentration of salts such as KCl into the cell, gives balance between external and internal osmolarities. Thus, biochemical adaptation of halophilic proteins in high-salt conditions have been evolved. Proteins from halophile are stable and functional in high-concentrations of 2 – 3 M salts. In addition, almost halophilic proteins are acidic, because high-percentages of acidic amino acids such as glutamate and aspartate are contained in the proteins. Generally, ribosomal proteins are found as typical basic proteins, because of their interactions with several rRNAs, however acidic ribosomal proteins from the extreme halophile are observed, showing questions and mechanisms for molecular interactions between acidic proteins and nucleic acids (rRNAs) in high-salt conditions. It also suggests mysterious biochemistry under high salt conditions, because these decreases water activity required for biochemical reactions in extreme halophile. In this study, Isolation and separation pattern of ribosomal proteins from extremely halophilic archaeonH. japonica were carried out. Because traditional methodologies (two dimensional gel electrophoresis; 2-D) in analyses of ribosomal proteins has been optimized for many basic proteins, method of 2-D was tried to modify for acidic ribosomal proteins from the halophile. For 2-D analysis, RFHR (radical-free and highly reducing) method was used. The method is modified version of Kalschmidt-Wittmann (K-W) method and it is improved quantitativity and resolution in protein analyses. Ribosomal proteins H.of japonica were successfully isolated and analyzed using the RFHR method. Isolation of the ribosomal proteins and improvements to analyze acidic proteins using the RFHR method will be reported and discussed.

272 abstracts book EXTREMOphiles_2012P140 Extremophilic enzymatic response for protection against UV-radiation damage Jenny M. Blameya,b, Bernardita Chirinoa, María Teresa Monsalvesa, Freddy Boehmwalda

aFundación Científica y Cultural Biociencia, Santiago, 7750132 Chile bDepartment of Chemistry and Biology, Universidad de Santiago, Santiago, Chile E-mail: [email protected]

Ultraviolet (UV) radiation severely harms biological systems, producing DNA damage and detrimental changes in the molecular structure of the cellular metabolic machinery. Exposing cells to radiation generates a range of harmful molecules called reactive oxygen species (ROS). The cells employ a series of enzymatic protective mechanism to defend themselves against these highly reactive species. Extremophiles in their natural environments are exposed to several conditions that favour ROS generation; however these microorganisms have enzymatic systems that can counteract this effect. Two important enzymes involved in this defense mechanism are catalase (KAT) and superoxide dismutase (SOD). To study these enzymes, we selected two novel radiation-resistant extremophiles: a thermophilic bacterium belonging to the genusGeobacillus isolated from a sterilization oven GWE1 and a psychrophilic bacterium isolated from Antarctica. In order to study protection effects of sod gene on a mesophilic microorganism, sod gene was selected to be incorporated into Escherichia coli. The sod gene from GWE1 was successfully cloned into E. coli obtaining the complete gene sequence. By bioinformatics analysis we predicted the three dimensional structure of SOD protein. In addition, we were able to functionally expresssod gen into E. coli. Furthermore, SOD recombinant protein was purified and biochemical analysis was performed. Activity, thermostability and optimal pH of the recombinant enzyme were compared to the native one. Through this work insertion and expression of an extremophilic sod gene into a mesophilic organism has been successfully achieved.

Acknowledgements AFOSR, Air Force Office for Scientific Research, USA, Grant Nº FA9550-09-1-0349

Reference · Z. Yuanyuan and et al (2011). Identification of three superoxide dismutase genes from aGeobacillus sp. Protein J 30: 66-71. · Y.Z. He and et al. (2007). Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring. Appl Microbiol Biotechnol 75: 367-376. · J. Sambrook and D.W. Russell (2002). Molecular cloning: a laboratory manual. In: Huang PT (ed). Science Press, Beijing, pp 1228-1232.

abstracts book 273 P141EXTREMOphiles_2012 A novel compensatory splicing mechanism for the Thermococcus kodakarensis Tko CDC21-1 intein without the highly conserved histidine Kazuo Toriab, Manoj Cheriyanb, Chandra Sekhar Pedamalluc, Marleny A. Contrerasb and Francine B. Perlerb

aLaboratory for Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka, 812-8581, Japan bNew England BioLabs, Ipswich, MA, 01938, USA cDana-Farber Cancer Institute, Boston, MA, 02115, USA E-mail: [email protected]

Inteins and other self-cleavage enzymes initiate autocleavage by converting a peptide bond to a (thio)ester bond when a Ser, Thr or Cys undergoes a N-[S/O] acyl migration assisted by residues within the precursor protein. Histidine in the conserved Block B motif of intein is an essential assisting residue for the initial acyl migration. This His is the most highly conserved intein residue and is present in almost all inteins. Previous studies identified polymorphic inteins that lack conserved catalytic or assisting residues, which compensate for the missing residue(s) by either using other amino acids to perform the same catalytic reaction or to perform novel, modified protein splicing reactions. It was found thatThermococcus kodakarensis CDC21-1 possessed the intein without the conserved histidine residue, thus the activity and function of this intein were investigated. The study on this intein demonstrated that the Tko CDC21-1 intein was fully active despite the absence of this conserved His. Also, it was found that a conserved ortholog-specific basic residue (Lys58), which is present outside the standard intein motifs, compensated for the role of His normally present within the Block B. We propose a novel compensatory mechanism, in which Lys58 catalyzes the initial N-S acyl migration by stabilizing the thiazolidine-tetrahedral intermediate, allowing it to be resolved by water-mediated hydrolysis rather than by protonating the leaving group as His is theorized to do in many other inteins. Autoprocessing enzymes may have more flexibility in evolving catalytic variations, since high reaction rates are not required when performing single turnover reactions on ‘substrates’ that are covalently attached to the enzyme. Consequently, it is expecting that inteins have more flexibility to sample catalytic mechanisms, which should provide insight into various strategies that enzymes use to accomplish catalysis.

Reference · Tori K, et al. (2012). The Thermococcus kodakaraensis Tko CDC21-1 intein activates its N-terminal splice junction in the absence of a conserved histidine by a compensatory mechanism. Biochemistry 50: 2496-505.

274 abstracts book EXTREMOphiles_2012OL1

session 5 genetics and gene expression

abstracts book 275 OL1EXTREMOphiles_2012

276 abstracts book EXTREMOphiles_2012P142 Differential utilization of multiple Orc/Cdc6- determined replication origins govern polyploidy in Haloarcula hispanica Zhenfang Wu, Jingfang Liu, Hailong Liu, Xiaoqing Liu and Hua Xiang

Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, P. R. China E-mail: [email protected]

Multiple replication origins have been mapped in several model archaea, while little is known about their utilization in vivo, in particular what roles they play in regulating polyploidy in haloarchaea. We have previously combined bioinformatic and genetic approaches to identify DNA replication originsHaloarcula in hispanica, and investigated the diversity and evolution of multiple replication origins in haloarchaea. Here, knockout analysis suggested that one functional origin per replicon was necessary for cell viability. Genetic studies suggested that characteristic diversity of the five functional replication origins, which were discriminated by means of proximity of distinctorc/cdc6 genes to their specific origins, is indicative of diverse Orc/Cdc6-determined replication origins in H. hispanica. Surprisingly, ARS plasmids with the two origins from mini-chromosome were significantly less stable than those with other origins, which might be relative to the poor stability of this mini-replicon. We delineated the cis elements at oriC1-cdc6A, and find that haloarchaea-specific «G-string» located at the end of ORB sequences is essential for ARS activity, and a pair of inverted G-rich region proximal to ORB sequences function as an enhancer for ARS activity. We employed genome-wide marker frequency analysis (MFA) to map replication origin activationin vivo in H. hispanica, finding that replication of chromosome was initiated from two separate origins, oriC1-cdc6A and oriC2-cdc6E. In particular, differential usage of the origins, even those in the same replicon, was observed in vivo. These findings not only highlighted mechanism(s) of replication initiation of polyploidy, but also demonstrated their roles in coordinating copy number of multi-replicons inH. hispanica.

abstracts book 277 P143EXTREMOphiles_2012 Transcriptional and functional analyses of Cas/CRISPR system in Haloferax mediterranei Ming Li, Zhenfang Wu, Hailong Liu, Jing Han and Hua Xiang

Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, P. R. China E-mail: [email protected]

The recently discovered CRISPR/Cas system has proved to be a prokaryotic antiviral machinery and probably involved in other functions (such as DNA repair). CRIPSR arrays coupled with Hmari-subtypecas genes are widely and nearly exclusively present in haloarchaeal species. Here, we dissected transcriptional elements from their unique GC-rich leader sequences, as well as the cis- and trans- elements involved in the pre-crRNA processing pathway, that is, the conserved cleavage site within repeat sequences and the catalytic Cas proteins. Additionally, we discovered a provirus seemed to be more active and prone to excise itself from the chromosome when some cas genes, but not cas3, were individually knocked out. Thus, this result indicated the presence of an unknown mechanism other than the newly characterized targeting pathway featured by Cas3, and the Hmari-subtype cas genes may be involved in the genome stability.

278 abstracts book EXTREMOphiles_2012P144 Identification and characterization of the function and regulation of a sugar phosphotransferase system gene cluster in Haloferax mediterranei Lei Cai, Shuangfeng Cai, Xiaoqing Liu, Dahe Zhao, Jian Zhou and Hua Xiang

Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, P. R. China E-mail: [email protected]

Sugar phosphotransferase system (PTS) transports sugars into cells using phosphoenolpyruvate (PEP) as the phosphoryl donor to phosphorylate sugars, which is widely spread among bacteria. Typical PTS contains five proteins, PtsI, Hpr, PtsA, PtsB, and PtsC. Analysis of 17 haloarchaeal genomes revealed that 6 haloarchaeal strains encode all of the five PTS-related proteins. In the genome ofH. mediterranei, these five genes are clustered and co-transcribed. The transcription start site of this pts-gene cluster is located 17 bp upstream of the ptsC start codon. The PTS knockout mutant can hardly utilize fructose as the sole carbon to grow up. RT-PCR, microarray analysis and a modified GFP reporter system indicated that this PTS gene cluster was significantly induced by fructose and F-1-P, whereas F-1,6-2P repressed this fructose induction. Detecting the PTS promoter activity of the 5’ flanking deletion mutants and site-direct mutantsvia the reporter system assay revealed that two regions of the PTS promoter, from –45 to -59 and -69 to -74, are related to the fructose induction. In silico analysis of the promoters of PTS gene cluster in those 6 haloarchaeal strains showed a conserved DNA sequence pattern, including these two regions and a predicted TATA box and BRE. Besides, a gene named deoR2 encoding a putative DeoR family transcriptional regulator, located upstream of the PTS gene cluster, was found to be involved in the regulation of this pts-gene cluster in H. mediterranei.

abstracts book 279 P145EXTREMOphiles_2012 Identification and characterization of the cognate anti-sigma factor and specific promoter elements of a Thermoanaerobacter tengcongensis ECF sigma factor Jingfang Liu, Jie Li, Zhenfang Wu, Huadong Pei, Jian Zhou and Hua Xiang

Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, P. R. China E-mail: [email protected]

Extracytoplasmic function (ECF) σ factors, the largest group of alternative σ factors, play important roles in response to environmental stresses, such as periplasmic stress, heat shock response, iron transport etc. Thermoanaerobacter tengcongensis belonging to the phylum Firmicutes, is an anaerobic, rod shaped, and low G+C content (33%) thermophilic bacterium, which was isolated from a freshwater hot spring in China and grows between 50-80°C, with an optimum temperature of approximately 75°C. The genome has been completely sequenced around 2000, in which, Tt-RpoE1 is annotated as an ECF σ factor. In this study, we revealed that the Tt-tolB gene located downstream of the Tt-rpoE1 gene encoded the cognate anti-σ factor, which could inhibit the transcription activity of Tt-RpoE1 by direct interaction with Tt-RpoE1 via its N-terminal domain. By in vitro transcription assay, the auto-regulation ability of Tt-RpoE1 was determined, and band shift assay showed that Tt-RpoE1 preferred to bind a fork-junction promoter DNA. With truncation or base-specific scanning mutations, the contribution of the nucleotides in -35 and -10 regions to interaction between Tt-RpoE1 and promoter DNA was explored by band shift assay, and the promoter recognition pattern of Tt-RpoE1 was determined as 52 tGTTACN16CGTC 32 , which was further confirmed byin vitro transcription assays. This result showed that the Tt-RpoE1-recognized promoter possessed a -10 motif (-13CGTC-10) as the specific recognition determinant. Site-directed mutagenesis in Region 2.4 of Tt-RpoE1 indicated that the «D» residue of DXXR motif was responsible for recognizing the -12G nucleotide. Our results suggested that distinct -10 motif may be an efficient and general strategy used by ECF σ factors in adaptive response regulation of the related genes.

280 abstracts book EXTREMOphiles_2012P146 Novel targets and cofactors for LysM from Sulfolobus solfataricus, a transcriptional regulator of the Lrp-family Ningning Songa , Trong Nguyen Ducb,c, Liesbeth van Oeffelena, Serge Muyldermansb,c, Eveline Peetersa and Daniel Charliera

aResearch group of Microbiology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium bLaboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium cDepartment of Structural Biology, VIB, Brussels, Belgium E-mail: [email protected]

LysM is an Lrp-like transcriptional regulator from S. solfataricus that was previously shown to bind to the control region of the lysWXJK operon, and was proposed to act as a leucine-sensitive co-activator for thelysW promoter, the sole known target of LysM (Brinkman et al., 2002). To identify LysM binding sites on the genome scale we have performed LysM-specific nanobody-based ChIP- chip experiments with S. solfataricus cells grown with and without L-lysine. In total the data revealed 80 ChIP enriched regions (chers; at a cut-off = 2-fold enrichment), 42 of which are common in both growth conditions. In vitro binding assays with a selected subset of chers (EMSA, footprinting) and in silico prediction of binding sites with a position weight matrix derived from a saturation mutagenesis of one half of the symmetrical consensus LysM box were used to validate the data. High affinity targets were found in both intergenic control regions and in ORF’s, and both classes comprise sites that are efficiently bound in vitro and in vivo, whereas others are only bound efficientlyin vivo. In vitro transcription and in vitro binding assays performed with LysM in combination with the general transcription factors TBP and TFB and the RNA polymerase indicate that LysM significantly stimulates transcription initiation by promoting the binding of TBP/TFB. Therefore, LysM is an activator in its own right. The identification of novel targets for LysM involved in various amino acid biosynthetic pathways prompted us to re-analyze the cofactor specificity of the regulator. Previously it was shown that L-lysine reduces but does not completely abolish LysM binding to the lysW operator (Brinkman et al., 2002). We confirmed that L-lysine (but also D-lysine) is the major effector molecule of LysM, but that arginine, homoarginine, glutamine, leucine, isoleucine, valine and methionine also exert a significant negative effect on LysM-operator DNA binding.

References Brinkman, A.R., Bell, S.D., Lebbink, R.J., de Vos, W.M, van der Oost, J. (2002). The Sulfolobus solfataricus Lrp- like protein LysM regulates lysine biosynthesis in response to lysine availability. J Biol Chem 277: 29537-29549.

abstracts book 281 P147EXTREMOphiles_2012 Involvement of universally conserved genes, ygjD and yeaZ orthologs, in DNA repair of Deinococcus radiodurans Takefumi Onoderaa,b,c, Katsuya Satoha, Toshihiro Ohtab and Issay Narumia

aIon Beam mutagenesis Research Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, Takasaki, Gunma 370-1292, Japan bLaboratory of Environmental Molecular Biology, Graduate School of Life Sciences, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan cResearch Fellow of the Japan Society for the Promotion of Science, Tokyo 102-8472, Japan E-mail: [email protected]

The ygjD and yeaZ orthologs are highly conserved in eubacteria, and the former is also widely found in the genomes of archaea and eukaryotes. It was recently reported that these orthologs were related to DNA metabolism in Escherichia coli (Handford et al., 2009), mitochondrial DNA maintenance and transcriptional regulation in Saccharomyces cerevisiae (Oberto et al., 2009). However, its actual function is still poorly understood. Deinococcus radiodurans, well known as a radioresistant bacterium, possesses the both orthologs, hereafterDrygjD and DryeaZ. In this study, DrygjD and DryeaZ deletion mutants were generated and challenged to various DNA damaging agents for investigation of gene disruption effect inD. radiodurans. DrygjD and DryeaZ deletion mutants were generated by targeted gene replacement. The deletion mutants were treated with mitomycin C (MMC), cisplatin, ultraviolet (UV), gamma rays, hydrogen peroxide and N-methyl- N’-nitro-N-nitrosoguanidine (MNNG). After treatments, cells were diluted with phosphate buffer, dropped onto agar medium and incubated at 30°C for 3 days prior to the enumeration of colonies. Reduction in viability after exposure to cisplatin, UV, gamma rays, hydrogen peroxide and MNNG was marginal in the deletion mutants compared to the wild-type strain. Meanwhile, the deletion mutants exhibited extreme sensitivity to MMC, which mainly causes interstrand DNA crosslinking. These results suggest that DrygjD and DryeaZ are involved in DNA repair and namely play a critical role in the interstrand DNA crosslinking damage repair in D. radiodurans. The involvement of DrygjD and DryeaZ in forward mutations at the rpoB locus will also be discussed.

References · J. I. Handford et al. (2009). Conserved network of proteins essential for bacterial viability. J Bacteriol 191: 4732-4749. · J. Oberto et al. (2009). Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance. Nucleic Acids Res 37: 5343-5352.

282 abstracts book EXTREMOphiles_2012P148 Functional analysis of multiple general transcription factors in Sulfolobus acidocaldarius Bernadette Rauch and Bettina Siebers

Molecular Enzyme Technology and Biochemistry, Biofilm Centre, University Duisburg-Essen, Germany E-mail: [email protected]

Archaea exhibit unique features as well as share characteristics with Bacteria and Eukaryotes. Archaeal cells have a bacterial morphology, they lack a nucleus, possess a relatively small circular chromosome and genes are organized in operon structures. In contrast to that, information processing (e.g. replication, transcription and translation) resembles respective eukaryal processes. The archaeal transcription machinery encompasses one multi-subunit RNA-Polymerase (RNAP), resembling the RNAP II of Eukarya, homologues of the TATA-binding protein (TBP), transcription factor TFIIB (TFB) and TFIIEá (TFE). RNAP, TBP and TFB are solely sufficient for transcription of archaeal promoters in vitro (Bell et al., 2001a; 2001b; Geiduschek & Ouhammouch, 2005). Therefore archaeal transcription is generally regarded as a simpler model of the more complex eukaryal processes. Interestingly, Archaea possess multiple copies of general transcription factors (GTFs) however, the distribution is species-dependent. Whereas the function of multiple GTFs has been addressed in different Euryarchaeota (e.g. P. furiosus, Halobacterium NRC-1) the role in Crenarchaeota is still unclear, however, a function comparable to bacterial σ-factors has been predicted. The current in vivo and in vitro studies focus on the thermoacidophilic Crenarchaeote Sulfolobus acidocaldarius (80°C, pH 2-3). S. acidocaldarius is the emerging model organism within the Archaea, since it is easy to grow and one of the few genetically tractable Archaea. This organism encodes one TBP and three TFBs. TFB1 and TFB2 are full-length proteins with an N-terminal Zn-domain and a C-terminal imperfect direct repeat. TFB3 is only half-size and lacks the B-finger and DNA-binding domain (Bell et al., 2001a). TFB1 was shown to be the typical TFIIB homologue in vitro and TFB3 was highly upregulated in microarray studies following UV- exposure and acts as a co-activator in the presence of TFB1 (Götzet al., Paytubi & White, 2009) whereas TFB2 has not been characterized so far. First insights into the function of multiple TFBs will be presented.

References · Bell SD, Magill CP, Jackson SP. (2001a). Basal and regulated transcription in Archaea. Biochem Soc Trans 29: 392-395. · Bell SD, Brinkman AB, van der Oost J, Jackson SP. (2001b). The archaeal TFIIEalpha homologue facilitates transcription initiation by enhancing TATA-box recognition. EMBO Rep 2:133-138. · Geiduschek EP and Ouhammouch M. (2005). Archaeal transcription and its regulators.Mol Microbiol 56: 1397-1407. · Götz D, Paytubi S, Munro S, Lundgren M, Bernander R, White MF. Responses of hyperthermophilic crenarchaea to UV irradiation.Genome Biol 8: R220. · Paytubi and White (2009), The crenarchaeal DNA damage-inducible transcription factor B paralogue TFB3 is a general activator of transcription. Mol Microbiol 72: 1487-1499.

abstracts book 283 P149EXTREMOphiles_2012 Involvement of two putative cyclopropanic fatty acid synthases in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens Francine Piubelia, Aggeliki Katsifab, Montserrat Argandoñaa, Joaquín J. Nietoa, Manuel Salvadora, Rosa García-Valeroa, Anna I. Koukkouc and Carmen Vargasa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain bInstitute of Immunology, Biological Sciences Research Center ‘Alexander Fleming’, Athens, Greece cSector of Organic Chemistry and Biochemistry, Department of Chemistry,University of Ioaninna, Greece E-mail: [email protected]

Chromohalobacter salexigens is a halophilic bacterium with an extremely broad salinity range for growth. Its two main osmoadaptation mechanisms are the synthesis of compatible solutes (ectoine and hydroxyectoine) and the adaptation of membrane composition. Expression of the ectoine synthesis genes is partially dependent on the the general stress factor sS (Calderón et. al, 2004). In a previous work, we showed that under high salinity conditions, C. salexigens membrane lipids contained a higher percentage of cyclopropanic fatty acids (CFA), whose synthesis is catalysed by the enzyme CFA synthase. The content of CFA was lower in an ectoine-reduced mutant than in the wild type, but it was restored to wild type levels after addition of exogenous ectoine. These findings suggested a connection between both osmostress responses in C. salexigens (Vargas et al., 2008). In this work we have investigated the transcriptional regulation of CFA synthesis C.in salexigens. The in silico analysis showed two putative cfa gene homologues (cfa1, cfa2) within the C. salexigens genome sequence. Transcriptional fusions of promoters of bothcfa genes (Pcfa1 and Pcfa2) and the reporter gene gfp were analyzed in the wild type strain at different salinities. ActivitiesPcfa1 of and Pcfa2 were growth-dependent, and displayed their highest values at late exponential or early stationary phase of growth. Second, we determined whether sS is involved in the regulation ofcfa transcription. For this purpose, we measuredPcfa1 and Pcfa2 expression in an rpoS mutant background (sS), and compared it with the wild-type strain. This was performed at different salinities and in the absence and presence of exogenously added ectoine. In absolute values, Pcfa1 expression was higher than Pcfa2 expression at any salinity tested. Interestingly, Pcfa1 expression was highest at low salinity (0.75 M NaCl), whereas Pcfa2 expression was osmoregulated, showing its highest values at 2.5 M NaCl. It was also showed that, under conditions of high salinity, sS is involved in the down- and up-regulation ofcfa1 and cfa2 transcription, respectively. Besides, an ectoine-induced upregulation observed incfa2 was shown to be rpoS dependent. Analysis of CFA activity incfa1 (cfa1::W) and cfa2 (cfa2::Km) single mutants and cfa1cfa2 (cfa1::W cfa2::Km) double mutant were also developed to rule out the involvement of both CFA synthases in membrane osmoadaptation.

References · M.I, Calderón, C. Vargas, F. Rojo, F. Iglesias-Guerra, L.N, Csonka, A. Ventosa, J.J. Nieto (2004). Microbiology 150: 3051-3063. · C. Vargas, A. Kallimanis, A.I, Koukkou, M.I Calderón MI, D. Canovas, F. Iglesias-Guerra, C. Drainas, A. Ventosa, J.J. Nieto (2005). Syst Appl Microbiol 28: 571-581. This research was financially supported by grants from the Spanish Ministerio de Ciencia e Innovación (BIO2011-22833), and Junta de Andalucía (P08-CVI-03724).

284 abstracts book EXTREMOphiles_2012P150 Molecular characterization of a hybrid histidine- kinase involved in osmoadaptation in the halophilic bacterium Chromohalobacter salexigens Carmen Vargas, Rosa García-Valero, Montserrat Argandoña, Manuel Salvador, Francine Piubeli and Joaquín J. Nieto

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain E-mail: [email protected]

Chromohalobacter salexigens is a moderately halophilic bacterium able to grow in a wide range of salinities (0.5 to 3 M NaCl in minimal medium) and up to 45 ºC. Its main osmo- and thermo- adaptation strategy is the cytoplasmic accumulation of the biostabilizers ectoine and hydroxyectoine, two compounds with great industrial interest. In order to coordinate its processes of osmo and thermoregulation, cells are equipped with different systems and mechanisms of sensing physical stimuli correlated to changes in the external medium (sensing) and pathways to transduce these stimuli into useful signals which can be processed in the cell (signal transduction). These different signal transduction pathways have not yet well been elucidated in halophilic bacteria. We have previously reported the first example of the involvement of a two-component response regulator, EupR, in the osmoadaptation of a true halophilic bacterium (Rodriguez-Moyaet. al., 2010). This regulator was implicated in the transcriptional regulation of ectoine(s) transport and its disruption led to an osmosensitive phenotype. Its putative cognate histidine kinase (Csal0869) was predicted to be a hybrid histidine kinase with both transmembrane and cytoplasmic sensor domains, suggesting that it could sense both external and internal conditions. In this work, we have molecular characterized this hybrid histidine kinase. By using RT-PCR, it was found that csal869 is not co-transcribed with its adjacent genes, forming a single transcriptional unit. A knock out of this hybrid histidine kinase was also constructed by an insertion of a Km cassette (csal0869::Km), whose preliminary phenotypic characterization will be shown. Anin silico functional analysis of the 21 histidine kinases present inC. salexigens genome and its associated putative response regulators is also presented as a preliminary description of the two-component systems occurring in C. salexigens. These data pave the way to a further elucidation of the signal transduction pathways involved in the osmostress responses inC. salexigens.

References · J. Rodriguez-Moya, M., Argandoña, M., Reina-Bueno, J.J., Nieto, F., Iglesias-Guerra, M., Jebbar and, C., Vargas, (2010). BMC Microbiol 10: 256. This research was financially supported by grants from the Spanish Ministerio de Ciencia e Innovación (BIO2011-22833), and Junta de Andalucía (P08-CVI-03724).

abstracts book 285 P151EXTREMOphiles_2012 Molecular characterization of the uptake of ectoines in Chromohalobacter salexigens J. Nietoa, Javier Rodríguez-Moyaa, Montserrat Argandoñaa, Fernando Iglesias-Guerra b and Carmen Vargas a

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain bDepartment of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seville, Spain E-mail: [email protected]

Ectoines (ectoine and hidroxiectoine) are organic compatible solutes that are accumulated in the cytoplasm of halophilic bacteria in response to osmotic and heat stress. They have important biotechnological applications as biostabilizers of molecules, membranes and whole cells. It is commonly accepted that uptake of exogenous compatible solutes is preferred over their synthesis de novo, as it is energetically less costly. In the halophilic bacterium Halomonas elongata, a deletion of the transporter large transmembrane protein TeaC, of the TeaABC ectoine(s)’s uptake system, led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations. In this work, we have molecular characterized the TeaABC uptake system of the ectoines-producer halophilic bacterium C. salexigens, and constructed mutants to use them as a genetic background for improving ectoine(s) production. The orthologs to teaA, teaB and teaC of C. salexigens are differently organized (teaA and teaBC in the complementary strand) than those of H. elongata, and controlled by two different promoters, teaAp and teaBCp. The phenotypic characterization of a teaC::Ù mutant showed that TeaABC is the C. salexigens main uptake system for external ectoine(s). In addition, in the presence of glucose ateaC mutant strain maintained over a 20-50% of the ectoine(s) uptake, suggesting the presence of an alternative, probably ATP-dependent, transporter. Bacterial milking experiments with this strain led to a 33 % increased of ectoine production. Expression experiments using transcriptional fusions ofteaA and teaBC promoters with the reporter gene lacZ, revealed that both promoters are activated in the presence of ectoine. Moreover, the response regulator EupR, belonging to a two-component system previously characterized in this halophile, was the main responsible for the inhibition of of teaA expression under low salinity conditions. This research was financially supported by grants from the Spanish Ministerio de Ciencia e Innovación (BIO2011-22833), and Junta de Andalucía (P08-CVI-03724).

286 abstracts book EXTREMOphiles_2012P152 New host-vector system for Deinococcus geothermalis Issay Narumia, Takefumi Onoderaa,b and Katsuya Satoha aIon Beam Mutagenesis Research Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, Takasaki, Gunma 370-1292, Japan bLaboratory of Environmental Molecular Biology, Graduate School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0932, Japan E-mail: [email protected]

Deinococcus geothermalis DSM11300T is an extremely radiation resistant, moderately thermophilic bacterium closely related to the mesophilic Deinococcus radiodurans (Ferreira et al., 1997). It has been reported that the D. geothermalis genome was comprised of a chromosome (2.47 Mb) and two plasmids (574 kb and 206 kb) (Makarova et al., 2007). In this study, we discovered that the D. geothermalis genome contained a small cryptic plasmid pGEC1 (1,957 bp) that had not been reported so far. pGEC1 possessed two open reading frames, one encoding 78 amino acid residues (pGEC Orf1) and the other encoding 298 amino acid residues (pGEC Orf2). The predicted amino acid sequence encoded by pGEC Orf1 exhibited similarity to the N-terminal region of hypothetical proteins upstream of the replication initiation factor encoded by plasmidsShewanella of species. On the other hand, the predicted amino acid sequence encoded by pGEC1 Orf2 exhibited weak homology to replication initiation factors encoded by plasmids of thermophilic Bacillus sp. TK015 and Synechocystis sp. PCC6803. Development of a new D. geothermalis host-vector system consisted of three stages: (i) construction of a new shuttle vector pGEC6 based on pGEC1 and pKatCAT5 (Satoh et al., 2009), (ii) plasmid curing to isolate a pGEC1-free derivative ofD. geothermalis, and (iii) transformation of the pGEC1-freeD. geothermalis with pGEC6. The D. geothermalis host-vector system developed in this study could be used to examine the functionality of pprA gene (Narumi et al., 2004) in the radioresistance of D. geothermalis through gene disruption and plasmid complementation experiments.

References · A. C. Ferreira et al. (1997). Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs.Int J Sys Microbiol 47: 939-947. · K. S. Makarova et al. (2007). Deinococcus geothermalis: the pool of extreme radiation resistance genes shrinks. PLoS One 26: e955. · K. Satoh et al. (2009). Development of versatile shuttle vectors for Deinococcus grandis. Plasmid 62: 1-9. · I. Narumi et al. (2004). PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Mol Microbiol 54: 278-285.

abstracts book 287 P153EXTREMOphiles_2012 Role of the global regulator rpoS in the long- and short- term osmo- and thermoadaptation, in Chromohalobacter salexigens Manuel Salvadora, Jose M. Pastorb, Montserrat Argandoñaa, Vicente Bernalb, Francine Pibuelia, Rosa García-Valeroa, Manuel Cánovasb, Joaquín J. Nietoa and Carmen Vargasa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain bDepartment of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, 30100 Murcia, Spain E-mail: [email protected]

The halophillic bacterium Chromohalobacter salexigens tolerates one of the widest range of salinities found in nature (0,1- 4 M NaCl in complex medium) mostly due to the cytoplasmatic accumulation of the compatible solutes ectoine and hydroxyectoine. Our research group demonstrated that the genes involved in the ectoine (ectABC) and hydroxyectoine (ectD y ectE) production are subjected to a complex transcriptional regulation by several global (sS, s70, s32, Fur) and specific (EctZ) transcriptional factors. In addition, strong post-translational mechanisms operate, in response to environmental variations such as osmotic stress and temperature (Vargaset. al., 2008). In a previous work, the involvement of the global stress regulator rpoS (which codifies sS) in the regulation of theect genes was analyzed by using transcriptional fusions PectA::lacZ in an Escherichia coli rpoS mutant. This makes necessary its confirmation and a deeper analysis in a C. salexigens mutant. In the present study, an rpoS mutant of C. salexigens has been generated and its implication in long term adaptation by ectoines synthesis has been analyzed at 37ºC under low (0.6 M and 0.75M NaCl) and high (2.5 M and 3 M NaCl) salinity, and under high salinity and high temperature (2.5 M NaCl at 45ºC). Consumption of glucose and ammonium were measured in wild-type and rpoS strains, and related to intracellular ectoines production. To analyze the involvement or rpoS in the short term adaptation of ectoines synthesis, similar measurements were made during osmotic upshift (from 0.75 M to 3 M NaCl at 37ºC) and heat upshift shock (from 37ºC to 45ºC at 2.5 M NaCl). Expression of ectA, ectB and ectD transcripts was also measured by qPCR. The differences observed between the mutant rpoS and the C. salexigens parental strain will be presented and discussed.

References · Vargas C, Argandoña M, Reina-Bueno M, Rodriguez-Moya J, Fernandez-Aunion C, Nieto JJ. (2008). Saline Systems 4:14. Research was financially supported by grants from the Spanish Ministerio de Ciencia e Innovación (BIO2011- 22833), and Junta de Andalucía (P08-CVI-03724).

288 abstracts book EXTREMOphiles_2012P154 The array of transcriptional factors sequences in the genome of D. hansenii reveals how this yeast orchestrates a complex response to salt stress Marissa Calderón-Torres, Miguel Murguía-Romero, Daniela E. Castro and Antonio Peña

Universidad Nacional Autónoma de México, México E-mail: [email protected]

Debaryomyces hansenii, a yeast of biotechnological interest for agriculture and medicine, is characterized by a high tolerance to sodium (4.1 M NaCl) and alkaline pH. D. hansenii has an osmoregulation strategy as the principal adaptation to salt stress. The strategy involves mainly the expulsion of sodium by transporters like Ena1p ATPase and synthesis of organic compatible compounds such as glycerol or trehalose. Recently were found some of the genes that participate in the osmoregulation (HOG1, ENA1, ENA2, GPD1, GPP, NHX1 and KHA1); however these genes are insufficient to have a complete model of osmoregulation D.in hansenii, together with this, for this yeast there are few massive analysis of gene expression and its regulation. In a comparison study of the stress signaling pathways in fungi, in which was used the sequence of signaling pathway proteins of Sacharomyces cerevisiae and by reciprocal BLASTP was found the functional orthologues in diverse fungal species, for D. hansenii, it was determined that central components of osmotic, oxidative and cell wall stress signaling pathway are well conserved (Nikolaou et al., 2009); another important study is the microarray of genome wide expression of D. hansenii exposed to hypersaline shock of Arroyo-González et al.(2009), they found known genes that are expressed under osmotic stress, genes related to protein synthesis/maintenance of the cell, and remarkably genes associated to mitochondrial function. For having a more compressive model of the response of D. hansenii to salt stress, in this work we evaluated the correlation of diverse transcriptional factors (salt, oxidative, temperature, pH, amino acid and nitrogen starving, lipids and cell wall stress), by in silico analysis of distribution and frequency of its corresponding binding sites in every gene promoter region of the D. hansenii genome. To validate the in silico results and considering that most binding site sequences are shared with those of S. cerevisiae, we include the analysis of a heterologous DNA microarray between cDNA of D. hansenii and DNA of S. cerevisiae (unpublished data) and the results of microarray obtained by Arroyo- González et al. (2009). From in silico analysis, high percentages of the frequency observed for each binding site sequence in the D. hansenii genome, were those related to transcriptional factors of heat shock, oxidative and salt stress and nitrogen metabolism. In reviewing the respective gen ontology, surprisingly we found almost the same cellular location, function and processing. In a first comparison of our in silico results with data of the heterologous microarray of D. hansenii exposed to 1.2 M NaCl for 30 min and results of Arroyo-González et al. (2009) for hypersaline stress (2 M NaCl for 30 min) we found that frequency percentages of binding site sequences of heat shock, oxidative stress and nitrogen metabolism are quite similar to those found with thein silico analysis. At this point we propose that in silico analysis of binding sites in every gene promoter region is a valuable tool to predict the differential gene expression profile, not only during salt stress, but also in diverse kind of stresses. Additionally, this analysis strategy can be useful before doing in vitro and in vivo studies.

References · Arroyo-Gonzalez N, Vázquez A, Ortiz Zuazaga HG, Sen A, Luna Olvera H, Peña de Ortiz S, and Govind NS (2009). Genome-wide expression profiling of the osmoadaptation response of Debaryomyces hansenii. Yeast 26: 111-124. · Nikolaou E, Agrafioti I, Stumpf M, Quinn J, Stansfield I, and Brown AJP (2009). Phylogenetic diversity of stress signalling pathways in fungi. BMC Evolutionary Biology 9: 44.

abstracts book 289 P155EXTREMOphiles_2012 Isolation and properties of acidophilic bacteria and the cryptic plasmid from acidic environments Tsubasa Satoa, Kazuo Kamimurab and Tadayoshi Kanaob

aDivision of Bioscience, Graduate School of Natural Science and Technology, Okayama University, 700-8530 Okayama, Japan bDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, 700-8530 Okayama, Japan E-mail: [email protected]

Two heterotrophic acidophilic bacteria have been isolated from low pH environments such as an acid mine drainage and a brook from acidic hot springs. Both strains showed vigorous growth (more than 2.0 of OD660nm) in the heterotrophic basal salts medium adjusted pH 3.0 by sulfuric acid and containing 15 mM sucrose as a carbon source. One isolated strain TKN1 from Yanahara acid mine drainage was Gram-negative, aerobic and rod shaped, which shared 96.7% 16S rDNA identity with the type strain Acidocella aluminiidurans. The other isolated strain TKN2 from an acid brook in Tsukahara hot springs district was closely related to the type strain Acidocella facilis (99.8% 16S rDNA identity). A cryptic plasmid was obtained from the strain TKN1. The plasmid, designated pTKN1, consists of 2299 nucleotides involving an open reading frame (ORF). The ORF was identified as a putative rep gene, encoding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. On the other hand, no extrachromosomal DNA could be detected in the cell-free extract of the strain TKN2. Minimum inhibitory concentrations (MIC) were determined for both of the strains. Ampicillin inhibited their growth at 50 mg/ml. The cryptic plasmid pTKN1 was ligated with pUC18 which involved ampicillin resistance gene and replicated in Escherichia coli cells. The hybrid plasmid, designated pUCTN1, was constructed and introduced into the strain TKN2 by electroporation. The recombinant strain TKN2 harboring pUCTN1 showed ampicillin resistance. The plasmid was maintained in the strain TKN2 cells at least 5 generations under selective condition with 50 m/ml ampicillin. These results suggested that the hybrid plasmid derived from pTKN1 was able to replicate in the strain TKN2 and E. coli - Acidocella shuttle vector was successfully constructed.

290 abstracts book EXTREMOphiles_2012P156 In vivo evolution research with chimeric Rpb5/ RpoH subunits of the RNA polymerase in Pyrococcus furiosus Ingrid Waegea, Bettina Sommerb, David Pöllmanna, Winfried Hausnera, Reinhard Sternerb and Michael Thomma

aDepartment of Microbiology and Archaeacentre, Faculty of Biology, University of Regensburg, 93053 Regensburg, Germany bDepartment of Biophysics and Biochemistry, Faculty of Biology, University of Regensburg, 93053 Regensburg, Germany E-mail: [email protected]

The archaeal RNA Polymerase (RNAP) and the entire archaeal transcription machinery exhibit strong similarity to the eukaryotic transcription system. Despite the increase in complexity in Eukaryotes, for most RNAP II subunits homologous components can be found in Archaea. Furthermore comparisons of the structure of archaeal and eukaryotic RNAP (Hirata et al., 2008; Cramer et al., 2001) provide evidence that the basic features, which are important for activity and interactions of the subunits, are preserved, indicating an evolutionary conservation. The subunit RpoH is a homologue of the C-terminal domain of Rpb5, the eukaryotic subunit of RNAP II that seems to influence the stability of the active center (Zaroset al., 2007). To provide evidence for the evolutionary relationship of the archaeal and eukaryotic system it was investigated whether a direct substitution of RNAP subunits between the domains is possible. A chimeric fusion protein of eukaryotic Rpb5 N-terminus and the RpoH C-terminal domain was created and its function in archaeal and eukaryotic RNAP was analyzed (Thommet al., 2009). Although the function of Rpb5 and RpoH could be substituted respectively in vitro by the hybrid, the in vivo complementation of Rpb5 in Saccharomyces cerevisiae was not possible. For that reason error-prone PCR was used to select Rpb5/RpoH hybrids able to complement Rpb5 deletion mutants in yeast. Exchange of a single amino acid in the C-terminal part of the chimeric protein led to viable cells. Recent experiments using the genetic system of Pyrococcus furiosus suggest that hybrid Rpb5/RpoH variants can replace the archaeal subunit RpoH in vivo. These strains will be used to analyze the influence of the mutations on the phenotype. Furthermore, archaeal Polymerases containing modified Rpb5/RpoH subunits will be purified and the role of these subunits in various steps of the transcription cycle will be analyzed in cell free transcription assays.

References · Cramer, P., Armache, K-J., Baumli, S., Benkert, S., Brueckner, F., Buchen, C., Damsma, G., Dengl, S., Geiger, S., Jasiak, A., Jawhari, A., Jennebach, S., Kamensski, T., Kettenberger, H., Kuhn, C., Lehman, E, Leike, K., Sydow, J. and Vannini, A. (2008) Structure of eukaryotic RNA polymerases. Annu. Rev. Biophys. 37: 337-352. · Hirata, A., Klein, B.J., and Murakami, K.S. (2008) The X-ray crystal structure of RNA polymerase from Archaea. Nature 451: 851-854. · Thomm, M., Reich, C., Grünberg, S., Naji, S (2009) Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases. Biochem. Soc. Trans. 37: 18-22. · Zaros, C., Briund, J., Boulard, Y., Labarre-Mariotte, S., Garcia-Lopez., C., Thuriaux, P. and Navarro, F. (2007) Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases.Nucleic Acids Research 35: 634-647.

abstracts book 291 P157EXTREMOphiles_2012 Quorum sensing in Halomonadaceae Melanie Schwab, Ali Tahrioui, Emilia Quesada and Inmaculada Llamas

Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain E-mail: [email protected]

N-acyl homoserine lactones (AHLs) are used as extracellular signals by a variety of Gram-negative bacteria. These signals, called autoinducers, control gene expression in response to bacterial cell density in a process known as quorum-sensing (QS). In previous studies we detected the production of AHL signal molecules inHalomonas anticariensis FP35T, a moderately halophilic bacterium (Llamas et al., 2005) and since then have been able to identify and characterize the QS genes involved in the synthesis of these AHLs in this bacterium (Tahrioui et al., 2011). The next obvious step was to see whether the QS system exists in other members of the Halomonadaceae family. To this end we conducted assays with three biosensor strains, Agrobacterium tumefaciens NTL4 (pZLR4), Chromobacterium violaceum CV026 and C. violaceum VIR07 to detect the presence of AHLs in 45 halophilic species belonging to the genera Carnimonas (1), Chromohalobacter (1), Cobetia (1), Halomonas (35), Halotalea (1), Kushneria (3), Modicisalibacter (1) and Salinicola (2). To further confirm the production of AHLs, culture extracts from all the species were analysed by TLC. We also confirmed the presence of QS genes in the species in question by Southern blot hybridization and PCR amplification. The information resulting from this study is a prior requirement for future research into the role of cell- density-dependent gene regulation in extreme environments such as those inhabited by theHalomonadaceae family.

References · A. Tahrioui, E. Quesada, and I. Llamas (2011). The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium. Microbiology 157: 3378-3387. · I. Llamas, E. Quesada, M. J. Martínez-Cánovas, M. Gronquist, A. Eberhard, and J. E. González, (2005). Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide- producing species of Halomonas. Extremophiles 9: 333-341.

292 abstracts book EXTREMOphiles_2012P158 Interference in the quorum-sensing system of pathogenic vibrios for fish and molluscs M. Torresa, S. Pradob, J. Dubertb, A. Tahriouia, M. Romerob, E. Quesadaa and I. Llamasa

aDpto. Microbiología. Facultad de Farmacia. Universidad de Granada, Spain bDpto. Microbiología y Parasitología. CIBUS - Facultad de Biología. Universidad de Santiago de Compostela, Spain E-mail: [email protected]

It is known that the production of virulence factors by many microorganisms pathogenic to fish and shellfish, such as Vibrio, Aeromonas, Yersinia and Edwarsiella, depends upon an intercellular communication system known as quorum sensing, which involves the production of signal molecules known as autoinducers (Natrah et al., 2010). The quorum-sensing system in the Gram-negative Proteobacteria involves the accumulation of N-acylhomoserine lactone (AHL) molecules in the extracellular medium until a critical value is reached, at which point they bind a transcriptional activator, which triggers the expression of target genes and consequently the production of virulence factors (González and Keshavan, 2006). In response to this, some bacteria have developed a strategy via a contrary process known as quorum quenching (QQ), which involves degrading the AHL signalling molecules via enzymes such as acylases and lactonases (Defoirdt et al., 2008). The Microbial Exopolysaccharide Research Group of the University of Granada is working with a Research Group of the University of Santiago de Compostela to develop a way of using natural bacterial responses to fight infectious diseases that affect fish and molluscs in aquaculture. Ten strains were selected from a total of 146 isolates obtained from bivalve hatcheries in Galicia due to their capacity to degrade short, medium and long-chain AHLs (C4-HSL to C12-HSL) by acylase activity (Torres et al., 2012). We describe the assays carried out in this work that have confirmed the quorum-quenching capacity of one of these bacteria, strain 459 of Thalassomonas sp., against AHLs produced by different microorganisms, including some pathogenic vibrios.

References · F. M. I. Natrah, T. Defoirdt and P. Sorgeloos (2011). Disruption of bacterial cell-to-cell communication by marine organisms and its relevance to aquaculture. Marine Biotechnol 13: 109-126. · J. E González and N. D Keshavan (2006). Messing with bacterial quorum sensing. Microbiol Mol Biol Rev 70: 859-875. · T. Defoirdt, N. Boon, P. Sorgeloos, W. Verstraete and P. Bossier (2008). Quorum sensing and quorum quenching in Vibrio harveyi: lessons learned from in vivo work. ISME J 2: 19-26. · M. Torres, M. Romero, S. Prado, J. Dubert, A. Tahrioui, A. Otero and I. Llamas (2012). N-acylhomoserine lactone-degrading bacteria isolated from bivalve hatcheries. Marine Biotechnol (en revisión).

abstracts book 293 P159EXTREMOphiles_2012 The archaeal Holliday junction resolvase Hje has a regulatory role and is involved in DNA repair Yansheng Lia, Qihong Huanga, Chaoning Zenga, Yanze Lia, Jinfeng Nia, Qunxin Sheb and Yulong Shena aState Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Rd., Jinan, 250100, P. R. China bArchaea Centre, Department of Biology, University of Copenhagen, Ole MaaløesVej 5, Copenhagen Biocenter, DK-2200 Copenhagen N, Denmark E-mail: [email protected]

Holliday junction (HJ) resolvases catalyze the cleavage reaction of Holliday junction, the hallmark of DNA recombinational repair pathway and play critical roles in maintaining genome integrity in all organisms. The model hyperthermophilic crenarchaea Sulfolobus usually have two HJ resolvases, Hjc and Hje. Although the biochemical properties and structures of both enzymes have been reported, the physiological roles in the cell are still speculative. Here, we studied the in vivo functions of Hje and Hjc, two HJ resolvases in the hyperthermophilic archaeon Sulfolobus islandicus using recently-established genetic system. We constructed deletion and ectopic expression strains of hjc and hje and analyzed the phenotypes by DNA damage agent agent-sensitivity assay, flow cytometry, microscopy, and transcriptomic approaches. We demonstrate that Dhje, but not Dhjc, was highly sensitive to DNA-damaging agents hydroxyurea (HU), cisplatin, and methyl methanesulfonate (MMS). In the presence of HU, the Dhje strain produced abnormal cells and displayed severe cell aggregation, suggestive of a DNA repair deficiency in the mutant. In the absence of DNA damaging agents, Hje overexpression led to a growth defect and accumulated large cells with more than two genome equivalents. Surprisingly, overexpression of nuclease- deficient Hje conveyed the same growth defect as overexpression of the wild type enzyme. We found that Hje interacted with a homologue of FK506 binding proteins-type peptidylprolyl isomerases, a protein family participating in diverse pathways including cell cycle regulation and signal transduction in eukarya and bacteria. Furthermore, the Hje overexpression strain exhibits a drastic change in the abundance of transcripts of genes involved in DNA replication, repair, transcription regulation, and cell division, as revealed by whole genome microarray analysis. Our findings collectively suggest that Hje, but not Hjc, is primarily involved in DNA repair and Hje functions as a regulator of DNA repair in maintaining genome integrity.

294 abstracts book EXTREMOphiles_2012P160 An Hjc-interacting nuclease-ATPase in Sulfolobus islandicus is essential for cell viability Binyuan Zhai, Yanze Li, Mingzhu Chu, Jinfeng Ni and Yulong Shen

State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Rd., Jinan, 250100, P. R. China E-mail: [email protected]

Holliday junction (HJ) is an important intermediate in the DNA homologous recombination repair pathway. It must be processed by enzymes including HJ0resolvase, the structure-specific endonuclease, to avoid genomic instability. The model hyperthermophilic crenarchaea Sulfolobus usually have two HJ resolvases, Hjc and Hje. Although the biochemical properties and structures of both enzymes have been well ducumented, the physiological roles in the cell are still speculative. To probe the function of Hjc, a His-tagged Hjc was firstly over-expressed inSulfolobus islandicus. Among the co-purified proteins, a putative nuclease-ATPase was identified. The interaction between Hjc and the nuclease- ATPase was further confirmed by pull-down, western blot, and yeast two hybrid assay. It is composed of three conserved domains: the PIN domain, AAA+ (ATPase associated with diverse cellular activities) domain, and P-loop_NTPase domain. Bioinformatical analysis also revealed that the nuclease-ATPases are highly conserved across all archaeal phyla. Using the MIT (marker insertion and target gene deletion) method, we attempted to delete the gene, but we could not obtain the deletion mutant strain of the gene (Zhanget al., 2010). We confirmed the essentiality of gene by the mutant propagation assay and supplementing the gene on the simvastatin selection based vector (Zhang et al., 2012; Zheng et al., 2012). We then constructed an over-expression strain of the nuclease- ATPase of S. islandicus and found that the over-expression of the gene strongly inhibited the growth of the cells. Intriguingly, the DNA helicase HerA, which was an essential enzyme in Sulfolobus found based on our previous study, was co-purified with the His-tagged nuclease-ATPase expressed in situ. It is assumed that the nuclease-ATPase has at least two partners, Hjc and HerA, suggesting that it plays multiple functions in DNA metabolism. The detailed role of the enzyme will be discussed.

References · Zhang, C. et al. (2010) Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method. Microbiology 156: 3386-3397. · Zheng, T. et al. (2012) Development of a simvastatin selection marker for a hyperthermophilic acidophile, Sulfolobus islandicus. Appl Environ Microbiol 78: 568-574.

abstracts book 295 P161EXTREMOphiles_2012 Clone library as tool for detection of multiple copies of 16S rRNA in Halobacteriaceae archaeon Leonardo H. Pintoa, Gigliola R. B. Sallôtob, Joyce Lemosa, Ricardo P. Vieiraa, Orlando B. Martinsa and Maysa M. Clementinob

aInstituto de Bioquímica Médica, IBqM - Universidade Federal do Rio de Janeiro, Brazil bInstituto Nacional de Controle da Qualidade em Saúde, INCQS – Fiocruz, Brazil E-mail: [email protected]

The prokaryotic taxonomy has been determined by phenotypic and genotypic characteristics, which 16S rRNA gene is its phylogenetic universal marker in the tree of life. For prokaryotes, the multiple non-identical copies of this gene have been described, leading difficulties to identification and classification. Some species of Halobacteriaceae family with complete or partial genome have show multiple-copies of this gene. Due the high 16S rRNA sequences similarity between species, the haloarchaeal taxonomy have been showing many difficulties to classification, intensified by non-identical 16S rRNA multiple copies. After culturing and isolating steps, eight archaeal isolates classified Halobacteriaceaein family were selected to DNA extraction followed by 16S rRNA amplification by PCR using 21FA and 1492RAB primers. After E. coli DH10b electro-transformation with pGEMt, a clone library for each isolate was made and 12 clones were selected for sequencing. All sequences were submitted by BLAST for comparative analysis with NCBI genebank and EzTaxon, showing high similarities (>97%) with Haloarcula and Haloferax genera. A comparative analysis between different clones for each isolate show high similarity on nucleotide sequences. The library construction also enabled the detection of two different genera from the same isolate. This results show that clone library can detected multiple copies 16S rRNA gene in pure culture isolate being a promising technique to help the genotypic characterizations of prokaryotes.

296 abstracts book EXTREMOphiles_2012P162 Genetic engineering of Pyrococcus furiosus to use chitin as a carbon source Martina Kreuzer, Michael Thomm andWinfried Hausner

Lehrstuhl für Mikrobiologie, University of Regensburg, 93053 Regensburg, Germany E-mail: [email protected]

Genome analysis of the genes coding for the chitinase inPyrococcus furiosus and Thermococcus kodakaraensis revealed that most likely a one nucleotide insertion in Pyrococcus caused a frame shift in the chitinase which splits the enzyme into two genes in comparison to Thermococcus (Oku and Ishikawa, 2006). Furthermore, our attempts to grow the wild type strain ofPyrococcus on chitin were negative. From these data we assume that Pyrococcus is most likely not able to use chitin as a carbon source. Using a recently described genetic system for Pyrococcus we removed the frame shift by deleting the additional nucleotide within the genome ofPyrococcus (Waege et al., 2010). The resulting chitinase in the mutant is a single subunit enzyme and is in perfect agreement with the Thermococcus enzyme. A detailed analysis of the wild type and the mutant using counted cell numbers and ATP and acetate production as growth indicators revealed that only the mutant is able to use chitin as a carbon source. Therefore, we assume that Pyrococcus is most likely not able to use chitin as a carbon source in the natural biotope.

References · T. Oku and K. Ishikawa (2006). Analysis of the hyperthermophilic chitinase fromPyrococcus furiosus: activity toward crystalline chitin. Biosci Biotechnol Biochem 70: 1696-1701. · I. Waege, G. Schmid, S. Thumann, M. Thomm and W. Hausner (2010). Shuttle vector-based transformation system for Pyrococcus furiosus. Appl Environ Microbiol 76: 3308-3313.

abstracts book 297 P163EXTREMOphiles_2012 N-glycosylation in Haloferax volcanii involves two distinct biosynthetic pathways Lina Kaminskia, Ziqiang Guanb, Sophie Yurist-Doutscha and Jerry Eichlerb

aDepartment of Life Sciences, Ben Gurion University, Beersheva 84105, Israel bDepartment of Biochemistry, Duke University Medical Center, Durham NC 27710, USA E-mail: [email protected]

While observed in members of all three domains of life, N-glycosylation in Archaea presents aspects of this universal post-translational modification not seen in either Eukarya or Bacteria. For example, in the halophilic archaeon, Haloferax volcanii, the surface (S)-layer glycoprotein, a reporter of N-glycosylation in this species, is simultaneously modified by two different N-glycans when grown in the presence of 1.75 but not 3.4 M NaCl. Specifically, when grown at the lower salinity, Asn-13 and Asn-83 are modified by a previously characterized pentasaccharide (comprising a hexose, two hexuronic acid, a methyl ester of hexuronic acid and mannose), whereas Asn-498 is modified by a tetrasaccharide of distinct composition (comprising a sulfated hexose, two hexoses and rhamnose). Deletion of genes encoding components of the pentasaccharide assembly pathway had no effect on the biosynthesis of the tetrasaccharide bound to Asn-498. However, deletion of other genes currently annotated as serving sugar-processing-related roles interfered with the assembly of the Asn-498- linked tetrasaccharide. As such, in Hfx. volcanii, two distinct N-glycosylation pathways operate on a single target protein, the S-layer glycoprotein. These results thus provide the first example of N-glycosylation relying on a process so complex.

298 abstracts book EXTREMOphiles_2012OL1

session 6 genomics and metagenomics

abstracts book 299 OL1EXTREMOphiles_2012

300 abstracts book EXTREMOphiles_2012P164 High through put mapping of transposon insertions for the identification of essential genes of the methanogenic archaeon Methanococcus maripaludis Felipe Sarmiento B., Jan Mrazek and William B. Whitman

Department of Microbiology, University of Georgia, 541 Biological Science Building, Athens, GA 30602-2605, USA E-mail: [email protected]

Two libraries of transposon mutants were generated for the hydrogenotrophic methanogenic archaea Methanococcus maripaludis S2 using a derivative of the Tn5 transposon. These libraries were analyzed by High Through put Mapping of Transposon insertions (HTMT), which mapped about 89,000 unique insertions to the genome following enrichment of DNA fragments comprising the transposon-chromosomal DNA junctions and Illumina sequencing technology. This approach identified genes essential and non-essential for the growth of M. maripaludis in rich and minimal media. Four hundred thirty-one genes were essential and one hundred ten genes were likely to be essential for growth in rich medium, which correspond to approximately 30% of the total number of genes in the genome. Many of the essential genes encoded for proteins involved in fundamental processes such as methanogenesis, replication, transcription and translation. However, 83 essential genes and 39 likely to be essential genes encoded for hypothetical ORFs, which await further characterization. In addition, 158 extra genes were essential or likely to be essential and 12 become unassigned or non-essential in minimal medium. The results demonstrate an efficient methodology to identify essential genes in archaea.

abstracts book 301 P165EXTREMOphiles_2012 Towards an improved Sulfolobus acidocaldarius expression system useful for functional metagenome analysis J. Korta, A. Wagnerb, S.V. Albersb and B. Siebersa

aMolecular Enzyme Technology and Biochemistry (MEB), Biofilm Centre, University of Duisburg-Essen, Universitätsstr. 5, 45141 Essen, Germany bMolecular Biology of Archaea, Max-Planck Institute for terrestrial Microbiology, Karl-von-Frisch-Str. 10, 35043 Marburg, Germany E-mail: [email protected]

Archaea offer exciting potential for biotechnological applications, since their proteins, so called «extremozymes», are active under harsh conditions. Unfortunately, the functional expression of many archaeal (hyper)thermophilic proteins in mesophilic or even thermophilic bacterial hosts is limited. Even more severe is the choice of expression hosts in functional metagenomics. Since Archaea harbor a unique transcription machinery, the use of bacterial expression systems might lead to a pre-selection in current metagenomic approaches. The establishment of an expression platform with a variety of host organisms, including Archaea, will help to capture the natural diversity.

Sulfolobus acidocaldarius is a well characterized thermoacidophilic, obligate aerobic Crenarchaeon that grows optimally at 78°C and pH 2-3. It is genetically tractable and a vector system for protein expression has been established.

For the expression in S. acidocaldarius the promoter of the maltose binding protein malE is employed. Extensive mutational analysis of different parts of themalE resulted in a hybrid promoter that had 5-fold higher expression levels than the wild type promoter. The insertion of the regulator that binds themalE promoter, termed MRP (maltose regulatory protein) into the optimized expression vector lead to a more than 4-fold increase of expression levels. First results about the expression of archaeal enzymes of biotechnological interest will be presented. Furthermore, preliminary results about the use of the vector for the expression of metagenomic libraries from hot environments for identifying new and industrially relevant enzymes will be discussed.

References · S. Berkner, A. Wlodkowski, S.V. Albers, G. Lipps (2010). Extremophiles 14: 249-259.

302 abstracts book EXTREMOphiles_2012P166 New abundant microbial lineages in hypersaline systems Ana B. Fernandeza, Rohit Ghaib, Ana Belen Martin-Cuadradob, Cristina Sanchez-Porroa, Francisco Rodriguez- Valerab and Antonio Ventosaa

aDepartment of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain bEvolutionary Genomics Group, Division of Microbiology, University Miguel Hernandez, Alicante 03550, Spain

E-mail: [email protected]

Metagenomics coupled to pyrophosphate sequencing (pyrosequencing) appears to be an ideal tool as there is no need for prior sequence information, there is no PCR or cloning bias and it allows for accumulating a large amount of genomic data about a given environment. In this study we have used a metagenomic approach in an attempt to describe the microbiota in the water column of ponds of marine solar salterns. These consist of a series of shallow interlinked ponds where seawater is gradually concentrated to precipitate sodium chloride and other valuable salts. It has been shown that prokaryotic diversity varies throughout this environmental gradient with specialized populations of halophilic prokaryotes displacing marine populations at about four to five times the average seawater salinity. The saturated brine (crystallizer ponds) has been previously studied by several approaches including classical cultivation, single-gene sequence analysis and analysis of metagenomic fosmid library sequences obtained by dideoxy sequencing technique. The microbial diversity encountered was low with only two abundant species: the extremely halophilic square archaeon Haloquadratum walsbyi and the extremely halophilic bacterium Salinibacter ruber. We have studied a solar saltern located in Santa Pola, Alicante (Spain), namely concentrator ponds with total salt concentration of 13 % (w/v), 19 % (w/v) and 33 % (w/v), and a salt saturated crystallizer pond with total salt concentration of 37 % (w/v). After DNA extraction from the water samples taken from the saltern the DNA was sequenced by pyrosequencing (454 Life Sciences). The sequence data was analysed using classical BLAST searches and 16S rRNA analysis. The analyses of these metagenomes (nearly 1534 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. Metagenomic assembly revealed new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon and a high-GC euryarchaeon.

abstracts book 303 P167EXTREMOphiles_2012 Genome analysis of Halanaerobium saccharolyticum DSM 6643T, a halophilic hydrogen producing bacterium Anniina Kivistöa, Antti Larjob, Ville Santalaa, Christophe Roosb and Matti Karpa

aDepartment of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland bDepartment of Signal Processing, Tampere University of Technology, Tampere, Finland E-mail: [email protected]

Halanaerobium saccharolyticum is a halophilic fermentative bacterium belonging to the orderHalanerobiales (Kivistö and Karp, 2010). Although few halophilic fermentative bacteria have already been sequenced, the fascinating group of microorganisms remains still relatively unstudied. H. saccharolyticum is interesting due to its efficient hydrogen 2(H ) production, vitamin B12 dependent 1,3-propanediol (1,3-PD) producing pathway that competes with H2 production, and ability for utilizing unpurified raw glycerol (a by-product of traditional biodiesel industry) (Kivistö et al., 2010, 2011). The genome was sequenced using Illumina paired-end sequencing technology, assembled using Velvet (Zerbino and Birney, 2008) and annotated using RAST server (Aziz et al., 2008). The draft genome composed of 58 contigs is 2,854,061 bp long and was predicted to contain 2622 coding sequences of which 56 for RNAs (Aziz et al., 2008). The glycerol fermentation pathways ofH. saccharolyticum were re-constructed according to genome analysis. The sequence analysis revealed vitamin B12 dependent glycerol dehydratase (catalyses the first step of 1,3-PD production), Fe-hydrogenase operon, heavy metal resistance genes, and ion pumps (adaptation to the high salinity) which were characterized in more detail. The genome sequence will provide insight into the fermentative pathways as well as physiology and adaptation strategies of halophilic fermentative bacteria.

References · R.K. Aziz, D. Bartels, A.A. Best, M. DeJongh, T. Disz, et al. (2008). The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics 9: 75. · A. Kivistö, M. Karp (2011). Halophilic anaerobic fermentative bacteria (review). J Biotechnol 152: 114-124. · A. Kivistö, V. Santala, M. Karp (2010). Hydrogen production from glycerol using halophilic fermentative bacteria. Biores Technol 101: 8671-8677. · A. Kivistö, V. Santala, M. Karp (2011). Closing the 1,3-propanediol route enhances hydrogen production from glycerol by Halanaerobium saccharolyticum subsp. saccharolyticum. Int J Hydrogen Energy 36: 7074-7080. · D.R. Zerbino, E. Birney (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18: 821-829.

304 abstracts book EXTREMOphiles_2012P168 Unveiling microbial life and functional diversity in Mediterranean deep-sea hypersaline lakes María Alcaidea, Mercedes V. Del Pozoa, Álvaro Lafrayaa, Rafael Bargielaa, Jesús Tornésa, Tatyana N. Chernikovab, Violetta LaConococ, Olga V. Golyshinab, Michail M. Yakimovc, Peter N. Golyshinb and Manuel Ferrer

aCSIC–Institute of Catalysis, 28049 Madrid, Spain bSchool of Biological Sciences, Bangor University, LL57 2UW Gwynedd, UK cInstitute for Coastal Marine Environment (IAMC), CNR, Messina, Italy E-mail: [email protected]

Microbial ability to thrive in extreme ecological niches depends upon adaptation of their enzymatic machinery to physical-chemical environmental constraints in situ. Despite the importance of extremophiles for blue-skies’ and applied research, only a tiny fraction of them is able to grow under laboratory conditions. In this context, extremophiles are several orders of magnitude less amenable to cultivation than common microorganisms, which severely impairs our knowledge of microbial biodiversity and, in turn, hinders our appreciation of functional protein diversity. Recently, the development of metagenomics approaches has opened the window into new opportunities to explore richness of uncultured biodiversity, providing presumptive compositional and functional blueprints of the whole microbial community. Additionally, major surprises, such as the occurrence of new protein families in newly-studied ecosystems, the discovery of multi-kingdom Pfam domains that highlight new biological processes conserved through evolution or the identification of a number of genes coding for proteins representing yet unknown functionalities, has been achieved. On the basis of metagenomic features, we provide a view into the microbial ecology and functional diversity in Mediterranean deep-sea hypersaline anoxic lakes (DHALs). These brine lakes were formed by evaporite dissolution/brine seeps and are important model environments to provide insights into possible metabolism and distributions of microorganisms and enzymes on the early Earth (La Conoet al., 2011; Ferrer et al., 2012). Additionally, functional protein studies in a number of further extreme marine environments will also be discussed,

References · M. Ferrer, et al. (2012). Unveiling microbial life in the new deep-sea hypersaline Lake Thetis. Part II: a metagenomic study. Environ Microbiol 14: 268-281. · V. La Cono, et al. (2011). Unveiling microbial life in new deep-sea hypersaline Lake Thetis. Part I: Prokaryotes and environmental settings. Environ Microbiol 13: 2250-2268

Acknowledgements The authors gratefully acknowledge the financial support provided by the EU projects MAMBA, MAGICPAH and ULIXES (FP7-KBBE-2008-226977/2009-245226/2010-266473).

abstracts book 305 P169EXTREMOphiles_2012 Functional diversity of microbial (meta-) genomes in the Iberian Peninsula Mónica Martíneza, María Alcaidea, Mercedes V. Del Pozoa, Rafael Bargielaa, Iván Loresb, Celia Méndezb, Jesús Sánchezb and Manuel Ferrera

aCSIC – Institute of Catalysis, 28049 Madrid, Spain bIUBA, Área de Microbiología, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain E-mail: [email protected]

The Metagenome of the Iberian Peninsula is a project that brings together and focuses the concerted work and efforts of different research institutions, universities, and companies with the overall aim of unveiling the microbial diversity of the Iberian Peninsula and exploiting this biodiversity for biotechnological applications. Here, using metagenomic approaches we select, analyse and compare the population structure among distinct sites among the Iberian Peninsula based on the analysis of specific sequences, and identify new functional activities. The different contribution of specific microbes and functions in both extreme and non extreme environments are discussed to identify specific features that help integrating «extremozymes» in biotechnological settings.

References · M. Ferrer et al. (2011). Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, Central Spain). Microb Ecol 62: 824-837. Acknowledgements This research was supported by the Spanish CSD2007-00005 and by European Regional Development Fund (ERDF) funds.

306 abstracts book EXTREMOphiles_2012P170 Genomic insights into the life of the unusual extremophiles of the deep-sea brines of the Red Sea André Antunesa, Intikhab Alamb, Vladimir B. Bajicb and Ulrich Stingla

aRed Sea Research Center, King Abdullah University of Science and Technology (KAUST), Saudi Arabia bComputational Bioscience Research Center, King Abdullah University of Science and Technology (KAUST), Saudi Arabia E-mail: [email protected]

The deep-sea brines of the Red Sea are considered to be one of the most remote, challenging and extreme environments on Earth, while remaining one of the least studied. These biotopes are unique due to the combination of multiple extremes namely high salinity (7-fold increase in salinity), high temperature (up to 70ºC), high concentration of heavy metals (1,000- to 10,000-fold increase in concentration), high hydrostatic pressure and anoxic conditions. Despite this combination of multiple environmental extremes, they have been shown to harbor a very high biodiversity, with identification of several new phylogenetic lineages (Antuneset al., 2011a). Cultivation-based studies resulted in the isolation of several new extremophiles, including the description of Halorhabdus tiamatea (representing a new species; Antunes et al., 2008a) Salinisphaera shabanensis, and Haloplasma contractile (both representing new orders; Antuneset al., 2003; 2008b) These three microbes were selected as part of a genome-sequencing project due to their phylogenetic position, peculiar features and unique biotope, and revealed some interesting features. InHlr. tiamatea, genes related to P-limitation were detected, together with evidence for trehalose synthesis and a new fermentative pathway within the Halobacteriaceae (Antunes et al., 2011b). The genome of Ssp. shabanensis includes several genes associated with salinity and nutrient variability and heavy-metal resistance (Antunes et al., 2011c). As for Hlp. contractile, the number, relative position, and presence or absence of specific genes within its genome give us important hints on the genetic basis for its unusual morphology and cellular contractility (Antunes et al., 2011d). Analysis of these draft genomes provides us with a first glimpse on some of their unusual characteristics and the ways they cope with living in such a harsh environment. References · Antunes, et al. (2011a). Microbiology of the Red Sea (and other) deep-sea anoxic brine lakes. Environ Microbiol Rep, doi:10.1111/j.1758-2229.2011. · Antunes, et al. (2008a). Halorhabdus tiamatea sp. nov., a non-pigmented, extremely halophilic archaeon from a deep-sea, hypersaline anoxic basin of the Red Sea, and emended description of the genusHalorhabdus . Int J Syst Evol Microbiol 58: 215-220. · Antunes, et al. (2003). Salinisphaera shabanensis gen. nov., sp. nov., a novel, moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep, Red Sea. Extremophiles 7: 29-34. · Antunes, et al. (2008b). A new lineage of halophilic, wall-less, contractile bacteria from a brine-filled deep of the Red Sea. J Bacteriol 190: 3580-3587. · Antunes, et al. (2011b). Genome sequence of Halorhabdus tiamatea, the first archaeon isolated from a deep-sea anoxic brine lake. J Bacteriol 193: 4553-4554. · Antunes, et al. (2011c). Genome sequence of Salinisphaera shabanensis, a gammaproteobacterium from the harsh, variable environment of the brine-seawater interface of the Shaban Deep in the Red Sea. J Bacteriol 193: 4555-4556. · Antunes, et al. (2011d). Genome sequence of Haloplasma contractile, an unusual contractile bacterium from a deep-sea anoxic brine lake. J Bacteriol 193: 4551-4552.

abstracts book 307 P171EXTREMOphiles_2012 Isolation of sodium non-requirement alkaliphiles and its physiology predicted from comparative genome analysis with sodium dependent ones Makoto Fujisawaa,b, Kazuaki Odakurac, Manato Watanabec, Takuya Kamiyamac and Kazuya Nodac

aFaculty of Food Life Sciences, Toyo University, 1-1-1 Izumino, Itakura, Oura, Gumma, 374-0193Japan bBio-Nano Electronics Research Center, 2100 Kujirai, Kawagoe, Saitama, 350-8585, Japan cFaculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura, Oura, Gumma, 374-0193 Japan E-mail: [email protected]

Alkaliphilic bacterial organisms have an optimal growth pH above 9.0 (Horikoshi, 1999). It can maintain the cytoplasmic pH close to neutral pH under such high pH condition. In general, sodium transport cycles are coupled to the cytoplasm acidification.Bacillus pseudofirmus OF4, an exclusively studied facultative alkaliphile whose genome has been sequenced (Janto et al., 2011), can grow on malate by oxidative phosphorylation and it requires sodium ion at more than 10 mM at pH 10.5 (Krulwich et al., 2001). However, some of alkaliphiles such as Bacillus foraminis CV53T are assumed to be independent on sodium ion (Tiago et al., 2006). In addition, contribution of intracellular acid production to it has been suggested from the fact that B. pseudofirmus OF4 could maintain the intracellular pH at 9.2 on glucose without sodium when the cells were upshifted from pH 8.5 to 10.5 (Krulwich et al., 2001). Here, we have tried to isolate alkaliphilic bacteria which showed requirement or tolerance to lithium ion instead of sodium ion by cultivating cells in growth medium with lithium ion to find some new insights on alkaline environment adaptation. Fifty-six colonies have been isolated. Characterization of the isolates revealed that one of the isolates named KU50 had similarity to B. pseudofirmus OF4. Sequencing analysis of the draft genome of KU50 revealed that the strain had a couple of differences in sodium cycle proteins toB. pseudofirmus OF4 ones.

References · K. Horikoshi (1999) Alkaliphiles: some applications of their products for biotechnology.Microbiol Mol Biol Rev 63: 735-750. · B. Janto, et al. (2011). Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an exterenal pH range from 7.5 to 11.4. Environ Microbiol 13: 3289-3309. · T. A. Krulwich, M. Ito and A. A. Guffanti (2001). The Na+-dependence of alkaliphily in Bacillus. Biochim Biophys Acta 1505: 158-168. · I. Tiago, C. Pires, V. Mendes, P. V. Morais, M. S. da Costa and A. Verissimo (2006). Bacillus foraminis sp. nov., isolated from a non-saline alkaline groundwater. Int J Syst Evol Microbiol 56: 2571-2574.

308 abstracts book EXTREMOphiles_2012P172 Integrating high-throughput technologies for the discovery of novel thermostable lignocellulases Colin Ohlhoffa, Rob Huddya, Mariette Smarta, Bronwyn Kirbya, Inonge Mulakob, Ana Casanuevaa, Rolene Bauera, Marla Tuffina and Don Cowanc

aInstitute for Microbial Biotechnology and Metagenomics, Department of Biotechnology, University of the Western Cape, Cape Town, South Africa bInstitute for Plant Biotechnology, Department of Genetics, Stellenbosch University, Stellenbosch, South Africa cDepartment of Genetics, University of Pretoria, Pretoria, South Africa E-mail: [email protected]

Agricultural plant waste material, also known as lignocellulosic biomass, represents an alternative renewable feedstock for the production of various high-value products. It is primarily composed of varying proportions of cellulose, hemicellulose and lignin. Although these components can serve as valuable substrates in the bioconversion process, they are generally recalcitrant to enzymatic degradation. This hurdle may be overcome by high temperature processing combined with thermophilic hydrolysis. We aimed to identify and characterize novel enzymes for high temperature bio-degradation of lignocellulosic-based feedstocks. Metagenomic gene discovery provides an efficient approach for accessing the genomes and genes of uncultured extremophilic microorganisms with the potential of harnessing these for various industrial applications. In this study the environmental metagenome of thermophilic compost, captured in a fosmid library is presented. A total of 36 clones, displaying lignocellulosic activities, were subjected to 454 sequencing and analysed using a combination of software tools. Surprisingly, phylogenetic analysis of the library clones revealed a low representation of thermophile diversity although a significant number of the genes identified within these clones may be involved in carbohydrate metabolism including the metabolism of mono-, di-, oligo- and polysaccharides. Using this approach, a large number of novel gene sequences were identified and the gene products functionally characterized for hydrolysis of lignocellulosic biomass.

abstracts book 309 P173EXTREMOphiles_2012 Characterization of thermostable GH 9 cellulases from a metagenomic source Stephen Mackaya, Robert Huddya, Marla Tuffina, Donald Cowana,b and Rolene Bauera aInstitute for Molecular Biotechnology and Metagenomics, University of the Western Cape, Bellville, Box X17, 7535, South Africa bUniversity of Pretoria, Pretoria, South Africa E-mail: [email protected]

Thermophiles are an ideal source of thermostable enzymes with relatively high optimum temperatures. Numerous industrial processes require stable enzymes active at relatively high temperature.Therefore a metagenomic library was constructed using inhibitor-free high molecular weight DNA isolated in a single step from a thermal compost heap (≥ 80˚C) rich in humic acid. The metagenomic library was screened using a Q-Pix2 robot on several lignocellulosic substrates for a range of lignocellulose activities. Several novel enzymes from the library have been identified, sequenced and cloned. Two GH 9 cellulases have been fully characterized and described. Both of these enzymes had novel sequence identities, displaying 46 and 75% sequence identity relative to sequences on NCBI, and have been characterized for their catalytic activity, temperature and pH profiles and substrate specificities.

310 abstracts book EXTREMOphiles_2012P174 Bioleaching heaps provide special opportunities for studying the adaption, ecophysiology and evolution of extreme acidophiles Raquel Quatrinia,c and David S. Holmesb,c

aLaboratory of Microbial Ecophysiology, Fundación Ciencia y Vida, Santiago, Chile bCenter for Bioinformatics and Genome Biology, Fundación Ciencia y Vida, Santiago, Chile cDepto. Ciencias Biologicas, Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chile E-mail: [email protected]

Bioleaching heaps are man-made deposits of crushed rock that contain copper ore often embedded in an iron-sulfur matrix. Ore grade and rock size are non-uniform within the heaps, and efficient and uniform aeration and irrigation are difficult to achieve. The pH typically ranges from 1-3 and the oxygen content can vary dramatically giving rise to both aerobic and microaerophilc habitats. Consequently, heaps contain a considerable diversity of micro-environments that may be occupied by different microbial consortia. In spite of local differences, the heaps are initially populated by mesophilc acidophiles that exothermically oxidize the iron and sulfur present. As a consequence, the temperature within the heaps rises and the mesophiles are replaced by moderate thermophilic bacteria and eventually by thermophilic Archaea. These polyphyletic microbial associations interact symbiotically, parasitically, commensally and competitively depending on abioitic drivers such as acidity, O2 and CO2 availability, toxic ions and temperature. Bioleaching heaps represent a unique opportunity to study, within reasonable time-scales (months), the genetics and metabolism of extreme acidophiles in a dynamically evolving environment that is especially suited to studies of microbial ecophysiology and adaption. The genetic complexity of the microbial population is sufficiently diverse to allow the testing of hypotheses but not too complex as to be un-interpretable. Analysis of genetic and metabolic functions across more that 50 complete or draft genomes of bioleaching acidophiles has allowed us to construct integrated models suggesting how organisms share the workload of maintaining the nutrient and energy budgets of the community. Large scale comparative genomics has also begun to highlight the importance of lateral transfer of genes, genetic islands and whole metabolic units in the evolution of niche specificity and genome organization in these extreme acidophiles. Acknowledgements: Fondecyt 1090451 and 1100887.

References · J. Valdés, J. P. Cárdenas, R. Quatrini, M. Esparza, H. Osorio, F. Duarte, C. Lefimil, R. Sepulveda, E. Jedlicki and D. S. Holmes (2010). Hydrometallurgy 104: 471-476. · J. P. Cárdenas, J. Valdés, R. Quatrini, F. Duarte, and D. S. Holmes. (2010). Appl Micobiol Biotech 88: 605-620.

abstracts book 311 P175EXTREMOphiles_2012 Archaeal diversity in the geothermal field Los Azufres revealed by metagenomics Luis E. Servín Garcidueñas and Esperanza Martínez Romero

Department of Ecological Genomics, Center for Genomic Sciences, UNAM, National University of Mexico, Cuernavaca, Morelos, Mexico E-mail: [email protected]

Los Azufres is a geothermal field located in the Trans-Mexican Volcanic Belt. The field harbors hot springs with a rich and unique microbial diversity. The main objective is to describe the diversity and to explore the genetic content of microbial communities that thrive under extreme geochemical conditions at Los Azufres using high throughput sequencing of environmental DNA. Amplification and sequencing of ribosomal genes from environmental DNA and phylogenetic analyses were achieved to create the microbial census of communities. DNA samples were purified from a hot spring and were sequenced with Illumina-Solexa and 454 Titanium technologies. The hot spring pH is 2 - 3 and temperature around 65 °C. Analyses from the hot spring metagenome (729 Mb) revealed sequences from archaeophages, minor Vulcanisaeta and Thermoproteus archaeal populations from the orderThermoproteales and the almost complete genome of an archaea from the order Sulfolobales. One of the sequenced DNA samples (437 Mb) contained mainly DNA from Sulfolobales and the assembly yielded a genome of around 1.7 Mb. Phylogenetic and phylogenomic analyses from theSulfolobales archaeum indicated that it could represent a novel genus based on the 16S rRNA gene sequence and the whole genome shotgun assembly. The generated set of predicted genes has the potential to yield insights into the microbial mechanisms used to survive under extreme conditions.

312 abstracts book EXTREMOphiles_2012P176 Thermus thermophilus phage P23-77: archetype of a novel branch of an ancient viral lineage Alice Pawlowskia, Ilona Rissanena, Jonathan M. Grimesb,c, Karl Harlosb, David I. Stuartb,c and Jaana K.H. Bamforda

aDepartment of Biological and Environmental Science, University of Jyväskylä, 40014 Jyväskylä, Finland bDivision of Structural Biology, Welcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, England cScience Division, Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot OX11 0DE, England E-mail: [email protected]

Analysis of viral structural elements such as major capsid proteins (MCPs) revealed highly conserved protein folds that can be used to group viruses into a reasonably small number of lineages (Bamford et al., 2005). The main characteristic of one of these lineages is a MCP fold that contains a vertical double beta-barrel. Members of the lineage infect hosts from all three domains of life suggesting that they have a common ancestor existing even before the separation of the current domains of life. Recently, viruses from extreme environments have been discovered that appear to utilize unusual versions of the double beta-barrel fold. We crystallized the two MCPs of one of these viruses, P23-77, an icosahedral dsDNA bacteriophage infectingThermus thermophilus (Rissanen et al., 2012). High resolution structure analysis revealed a single beta-barrel core fold of the capsid proteins homologous to the canonical double beta barrel fold. Therefore, P23-77 represents the earliest branch of a beta-barrel super-lineage consisting of dsDNA viruses with vertical beta-barrel coat proteins for capsid assembly. We identified additional members of the P23-77 lineage, distributed equally on earth but so far exclusively found in thermophilic bacteria and halophilic archaea (Jalasvuori et al., 2010). Our findings show that extreme environments are an appealing domain for virus research aiming to identify ancient viral branches, linked to the most primordial prokaryotic populations and perhaps to the last universal common ancestor of cells.

References · D.H. Bamford, J.M. Grimes and D.I. Stuart (2005). What does structure tell us about virus evolution?.Curr Opin Struct Biol 15: 655-663. · I. Rissanen, A. Pawlowski, K. Harlos, J.M. Grimes, D.I. Stuart and J.K.H. Bamford (2012). Crystallization and preliminary analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. Acta Cryst F68: 580-583. · M. Jalasvuori, A. Pawlowski and J.K.H. Bamford (2010). A unique group of virus-related, genome-integrating elements found solely in the bacterial family Thermaceae and the archaeal family Halobacteriaceae. J Bac 192: 3231-3234.

abstracts book 313 P177EXTREMOphiles_2012 A constraint-based metabolic model designed to optimize ectoines production in Chromohalobacter salexigens Montserrat Argandoñaa, Francine Piubelia, Javier Rodríguez-Moyaa, Jose M. Pastorb, Manuel Salvadora, Vicente Bernalb, Angel Sevillab, Manuel Cánovasb, Joaquín J. Nietoa and Carmen Vargasa

a Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain b Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, 30100 Murcia, Spain E-mail: [email protected]

Ectoines (ectoine and hydroxiectoine) are compatible solutes which are accumulated by some halophilic bacteria in response to osmotic and heat stress. Due to their stabilizing nature, they have a great biotechnological potential in dermopharmacy, cosmetics, molecular biology, and biomedicine. C. salexigens is a halophilic bacterium which naturally produces ectoine and hydroxyectoine. Because of the complexity of the metabolic pathways for ectoines synthesis, and their regulation, a global and integrative approach such as Systems Biology is required in order to develop new genetic engineering strategies for the optimization of ectoines industrial production in C. salexigens. In this work, we present a partial refined metabolic reconstruction C.of salexigens and its associated model, in which C and N metabolism is connected with all pathways concerning transport, synthesis, degradation and efflux of C. salexigens main compatible solutes (choline, betaine, glutamate, ectoines, trehalose). By using as a basis the previous genome-based metabolic reconstruction of Chromohalobacter salexigens, iOA584 the complete metabolism of compatible solutes was included. In addition, analysis of central metabolism of C. salexigens and proteomics were performed in our laboratory in maximal and minimal conditions for ectoines production. They revealed some metabolic pathways directly involved in osmoadaptation and therefore related with ectoines synthesis. All significant metabolic pathways were revised, reannotated and included in the model after consulting literature data and developing additional searches in databases as BLAST, KEGG, CDD, BRENDA, BIOCYC and PathwayTools and up-to date NCBI genome annotation ofC. salexigens. In addition, we performed conserved domains comparison and phylogenetic analysis to correctly assignation of different isoenzymes to a specific reaction. In silico analysis of model connectivity and dead-end products were performed and also preliminary simulations are in progress. This research was financially supported by grants from the Spanish Ministerio de Ciencia e Innovación (BIO2011-22833), and Junta de Andalucía (P08-CVI-03724).

314 abstracts book EXTREMOphiles_2012P178 Metagenomic screening of extremophilic sediments for rhamnolipid production Wesley Trevor Williamsa, Johannes Küglerb, Marla Tuffina, Christoph Syldatkb and Don Cowanc

aInstitute for Microbial Biotechnology and Metagenomics, Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape-Town, South Africa bInstitute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology, D-76131, Karlsruhe, Germany cDepartment of Genetics, University of Pretoria, Pretoria 0002, South Africa E-mail: [email protected]

Rhamnolipids are biodegradable biosurfactants with low critical micelle concentrations, therefore are ideal for industrial and environmental applications (Desai & Banat 1997). Rhamnolipids are composed of one or two hydrophilic rhamnose molecules linked via a β-glycosidic bond to one or two hydrophobic β-hydroxy fatty acids to form an anionic biosurfactant. Currently, rhamnolipids are mainly produced by the opportunistic pathogen, Pseudomonas aeruginosa and production is coupled to the complex quorum sensing regulation. Production of rhamnolipids can be improved by avoiding the pathogenic production strains and decoupling the production from the quorum sensing system (Wittgens et al. 2011). Soil samples from two extremophilic environments were screened for rhamnolipid producing organisms: from underneath mummified seal carcasses in the Miers Dry Valley, Eastern Antarctica and hypersaline sediments from the Namib Desert, Western Namibia. Biosurfactant activity was detected by measuring the change in surface tension and rhamnolipids were screened by thin layer chromatography. To complement the culture based approach, metagenomic libraries were constructed from the same soils and functionally screened for biosurfactant producing genetic elements, notably the rhlABC operon. The culture dependent method has resulted in the isolation of several putative biosurfactant producing bacterial strains from the Miers Dry Valley mineral soils. Identification of new sources of rhamnolipids will improve our understanding of their regulation, ultimately enabling high levels of production in non-pathogenic heterologous hosts.

References · Desai, J.D. & Banat, I.M. 1997. Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews 61: 47-64. · Wittgens, A. et al. 2011. Growth independent rhamnolipid production from glucose using the non- pathogenic Pseudomonas putida KT2440. Microbial Cell Factories 10: 80.

abstracts book 315 P179EXTREMOphiles_2012 Functional metagenomics of thermophilic phage replisomes David Meada, Sally Floyda, Audrey Klingelea, Brian Hedlundb, Jeremy Dodsworthb, Ron Godiskaa, Michael Lodesa and Tom Schoenfelda

aLucigen Corporation, Middleton, WI, USA bUniversity of Nevada-Las Vegas, NV, USA E-mail: [email protected]

DNA polymerases can replicate a 4 Mb bacterial genome with extremely high fidelity in minutes. However, these capabilities have not translated to PCR amplificationin vitro, which is limited to sequences of ~10 kb and requires 5 to 6 hours, resulting in much lower fidelity. Notably, all commercially available thermostable DNA polymerases are derived from one of two very closely related groups of microbial polymerases (bacterial Pol I and archaeal Pol II), whose biological roles include DNA repair and lagging strand synthesis. Phage and viral DNA polymerases represent an alternative source of enzymes for sequencing and amplification as they are processive replicase enzymes that are significantly more diverse than those of their hosts1. They have distinct and potentially more useful biochemical properties compared to the host repair enzymes, such as higher fidelity, higher processivity and improved nucleotide analogue incorporation. These properties could be exploited to improve DNA amplification and sequencing by increasing sensitivity, speed, accuracy, and reliability. Very little is known about viral replicase enzymes from high temperature environments, due to the low number of cultivated viruses that have been discovered and even fewer replicase enzymes. Traditionally, new DNA polymerases have been identified by screening culturable microorganisms for enzyme activity. In this study, metagenomic libraries were screened for polymerases from viruses and bacteriophages to detect enzymes from viruses that have not been cultured in vitro. Viral metagenomes were isolated from Octopus2 and Black Pool hot springs (Yellowstone), as well as Great Boiling Spring (Nevada). Analysis of the assembled sequence data revealed at least three different classes of viral replicons, or operons containing DNA polymerases surrounded by putative accessory replication proteins. This contrasts with bacterial replication proteins that are seldom found in operons. In order to capture entire viral replisomes a linear cloning system capable of cloning and expressing 10-20 kb inserts was developed. A high throughput screen for functional thermophilic polymerases from one of the hot springs identified a new family of related polymerases and their surrounding accessory proteins. The goal is to expanded the search for new high temperature replicases to five geochemically distinct sites and compare the results for gene content and structure associated with geographical and temporal separation, replication strategies and to provide new biological reagents.

References · Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme (2012) PLoS ONE 7: e38371. · Assembly of viral metagenomes from Yellowstone Hot Springs. (2008) Appl Environ Microbiol 74: 4164.

316 abstracts book EXTREMOphiles_2012

session 7 applications

abstracts book 317 EXTREMOphiles_2012

318 abstracts book EXTREMOphiles_2012P180 Natrialba sp. strain E21, an extremely halophilic bacterium isolated from a solar saltern in Ain Salah (Algeria) able to produce biosurfactant by free and entrapped cells

Salima Kebbouche-Ganaa, Souad khemilia, Mohamed Lamine Ganab and Imene Feriounea

aVALCORE Laboratory, Department of Biology, University M’Hamed Bougara of Boumerdes, 1st November Avenue, 35000 Boumerdes, Algeria bCentre of Research and Development, Biocorrosion Laboratory, SONATRACH, Avenue of 1st November,35000 Boumerdes, Algeria E-mail: [email protected]

Halotolerant microorganisms able to live in saline environments offer a multitude of actual or potential applications in various fields of biotechnology. This is why some strains of Halobacteria from an Algerian culture collection were screened for biosurfactant production in a standard medium using the qualitative drop-collapse test and emulsification activity assay (kebbouche-Gana et al., 2009). An isolate, strain E21 was obtained from a solar saltern located close to Ain Salah in the south of Algeria. The strain had salt growth requirement that was always between 1.5 and 4.0 M, the optimum of salts is at about 2.54 M, it was grew optimally at 40 ºC and pH 5.5-9.0. Analysis of 16S rRNA gene sequence indicates that the strain has 93 % sequence similarity with the genus Natrialba. A similarity (based on partial 16RNAr sequence) significant for possible species relatedness (98%) is found with the two validly described species Natrialba aegyptia 40(99%) and Natrialba taiwanensis strain B1 (98%) (Hezayen et al., 2001; Xu et al., 2001). So, the biosurfactant synthesis was followed by measuring surface tension and emulsifying index E24 over 18 days at 40°C and at neutral pH. Some diffusional limitations in alginate beads affected the kinetics of biosurfactant production when compared to that obtained with free cells culture. The minimum values of surface tension were 27 mN m-1 and 30 mN m-1 achieved after 18 days with free and immobilized cells, respectively, while the corresponding maximum E24 values were 65.3 % and 62.3 %, respectively. The critical micellar concentration was found to be 280 mg l-1. This is the first report about production of biosurfactants in the extremes conditions by free and entrapped cells.

References · S.Kebbouche-Gana, M.L. Gana, S. Khemili, F. Fazouane-Naimi, N.A. Bouanane, M. Penninckx and H. Hacene (2009. Isolation and characterization of halophilic Archaea able to produce biosurfactants. J Ind Microbiol Biotechnol 36: 727-738S. · F. F. Hezayen, B.H.A. Rehm, B.J. Tindall and A. Steinbüchel (2001). Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp. nov. and description of Natrialba aegyptiaca sp. nov., a novel extremely halophilic, aerobic, non-pigmented member of the Archaea from Egypt that produces extracellular poly(glutamic acid). Int J Syst Evol Microbiol 51: 1133-1142. · Y. Xu, Z. Wang, Y. Xue, P. Zhou, Y. Ma, A. Ventosa, and W. D. Grant, (2001). Natrialba hulunbeirensis sp. nov. and Natrialba chahannaoensis sp. nov., novel haloalkaliphilic archaea from soda lakes in Inner Mongolia Autonomous Region, China. Int J Syst Evol Microbiol 51: 1693-1698.

abstracts book 319 P181EXTREMOphiles_2012 Antagonistic activity of an extremely halophilic bacterium Bacillus sp. nov. B21 obtained from an Algerian oil field against sulfate-reducing bacteria consortium Mohamed Lamine Ganaa and Salima Kebbouche-Ganab aCentre of Research and Development, Biocorrosion Laboratory, SONATRACH, Avenue of 1st November,35000 Boumerdes, Algeria bVALCORE Laboratory, Department of Biology, University M’Hamed Bougara of Boumerdes, 1st November Avenue, 35000 Boumerdes, Algeria E-mail: [email protected]; Algeria. [email protected]

Antagonist microorganisms have been successfully used in agriculture; this provides the basis for using this approach in other fields, such as combating microbiologically influenced corrosion (MIC). In this study, we attempted to evaluate the potential uses of an halophilic antagonists bacteria strain B21 to reduce the sulfate- reducing bacteria (SRB) activity and the sulfides (H2S) produced corrosiveness in order to protect, in such a way metals from biocorrosion. The present study enlightens the role of the antagonistic potential of nonpathogenic strain B21 obtained from water coming from In-Amenas production oil reservoir (Algeria) against SRB consortium. The inhibitor effects of strain B21 were compared with those of chemical biocide tetrakishydroxymethylphosphonium sulfate (THPS), generally, used in the Algerian petroleum industry. The biological inhibitor shows much better and effective. Growth of SRB in coculture with bacteria strain B21 antagonist has highlighted a decline in the growth of SRB, a reduction in the production of sulfides with the consumption of sulfate. The observed effect seems more important in comparison to the effect caused by the biocide THPS tested. The strain B21 was facultative aerobic specie, salt growth requirement is always above 5% (w/v) salts with an optimal concentration of 10–25%. Phylogenetic analysis based on 16S rRNA gene partials sequences showed that strain B21 was a member of the genus Bacillus (EMBL, FR671419)., being most closely related to Bacillus qingdaonensis DQ115802 (94.0% sequence similarity), Bacillus aidingensis DQ504377 (94.0 %) and Bacillus salarius AY667494 (92.2 %) Wang et al., (2007); Xue et al., (2008); Lim et al., (2006) and Nielsen et al., (1995). Comparative analysis of the partial 16S rRNA gene sequence data, physiological, biochemical and phenotypic features of the novel isolate and related species of Bacillus indicated that strain B21 may represents a novel species within the genus Bacillus, for which the name Bacillus sp.B21. So, the results of this study indicate the potential for application of Bacillus strain B21 as biocontrol agents for the fight against corrosion in the oil industry.

References · Q. Wang, W. Li, Y. Liu, H. Cao, Z. Li and G. Guo (2007). Bacillus qingdaonensis sp. nov., a moderately haloalkaliphilic bacterium isolated from a crude sea-salt sample collected near Qingdao in eastern China. Int J Syst Evol Microbiol 57: 143-1147. · Y. Xue, A. Ventosa, X. Wang, P. Ren, P. Zhou, Y. Ma (2008). Bacillus aidingensis sp. nov., a moderately halophilic bacterium isolated from Ai-Ding salt lake in China. Int J Syst Evol Microbiol 58: 2828-2832. · J.M. Lim, C.O. Jeon, S.M. Lee, J.C. Lee, L.H. Xu, C.L. Jiang, C.J. Kim (2006). Bacillus salarius sp. nov., a halophilic, spore forming bacterium isolated from a salt lake in China. Int J Syst Evol Microbiol 56: 373-377. · P. Nielsen, D. Fritze, F.G. Priest (1995). Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology 141:1745–1761.

320 abstracts book EXTREMOphiles_2012P182 A reconstituted Mn(II)-peptide complex of Deinococcus radiodurans preserves immunogenicity of lethally irradiated vaccines against viruses and Staphylococcus aureus Elena Gaidamakova,a Ian Mylesb, Dennis McDaniela, Cedar Fowlerb, Patricia Valdezb, Manoshi Gayena,c, Paridhi Guptaa,c, Anuj Sharmaa, Pamela Glassd, Radha Maheshwaria, Sandip Dattab and Michael Dalya

aUniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA bNational Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA cBirla Institute of Technology and Science, Pilani, Rajasthan-333031, India dUS Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, USA E-mail: [email protected]

Sterilization of pathogens with gamma-radiation is an attractive approach for development of inactivated whole-organism vaccines. However, the radiation doses required to ensure sterility also destroy immunogenic epitopes needed to mount a protective immune response. We report that genome damage and killing can be uncoupled from epitope damage using a reconstituted Mn(II) peptide complex of D. radiodurans, a radiation- resistant bacterium. The Mn(II) complex (Daly et al., 2010) preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus (VEEV), and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine protected mice against methicillin-resistantS. aureus (MRSA) by eliciting T cell- and B cell-dependent responses. We demonstrate that structural integrity of viruses and bacteria can be preserved at radiation doses far above those which abolish infectivity.This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.

References · Daly, M.J., Gaidamakova, E.K., Matrosova, V.Y., Kiang, J.G., Fukumoto, R., Lee, D.Y., Wehr, N.B., Viteri, G.A., Berlett, B.S., and Levine, R.L. (2010). Small-molecule antioxidant proteome-shields inDeinococcus radiodurans. PLoS One 5: e12570.

abstracts book 321 P183EXTREMOphiles_2012 Effect of the halophilic bacteria on wheat growth under water stress in saline soils Ahmad Ali Pourbabaeea, Maryam Talebia, Mehdi Shorafab and Seyed Alireza Salamic

aBiotech Lab., Department of Soil Science, Faculty of Agriculture Engineering and Technology, University of Tehran, Karaj, Iran bDepartment of soil science, University of Tehran, Karaj, Iran cDepartment of horticultural science, University of Tehran, Karaj, Iran E-mail: [email protected]

Salinity stress is of important in arid and semi-arid areas of the world due to its impact on reducing crop yield (Jalili et al., 2009). On the other hand, wheat is the crop which drought and salinity conditions can reduce its yield. In this study, 2 halophilic bacteria were selected (under name TP7 and TP5) among 35 isolates based on the growth in presence of 5% NaCl and production of exopolysaccharide from saline soil. Identification based on the 16S rRNA gene sequence indicated that the strain TP7 (halotolerant) most closely related to Bacillus subtlis (98.49% similarity) and TP5 (moderate halophilic) (is under review). To determine the effect of this bacteria on chlorophyll and relative water content of wheat, the study was carried out in factorial form with completely randomize design on wheat under greenhouse condition. The factors included: bacteria in four levels; without inoculation of bacteria (B1), isolate TP7 (B2), isolate TP5 (B3), both isolates (B4) and salinity of soils in four levels ( 2, 4, 8, 16 dSm-1) and soil moisture in two levels (75 and 25 percent of available water).Chlorophyll content and relative water content were determined (Songsri et al,2009). The results showed that inoculated treatments had higher chlorophyll content and relative water content compared to non inoculated treatment. Increase in the chlorophyll content has been remarkable in inoculated treatments with strain TP7 and in relative water content by strain TP5 in saline soils 4 and 16 dSm-1respectively. Inoculation with these bacteria can increase plant resistance to salt and drought stresses by improvement of soil structure and water holding capacity.

References · P. Songsri, S.Jogloy, C.C. Holbrook, T.Kesmala and N.Vorasoot (2009). Association of root specific leaf area and SPAD chorophyll meter reading to water use efficiency of Peanut under different available soil water. Agr Water Manage 96: 790-798. · F. Jalili, K. khavazi, E. Pazira, A. Nejati, H. Asadi, H. Rasuli and M Miransari (2009). Isolation and characterization of ACC deaminas-producing fluorescent Pseudomonas, to alleviate salinity stress on canola growth. J Plant Physiol 166: 667-674.

322 abstracts book EXTREMOphiles_2012P184 Enzymatic degradation of PrPSc by a protease secreted from Aeropyrum pernix K1 Marko Snaidera, Tanja Vilfana, Maja Cernilecb, Vladka Curin Serbecb and Natasa Poklar Ulriha

aBiotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia bBlood Transfusion Center of Slovenia, Šlajmerjeva 6, 1000 Ljubljana, Slovenia E-mail: [email protected]

An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrPSc) from different species (human, bovine, deer and mouse). Degradation of the PrPSc isoform by an R30 fraction and purified protease was evaluated using the 6H4 anti- PrP monoclonal antibody and by detection of fragments from the N-terminal and C-terminal of PrPSc, by Western blotting using the EB8 anti-PrP monoclonal antibody and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrPSc, the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry: the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 34 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optimum of enzymatic activity of the purified pernisine in the presence of 1 mM

CaCl2 was at 105°C ±0.5 °C and pH 6.5 ±0.2. Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest PrPSc.

abstracts book 323 P185EXTREMOphiles_2012 Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4 Sotaro Sanoa and Shinsuke Fujiwaraa,b

aDepartment of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan bResearch Center for Environmental Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan E-mail: [email protected]

Reverse transcriptase is the enzyme that catalyzes DNA polymerization using RNA as a template (RNA- dependent DNA polymerase). The enzyme is used in various genetic experiments such as a microarray analysis by synthesizing cDNA or the start-site mapping of transcript mRNA. In these experiments, reverse transcriptases play central roles, and the enzymes derived from retroviruses, such as the Moloney Murine Leukemia Virus (MMLV) or the Avian Myeloblastosis Virus (AMV), are mainly utilized. In terms of application, the use of retroviral reverse transcriptase has two major drawbacks. One is low fidelity because of the lack of 3’-5’ exonuclease (proofreading) activity. The other is instability because retroviral reverse transcriptases are generally thermolabile. In the present study, another approach to create thermostable reverse transcriptase has been tried using DNA polymerases from thermophilic bacteria. Family A DNA polymerase (K4PolI) from Thermotoga petrophila K4 was obtained as a recombinant form, and the enzyme characteristics were analyzed. K4PolI showed thermostable DNA-dependent DNA polymerase activity with 3’-5’ exonuclease activity but no detectable RNA-dependent DNA polymerase activity. Its tertiary structure was speculated byin silico modeling to understand the binding situation between K4PolI and template DNA. Nine amino acids in the 3’-5’ exonuclease domain are predicted to be involved in DNA/RNA distinction by steric interference with the 2’ hydroxy group of ribose. To allow K4PolI to accept RNA as the template, mutants were constructed focusing on the amino acids located around the 2’ hydroxyl group of the bound ribose. The mutants in which Thr326, Leu329, Gln384, Phe388, Met408, or Tyr438 was replaced with Ala (designated as T326A, L329A, Q384A, F388A, M408A, or Y438A, respectively) showed RNA-dependent DNA polymerase activity. All the mutants showed reduced 3’-5’ exonuclease activity, suggesting that gain of reverse transcriptase activity is correlated with loss of 3’-5’ exonuclease activity. In particular, the mutants enabled direct DNA amplification in a single tube format from structured RNA that was not efficiently amplified by retroviral reverse transcriptase.

References · S.Sano et al. (2012) J Biosci Bioeng 113/3, 315-321.

324 abstracts book EXTREMOphiles_2012P186 The potential biotechnological applications of the exopolysaccharides produced by moderately halophilic bacteria Hakima Amjres, Victoria Béjar, Inmaculada Llamas and Emilia Quesada

Microbial Exopolysaccharide Research Group, Department of Microbiology, Faculty of Pharmacy, Cartuja Campus, University of Granada, 18071 Granada, Spain E-mail: [email protected]

Industry is constantly looking for exopolysaccharides with novel functional properties to satisfy the needs of modern technology, particularly in the fields of foodstuffs, cosmetics, and environmental cleaning and waste management. There is also a pressing modern demand for industry to resort to non-toxic, biodegradable, environmentally friendly substances. Most of the microorganisms used so far for the industrial production of polysaccharides belong to a small number of non-halophilic, soil-dwelling, diazotrophic taxa that often live in association with plants. Nevertheless, in recent years little or no progress has been made towards identifying further strains of this type, nor in widening their potential range of applications. Therefore a new approach to encountering polysaccharides with novel properties might be called for, entailing the investigation of different environments, particularly extreme ones. Within this context, our microbial exopolysaccharide research group (BIO-188) has made numerous initial findings pointing to the fact that many halophilic bacteria produce extracellular polysaccharides offering numerous applications in various fields of medicine and industry. Thus we have deemed it worthwhile to undertake a wide research program, examining microorganisms from several kinds of hypersaline habitat in an attempt to find new EPSs with different characteristics and potential uses. We review here the EPSs produced by halophilic bacteria, mainly species belonging to the Halomonadaceae and Alteromonadaceae families, which have been discovered and described by our research group. They have different chemical compositions and functional properties from those currently being marketed and used by industry (Mata et al., 2006; 2008; Ruiz-Ruiz et al., 2011; Llamas et al., 2010; 2012).

References · I. Llamas, J. A. Mata, R. Tallón, P. Bressollier, M. C. Urdaci, E. Quesada and V. Bejar (2010). Characterization of the exopolysaccharide produced by Salipiger mucosus A3T, a halophilic species belonging to the alphaproteobacteria, isolated on the Spanish Mediterranean seaboard. Mar Drugs 8: 2240-2251. · I. Llamas, H. Amjres, J. A. Mata, E. Quesada and V. Béjar (2012). The potential biotechnological applications of the exopolysaccharide produced by the halophilic bacterium Halomonas almeriensis Molecules (in press). · J. A. Mata, V. Béjar, I. Llamas, S. Arias, P. Bressollier, R. Tallón, M. C. Urdaci and E. Quesada (2006). Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis. Res Microbiol 157: 827-835. · J. A. Mata, V. Béjar, P. Bressollier, R. Tallón, M. C. Urdaci, E. Quesada and I. Llamas (2008). Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae. J App Microbiol 105: 521-528. · C. Ruiz-Ruiz, G. K. Srivastava, D. Carranza, J. A. Mata, I. Llamas, M. Santamaría, E. Quesada and I. J. Molina (2011). An exopolysaccharide produced by the novel halophilic bacterium Halomonas stenophila strain B100 selectively induces apoptosis in human T leukemia cells.App Microbiol Biotechnol 89: 345-355.

abstracts book 325 P187EXTREMOphiles_2012 Fast technique for finding naphthalene-degrading haloalkaliphilic actinomycetes Ninfa Ramírez-Durána, Reyna del Carmen Lara Severinoa, Hugo Ramírez Saad and Horacio Sandoval Trujillob

aFacultad de Medicina, Universidad Autónoma del Estado de México. Paseo Tollocan y Jesús Carranza s/n Colonia Moderna de la Cruz, Toluca, C.P. 50180, México bUniversidad Autónoma Metropolitana-Xochimilco. Calzada del Hueso 1100, Colonia Villa Quietud, Coyoacán, C.P. 04960, México, D.F. E-mail: [email protected]

Polycyclic aromatic hydrocarbons (PAHs) are environment pollutants from principally anthropogenic activities. Due to their molecular structure these compounds remain for long periods of time in the environment without change and that is the direct cause for their permanence and resistance to microbiological degradation. Hypersaline environments as well as many more ecosystems have suffered contamination. Nevertheless, there is little information concerning the halophilic bacteria to degrade hydrocarbons present in these hypersaline environments. Also, there are few assays aimed at the rapid detection of bacteria degrading PAHs in a solid medium. The best known method for the isolation of PAHs degrading bacteria is the Kiyohara assay (Kiyohara et al., 1982) but it is not the best for actinomycete isolation due to slow bacterial growth. The aim of this research was the development of a fast assay to evaluate PAHs degradation by halophilic actinomycetes. The solid culture medium for PAHs degradation contained 2 g KCl, 1 g KNO3, 1 g (NH4)2H2PO4, 0.5 g KH2PO4, 0.01 g yeast extract, 20 g agar and 10% saline (enough for 1 L) at 7.3+/- 0.1 pH. Halophilic actinomycetes isolated from various hypersaline environments were used. At first the actinomycetes were inoculated in parallel streaks in a Petri dish with minimum culture medium and a strip of filter paper impregnated for 5 min in a 5% w/v in a naphthalene-alcohol solution was placed perpendicularly to the streaks. Bacterial growth was observed after 72 h incubation at 37ºC. The second phase of the experiment was confirmatory. The culture medium was prepared, sterilized and cooled at room temperature for 5 min. A 5% w/v naphthalene-alcohol solution was heated up to the temperature of the culture medium. The warm naphthalene solution and the culture medium were mixed until crystal suspension appeared (150 rpm) and then it was poured in dishes to solidify. The same actinomycetes strains used in the first part of the assay were inoculated in single streaks and incubated under the same conditions. After the 72 h period only the degrading strains showed growth next to the naphthalene impregnated filter paper. In the second phase the actinomycetes inoculated in the culture medium added with naphthalene suspension showed greater growth with clear halos around some colonies due to naphthalene consumption. The screening assay with the filter paper strip is found to be useful with great amounts of strains. However, the confirmatory assay with the culture medium-naphthalene suspension is a more reliable test for PAHs degradation since the uniform suspension of the naphthalene in the agar promotes a good growth of these slow growing bacteria and evidences the hydrocarbon consumption by the loss of turbidity around the bacterial colonies. We report 15 strains PAHs degrading belonging to the genera: Saccharomonospora and Nocardiopsis. All the strains are able to grow from 5 to 15% NaCl, with an optimum at 10% NaCl. The range of pH for their growth is from 8-10, with the optimum at pH 9.

References · Kiyohara, H., Naga, K. and Yana, K. 1982. Rapid screen for bacteria degrading water-insoluble, solid hydrocarbons on agar plates. Appl. Environ. Microbiol 43: 454-457.

326 abstracts book EXTREMOphiles_2012P188 Evaluation of the applicability of extremophiles for generation of EtOH and other valueable products from waste sulphite pulping liquor M. Weissgrama,b, H.K. Weberb and C. Herwiga

aDepartment of Bioprocess technology, Technical University Vienna, 1010 Vienna, Austria bKompetenzzentrum Holz GmbH, Altenberger Strasse 69, 4040 Linz, Austria E-mail: [email protected]

Spent sulfite liquor contains substances like xylane, galactomannane, degradation products of polysaccharide as well as by- and degradation products of monomeric sugars. These substances are promising substrate candidates for fermentation into valuable products like ethanol or organic acids by specialized microorganisms. Generally these waste flows leave the process at high temperatures, have a low pH value and contain considerable amounts of toxins. Without major changes (time consuming cooling steps, pH adaption and removal of toxins) growth and metabolism of normal mesophilic microorganisms is not possible in such an environment. Therefore, this study aims to elicit the potential of extremophile organisms to sustain a robust fermentation process of spent sulfite liquor to value-adding products. A variety of strains has been reported to be capable of fermenting the above mentioned substrates to ethanol or industrially interesting organic acids like succinic acid. In a first step the xylose-fermenting strainsA. succhinogenes, C. lactoaceticus, C. thermocellum,the arabinose- and xylan-fermenting strains T.saccharolyticum, T. lettingae, C. saccharolyticus and the cellobiose fermenting strain T. cellulolyticus, all of them thermophiles, were investigated. Stepwise altering of growth conditions from home medium (as indicated by the supplier DSMZ) to defined medium containing the substrates xylose, arabinose, xylan and cellobiose, as well as stepwise altering of pH values (from pH 5 to pH 2) and temperature (from 60°C to 80°C) was used to elucidate the viability limits of the used strains. Strains capable of considerable production of EtOH or organic acid were tested for their resistance against toxins commonly found in spent sulfite liquor. Changes in growth rates, as well as changes in production of EtOH and organic acids from constituents of spent sulfite liquor were investigated throughout the whole experimental setup in a qualitative approach.

abstracts book 327 P189EXTREMOphiles_2012 Health from extreme environments: searching for antimicrobial and antitumor activities in acidotolerant microalgae María Vázqueza, Francisco Navarrob, Alberto Toimilb, Sandra Morab, Miguel Ángel Castañod, Adriana Márquezd, Inés Garbayoc, Eduardo Forjánb and Carlos Vílcheza

aAlgal Biotechnology, CIECEM, University of Huelva, 21760 Matalascañas, Spain bEnvironmental Biology and Public Health, University of Huelva, 21007 Huelva, Spain cAlgal Biotechnology, Faculty of Sciences, University of Huelva, 21007 Huelva, Spain dHospital «Juan Ramón Jiménez», Servicio Andaluz de Salud, 21005 Huelva, Spain E-mail: [email protected]

Microalgae have been referred as interesting natural sources of valuable macromolecules, many of which exhibit multiple bioactivities with applications in human health, such as antioxidant, anti-inflammatory, antimicrobial, antiviral or antitumor activities. These bioactive molecules obtained from microalgae could also be called algaceuticals. The aim of this work is to investigate antimicrobial and antitumor activities synthesized and accumulated acidotolerant microalgae: Coccomyxa onubensis and Chlamydomonas acidophila. Both microalgae are extremophile microorganisms isolated from acid drainages of the Río Tinto mining area, and have been selected based on the highly expressed antioxidant response. This work includes (a) growth of the selected microalgae under suitable conditions to promote target compounds biosynthesis; (b) chemical assessment of crude extracts, and (c) testing selected chemical fractions from crude extracts for desired bioactivities. To assess the value of these microalgae as a source for antimicrobial and antitumor compounds, specifically designed methodology is being used including in-vitro and in-vivo assays for bioactivities detection and chemical characterization of bioactive compounds via HPLC and GC-MS. Microalgal extracts are being testedin vitro for their antimicrobial effects against several pathogen bacteria, fungi and yeast species including Pseudomonas, E. coli antibiotic resistant strains, Proteus, Staphylococcus, Candida and Aspergillus, among others. First very promising positive results were already obtained, particularly against Gram (-) bacteria. Microalgal extracts will also be tested for their antitumor properties on selected human cell lines. Since found bioactive compounds might also be supplied to the human body in the form of active compounds-enriched biomass, algal biomass toxicity on health will also be tested in vivo, being included in rat chow to assess food biosafety of potential algae-based food supplements.

Acknowledgements This work has been supported by grant AGR-4337 (Proyecto de Excelencia, Junta de Andalucía)

328 abstracts book EXTREMOphiles_2012P190 Coccomyxa onubensis, a novel acidotolerant microalga: identification and biotechnological value Isabel Vaqueroa, Mayca Márqueza, Stefan Girlichb, Juan Luis Fuentesd, Benito Mogedasa, Manuel González del Valled, Inés Garbayoa, Eduardo Forjána, Volker Hussc and Carlos Vílcheza

aAlgal Biotechnology Group, CIECEM, University of Huelva, 21760 Matalascañas, Spain bUniversity of Applied Sciences, Wiesenstraße 14, 35390 Gießen, Germany cUniversity of Erlangen, Staudtstr. 5, 91058 Erlangen, Germany dBioAvan S.L., Parque Empresarial Torneo, c/ Geología 97, 41015 Sevilla, Spain E-mail: [email protected]

A novel unicellular microalga was isolated from an acidic drainage in the mining area of Huelva, Spain. The isolated ribosomal 18S subunit rDNA sequence showed homology with known sequences from green microalgae, the closest sequence (98% homology) belonging to the genus Coccomyxa. It clustered with two environmental sequences as a distinct subgroup of the genusCoccomyxa . The isolated microalga therefore was an up to now uncultured microalga and was finally named asCoccomyxa onubensis. The microalga adapted to naturally grow on highly acidic conditions (pH 1.7–3.1), with high concentrations of dissolved metals, including Fe, Cu, Mn, Ni, and Al ions. The novel microalga contains a large chloroplast with presence of lipid droplets and a large number of starch bodies. The isolated microalga showed increased productivities when cultivated photoautotrophically on alternating high/low CO2 supply periods. Unlike other acidotolerant microalgae, Coccomyxa onubensis showed high growth rates (up to 0.6 d-1) in a suitable culture medium and can be successfully grown in continuous mode in flat panel photobioreactors and safe from noticeable external biological contamination due to the low pH. Microalgal growth and lutein accumulation –commercial value antioxidant- were shown to be iron and copper dependent. In particular, when cultivated under suitable temperature, light and nutrient conditions, lutein accumulated up to about 11 g.kg-1, by far the highest lutein accumulation ever reported in microalgae.

Acknowledgements This work has been done thanks to the financial support from Junta de Andalucia (Grant AGR-4337) and FEDER funds from »Programa Operativo de Cooperación Transfronteriza» Spain-Portugal 2007-2013 (POCTEP).

abstracts book 329 P191EXTREMOphiles_2012 Enhancement of polyunsaturated fatty acids and carotenoid production in the extremophile microalga Coccomyxa onubensis by UVA radiation Mª Carmen Ruiza, Živan Gojkovicc, Nora Janzerb, Mª José Dominguezc, Carlos Vilcheza, José M. Vegad and Inés Garbayoc

aAlgal Biotechnology Group, CIECEM, University of Huelva, 21760 Matalascañas, Spain bUniversity of Applied Science in Gießen (Germany) «Fachhochschule Giessen-Friedberg» cAlgal Biotechnology Group, Faculty of Sciences, University of Huelva. 21007 Huelva, Spain dFaculty of Chemistry, University of Sevilla, Avda. Prof. García González, 41012 Sevilla, Spain E-mail: [email protected]

Polyunsaturated fatty acids (PUFAs) have been shown to be beneficial in the prevention of human illnesses. Carotenoids are also of significant commercial interest in the food and cosmetics industries, and do play an important role in plants protecting photosynthesis from being damaged by UV light. The objective of this work was to study the production of PUFAs and carotenoids, in response to UVA treatment of the extremophile microalga Coccomyxa onubensis, a carotenoid-enriched unicellular microalga, isolated from an acidic river in Huelva, Spain. C. onubensis was subjected to 96h exposure of continuous UV-A radiation added to photosynthetically active radiation (PAR, 400– 700 nm). The results indicate that UV-A radiation produces large increases in total carotenoid levels (especially in lutein content) and did not adversely affect the rate of growth. The maintenance of increased carotenoid levels required continuous UV-A radiation supply. Significant increases of total PUFAs and lipid content was found during the UVA exposure period. Concentrations of individual PUFAs, including linoleic acid as well as total carotenoids, were significantly increased at different stages of the UVA treatment. The expected antioxidant response to the incident UV-A radiation consisted of increases in lutein (an antioxidant carotenoid) and PUFAs content. The results suggest that modulated UV-A radiation can be used as a tool to stimulate value antioxidant molecules accumulation in acidophile/acidotolerant microalgae.

Acknowledgements This work has been supported by grant AGR-4337 (Proyecto de Excelencia, Junta de Andalucía).

330 abstracts book EXTREMOphiles_2012P192 Thermostable fluorescent protein palette for the study of thermophilic metabolism and its regulation Yamal Al-Ramahi, José Berenguer and Aurelio Hidalgo

Centro de Biología Molecular Severo Ochoa (UAM-CSIC). Universidad Autónoma de Madrid 28049, Madrid, Spain E-mail: [email protected]

Thermus thermophilus is an excellent model organism for basic and applied research (1). Unlike other organisms Thermus thermophilus grows easily in the laboratory, can acquire exogenous DNA by means of an extraordinarily efficient mechanism of natural competence, and usually is the organism of choice for structural biology thanks to the ease of its enzymes and multiproteinic complexes to crystallize. Although some tools such as antibiotic resistance genes are being developed as molecular markers, there is still a shortage of tools to study the physiology of thermophiles and its regulation, especially in comparison to other models such as Escherichia coli. Fluorescent proteins constitute the basis of powerful tools providing solutions for the study of physiological processes and the development of biotechnological applications. Since the discovery of the green fluorescent protein fromAequorea victoria by Shimomura fifty years ago, great knowledge has been achieved regarding the three dimensional structure and the mechanisms for fluorescence emission, a broad color palette has been developed and the number of successful applications is rapidly increasing. There were no existing examples of the applicability of these techniques in thermophiles until recently (2). The results achieved by protein engineering of superfolder green fluorescent protein (the first thermostable variant efficiently used in Thermus thermophilus) evidence potential to broaden the spectrum of the thermoestable fluorescent protein palette and its future applications.

References · Cava et al. 2009. Extremophiles 13: 213-231. · Cava et al. 2008. Environmental Microbiology 10: 605- 610.

abstracts book 331 P193EXTREMOphiles_2012 Usage of halotolerant bacteria toward bioconversion containing hydrophilic ionic liquid Atsushi Kurata, Humiya Seno, Chiaki Matsuhara, Takuma Tatsunami, Yasuyuki Ikeda and Noriaki Kishimoto

Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara City, Nara 631-8505, Japan E-mail: [email protected]

Ionic liquids (ILs) are composed of a bulky asymmetrical cation and a small anion. Unlike conventional organic solvents, ILs are able to dissolve many compounds. Thus, ILs are useful as reaction media. Using a hydrophobic IL as reaction solvent, we developed a convenient procedure for enzymatic synthesis of 3-cyclohexylpropyl caffeate, which exhibits a strong antiproliferative activity on human tumor cells1) . However, we found difficulties in using hydrophilic ILs as reaction solvents in the enzyme reaction. It is suggested that hydrophilic ILs can cause changes in enzyme conformation and thereby enzyme denaturation 2). Thus, it would be desirable to develop enzymes that are stable and functional in hydrophilic ILs. The purpose of this research is to elucidate the mechanism of resistance to hydrophilic ILs in hydrophilic IL-tolerant microorganisms, and to identify enzymes that are stable in hydrophilic ILs. Mixed bacterial floras obtained from food, soil, sea water, or saltern samples were grown in the culture medium containing hydrophilic IL, namely, [BMIM][Cl]. Two bacterial isolates grew adequately in the broth containing 10% [BMIM][Cl], [BMIM][CF3SO3], or sodium chloride. The 16S rRNA gene sequences of the isolates had the highest similarities to those of bacteria belonging to the generaStaphylococcus and Bacillus. Using Staphylococcus sp. FSW1, the culture supernatant containing [BMIM][Cl] was analyzed by LC-MS. The [BMIM] cation was not degraded, indicating thatStaphylococcus sp. FSW1 is a hydrophilic IL ([BMIM][Cl]) -tolerant bacterium. Whereas, protease activity fromBacillus sp. CMW1 was detected in the culture supernatant containing [BMIM][Cl]. A proteinous substrate was degraded by the enzyme in the presence of 600 mM [BMIM] [Cl]. These results indicated that halotolerant bacteria could grew in the presence of hydrophilic ILs, and the enzyme from halotolerant bacteria would act in the presence of hydrophilic ILs. Hydrophilic ILs are able to dissolve cellulose, keratin, and lignin. Thus, using halotolerant bacteria and enzymes with hydrophilic ILs, a novel system for efficient conversion of these compounds could be constructed.

References · Kurata, A et. al. (2011) Applications of Ionic Liquids in Science and Technology, Scott Handy (ed.), Intech, Capter 2, pp. 22-44. · Anderson, JL et. al. (2002) J Am Chem Soc 124:14247-14254.

332 abstracts book EXTREMOphiles_2012P194 Analysis of the immunological effect of Halorubrum sodomense Han-Seung Leea, Seulki Parka, Ji Eun Ohb, Danbie Janga, Byoung-Chan Kimc, Dong-Woo Leec and Heung-Kyu Leeb

aDepartment of Bio-Food Materials, College of Medical and Life Sciences, Silla, University, Busan, Korea bGraduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea cKorea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea E-mail: [email protected]

Halophilic archaea thrive ubiquitously in environments of high salt concentration and some strains have been isolated from salty fermented foods. However, in recent years, DNAs of haloarchaea have been detected by molecular analysis from human intestinal mucosa and fecal samples (Oxley et al., 2010; Nam et al., 2008), which indicated haloarchaea might be members of human microbiome (Dridi et al., 2011). We tried to isolate and culture halophilic archaea from two hundred human fecal samples. After serial transfer of five grown samples, we amplified partial 16S rRNA genes using haloarchaea specific primers. Interestingly, phylogenetic analyses based on the partial 16S rRNA gene sequence comparison indicated all five strains were Halorubrum species and one strain’s sequence exactly identified to H. sodomense (100% 16S rRNA sequence identity). However pure culture is failed for unknown reason.

Some research represented halophilic archaea might have positive effects on fermented food’s safety (Aponte et al., 2010) but their role for human health and disease is still unknown. For safety issue, we tried to examine the ability of H. sodomense to induce the inflammatory responses by dendritic cells and macrophages derived from mouse bone marrow. Interestingly, H. sodomense did not lead to the production of proinflammatory cytokines such as IL-12, IL-6 and TNF-alpha, and the up-regulation of costimulatory molecules such as CD40, CD86 and MHC II after H. sodomense infection to dendritic cells and macrophages in vitro. We will further investigate whether H. sodomense could induce immune tolerance in intestine through the generation of regulatory T cells from naïve T cells in vivo.

References · A. P. Oxley et al. (2010). Halophilic archaea in the human intestinal mucosa.Environ Microbiol 12: 2398-2410. · Y. D. Nam et al. (2008). Bacterial, archaeal, and eukaryal diversity in the intestines of Korean people. J Microbiol 46: 491-501. · B. Dridi et al. (2011). Archaea as emerging organisms in complex human microbiomes. Anaerobe 17: 56-63. · M. Aponte et al. (2010). Could halophilic archaea improve the traditional salted anchovies (Engraulis encrasicholus L.) safety and quality? Lett Appl Microbiol 51: 697-703.

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Thermodynamics and structure & function relationship of α-amylases from thermophilic bacteria from Tulsi Shyam, Gujarat, India Bhavtosh A. Kikani and Satya P. Singh

Department of Biosciences, Saurashtra University, Rajkot – 360 005, Gujarat (India) E-mail: [email protected]; [email protected]

Thermophilic microorganisms are not extensively explored with respect to their diversity and biotechno- logical potential, as compared to mesophiles. Exploration of molecular diversity using culture dependent and metagenomic approaches along with biocatalytic potentials of thermophilic bacteria from Tulsi Shyam hot spring reservoir at Gujarat, India, are the major thrust of our research1. While dealing with the biocatalytic potentials, Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033)2, Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556) and Bacillus sp. TSSC-3 (GeneBank Number, EU710557) were screened as potent α-amylase producers. The α-amylases were purified by single step purification using hydrophobic interaction chromatography on Phenyl Sepharose 6FF with reasonably high yield and purification fold. The purified α-amylases were characterized with respect to substrate kinetics and stability2. Various thermody- namics parameters, such as ∆S*, ∆H*, E and ∆G* were computed and analyzed. The changes in secondary structure of the enzymes under various physicochemical conditions are analyzed by the Circular Dichroism (CD) spectroscopy. The attracting features of the study include easy and cost effective single step purification, high thermostability and broad pH stability of the enzyme. The calcium independent nature1,3 and resistance towards chemical denaturation point at the commercial applications.

References • Singh, S.P., Shukla, R.J. and Kikani, B.A. (2010). Molecular diversity and biotechnological relevance of thermophilic actinobacteria. In “Thermophiles in Environmental and Industrial Biotechnology” Springer Publication, UK (In Press). • B. A. Kikani and S. P. Singh (2011). Single step purification and characterization of a thermostable and calcium independent α-amylase from Bacillus amyloliquifaciens TSWK1-1 isolated from Tulsi Shyam hot spring reservoir, Gujarat (India), International Journal of Biological Macromolecules. 48: 676-681. • Kikani, B.A., Shukla, R.J. and Singh, S.P. (2010). Biocatalytic potential of thermophilic bacteria and actinomycetes. In “Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology” Formatex Research Center, Badajoz, Spain. Vol. 2: 1000-1007.

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index of authors index of authors index

abstracts book 335 EXTREMOphiles_2012

336 abstracts book EXTREMOphiles_2012

Adams, Michael W. W...... CL1; P81 Akita, Hironaga...... P101 Alavi, Sana...... P122 Albers, Sonja-Verena...... S2.1; P86; P165 Albuquerque, Luciana...... P6 Alcaide, María...... P168; P169 Allen, Eric...... S3.2 Alsafadi, Diya...... P93 Amils, Ricardo...... S1.2; P26; P29; P61; P62; P72; P90 Amoozegar, Mohammad A...... P3; P11; P28; P34; P37; P122 Anderson, Dominique...... P69 Andrei, Stefan Adrian...... P17 Anton, Josefa...... O21 Antranikian, Garabed...... O7; P96 Antunes, André...... P170 Aono, Riku...... P111; P112 Argandoña, Montserrat...... P149; P150; P151; P153; P177 Ascaso, Carmen...... O3; O23; P36 Atomi, Haruyuki...... O6; O30; P71; P111; P112 Averhoff, Beate...... P104 Babkin, Igor...... P20; P22 Bai, Wenqin...... P114 Banciu, Horia...... P17 Baricz, Andreea...... P17 Bartlett, Douglas...... S5.3 Bejar, Victoria...... P42; P47 Berenguer, José...... S2.2; P192 Birkeland, Nils-Kåre...... P124 Blamey, Jenny M...... P115; P127; P129; P140 Blesak, Karol...... P108; P109 Bonch-Osmolovskaya, Elizaveta...... O10; P4; P5; P14; P24; P52; P80; P87; P106 Borges, Nuno...... S6.2; O17; P81; P82; P123 Brockwell, David J...... O11; P113 Caglayan, Pinar...... P2 Cai, Lei...... P144 Calderón-Torres, Claudia Marissa...... P154 Callac, Nolwenn...... O8 Cárdenas, Juan Pablo...... P73 Cario, Anais...... O19; P74 Carré-Mlouka, Alyssa...... P32 Casamayor, Emilio O...... P9 Cavicchioli, Ricardo...... S5.2 Charlier, Daniel...... P146 Chernyh, Nikolai A...... P52; P87 Cockell, Charles...... S1.1 Coman, Cristian...... P17; P53 Corcelli, Angela...... P7 Corral, Paulina...... P7 Cowan, Don...... S3.1; O9; P43; P44; P51; P69; P172; P173; P178 Crespo, Carla...... P83; P84 Cunha, Sofia...... O26; P75 da Costa, Milton S...... O26; P6; P75

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d’Avó, Filipa...... O26; P75 da Silva Esteves, Ana M...... S6.2; P81 Daupan, Socorro Martha Meg-ay V.... P55 de la Fuente Vicenta...... P90 de la Haba, Rafael R...... P10; P23 de los Ríos, Asunción...... O3; P36 del Moral, Catalina...... P61 Didari, Maryam...... P37 DiRuggiero, Jocelyne...... O3; O23 Dougan, Lorna...... O11; P113 Doukyu, Noriyuki...... P56 Driessen, Arnold J. M...... O5; P86 Elleuche, Skander...... P96 Escudero, Lorena V...... P19; P60 Faramarzi, Mohammad Ali...... P126 Fazeli, Seyed A. S...... P3; P11; P34 Fedoy, Anita-Elin...... P124 Fernández, Ana B...... P16; P50; P166 Ferrara, Maria Carmina...... P110 Flores M., Patricio A...... P127 Forterre, Patrick...... S1.4 Franzetti, Bruno...... S4.1; P91 Fujinami, Shun...... P59 Fujisawa, Makoto...... P171 Fujiwara, Shinsuke...... P79; P185 Fukazawa, Risako...... P119; P120 Fukuda, Wakao...... P71 Gagen, Emma J...... P58 Gaidamakova, Elena...... P182 Gana, Mohamed Lamine...... P180; P181 García, María Teresa...... P54; P121 Gavrilov, Sergey...... P24; P80 Gerasimenko, Ludmila...... P30; P31 Ghai, Rohit...... S3.3; P166 Giudici-Orticoni, Marie-Therese...... O4; P125 Glaring, Mikkel Andreas...... P39; P138 Godfroy, Anne...... O8 Gogliettino, Marta...... P95 Gómez, Felipe...... S1.2; O1 Gonçalves, Luis G...... S6.2; O17 González, Roberto...... P25 González-Grau, Juan Miguel...... P1 Gouy, Manolo...... S4.1; S4.2 Grant, William D...... O7; P43 Guiliani, Nicolas...... P89 Gulecal, Yasemin...... P63 Gutiérrez, Carmen...... P7 Haenelt, Inga...... P136 Hagiuda, Emi...... P117 Hamajima, Yuki...... P130; P131 Hausner, Winfried...... P156; P162 Hidalgo, Aurelio...... S2.2; P192

338 abstracts book EXTREMOphiles_2012

O26; P75 Higashibata, Hiroki...... P57; P79 S6.2; P81 Hirata, Akira...... P97; P98 P55 Hoffmann, Toni...... O11; P113 P90 Holmes, David...... P73; P174 P10; P23 Horikoshi, Koki...... P33 O3; P36 Hriscu, Monica...... P133 P61 Huang, Li...... O13 P37 Infante, Carmen...... P13; P16 O3; O23 Inoue, Akira...... P41; P105 O11; P113 Ito, Masahiro...... S2.3; P38; P59 P56 Iwasaki, Toshio...... P117; P118; P119; P120 O5; P86 Iwata, Fumiya...... P128 P96 Janecek, Stefan Adrian...... P108; P109 P19; P60 Jang, Danbie...... P194 P126 Jebbar, Mohamed...... O18 P3; P11; P34 Jiang, Kai...... P64 P124 Johnsen, Ulrike...... P85 P16; P50; P166 Jones, Brian E...... P43 P110 Jorge, Carla D...... P123 P127 Kaminski, Lina...... P163 S1.4 Kanai, Tamotsu...... O30; P71 S4.1; P91 Kanao, Tadayoshi...... P116; P155 P59 Kang, Sung Gyun ...... S6.1; P78; P135 P171 Kanoksilapatham, Wirojne...... P1 P79; P185 Karp, Matti...... P167 P119; P120 Kato, Chiaki...... P38; P131 P71 Kawamoto, Jun...... P134 P58 Kawamura, Takuya...... P99; P97 P182 Kawarabayasi, Yutakawa...... O25; P100 P180; P181 Kebbouche-Gana, Salima...... P180; P181 P54; P121 Khomarbaghi, Zahra...... P28 P24; P80 Kikani, Bhavtosh A...... P195 P30; P31 Kim, Yun Jae...... S6.1; P135 S3.3; P166 Kivistö, Anniina...... P167 O4; P125 Kixmüller, Dorthe...... P67 P39; P138 Kojima, Mio...... P105 O8 Kort, Julia...... P165 P95 Krulwich, Terry A...... O16 S1.2; O1 Kublanov, Ilya V...... P14; P24; P80 S6.2; O17 Kurata, Atsushi...... P194 P25 Kushida, Takashi...... P79 P1 Labes, Antje...... O29 S4.1; S4.2 Lafraya, Álvaro...... P168 O7; P43 Lage, Claudia...... O2 P89 Lake, James A...... OL1 P63 Lauro, Federico M...... S5.2 P7 Lee, Hyun Sook...... S6.1; P78; P135 P136 Lee, Han-Seung...... P194 P117 Lentini. Valeria ...... P18 P130; P131 León, Mª José...... P13; P16; P50 P156; P162 Li, Ming...... P143 S2.2; P192 Littlechild, Jennifer...... O12; P39

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Liu, Jingfang...... P142; P145 Llamas, Inmaculada...... O27; P157; P158; P186 López-Hermoso, Clara...... P10; P13; P16 Lylloff, Jeanette Eva...... P138 Ma, Yanhe...... P64; P114; Maciel Paulo, Elinalva...... P132 Mackay, Stephen...... P173 Mackenzie, Roy...... P40 Madern, Dominique...... S4.1 Marín, Irma...... P66 Martínez, Mónica...... P169 Matsushita, Shinichi...... P117; P119; P120 Mead, David...... O24; P179 Mehrshad, Maliheh...... P34; P37 Mellado, Encarnación...... P54; P121 Merkel, Alexander Y...... P15; P24; P80 Miura, Takeshi...... P33; P41 Mohmmed, Abdolkader A...... P88 Moracci, Marco...... S4.3; P107; P110 Moreno, Mª de Lourdes...... P54 Morgan, Hugh...... P49 Morikawa, Masaaki...... P8 Morozova, Vera...... P20; P22; P21; Müller, Volker...... S6.3; P136 Nakasone, Kaoru...... P139 Narumi, Issay...... P79; P152; Nieto, Joaquin J...... P149; P150; P151; P153; P177 Oger, Philippe...... O18; O19; P74 Oggerin, Monike...... S1.2; P29; P61 Ohlhoff, Colin...... P172 Onodera, Takefumi...... P152; P147 Oren, Aharon...... O20; P65 Oueriaghli, Nahid...... P42; P47 Palmieri, Giana...... P95 Papke, R. Thane...... S3.4; P7; P10; P23 Pasic, Lejla...... S3.3; P46; Pawar, Sudhanshu...... P77 Pawlowski, Alice...... P176 Peralta, Rocío...... P115 Pereira, Jairo...... P129; P115 Perevalova, Anna...... P106 Pérez, Dolores...... P121 Perugino, Giuseppe...... O14; P107 Pinto, Leonardo H...... P161 Piubeli, Francine...... P54; P149; P150; P153; P177 Podar, Mircea...... O22 Podosokorskaya, Olga...... P14; P24; P80 Poklar Ulrih, Natasa...... P184 Pourbabaee, Ahman Ali...... P183 Prangishvili, David...... S2.4 Quesada, Emilia...... O27; P42; P47; P157; P158; P186 Rainey, Fred...... S1.3

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P142; P145 Ramirez Duran, Ninfa...... P45; P187 O27; P157; P158; P186 Ramirez Saad, Hugo...... P45; P187 P10; P13; P16 Rauch, Bernadette...... P148 P138 Rettberg, Petra...... S5.4 P64; P114; Rivas, Luis A...... P29; P35 P132 Rodrigues, Marta V...... S6.2; P82 P173 Rodriguez, Nuria...... S1.2; P26; P29; P61; P90 P40 Rodriguez-Valera, Francisco...... S3.3; P16; P50; P166 S4.1 Rohulya, Olha V...... P86 P66 Ron, Emanuel...... P84 P169 Rossi, Mosè...... S4.3; O14; P95; P107 P117; P119; P120 Rua, María Luisa...... P25 O24; P179 Ruiz, Mª Carmen ...... P191 P34; P37 Sahm, Kerstin...... O7 P54; P121 Saitoh, Yoshimoto...... P12 P15; P24; P80 Salvador, Manuel...... P149; P150; P153; P177 P33; P41 Salzer, Ralf...... P104 P88 Samylina, Olga...... P31 S4.3; P107; P110 San Martin, Patxi...... P72 P54 Sánchez-Andrea, Irene...... P26 P49 Sánchez-Porro, Cristina ...... P2; P10; P13; P16; P37; P50; P166 P8 Sánchez-Ruiz, José Manuel...... S4.4 P20; P22; P21; Santos, Helena...... S6.2; O17; P70; P81; P82; P123 S6.3; P136 Sanz, Jose Luis...... S1.2; P26 P139 Sarmiento, Felipe...... P129; P164; P79; P152; Sato, Takaaki...... P111; P112 P149; P150; P151; P153; P177 Satomura, Takenori...... P92 O18; O19; P74 Schönheit, Peter...... S6.4; P85 S1.2; P29; P61 Servin, Luis Eduardo...... P175 P172 Shen, Yulong...... P159; P160 P152; P147 Shin, Jae-Ho...... P103 O20; P65 Siristova, Lucie...... O28 P42; P47 Slobodkin, Alexander...... O10; P4; P5; P95 Slobodkina, Galina...... O10; P5; P4; S3.4; P7; P10; P23 Slutskaya, Elvira...... P102 S3.3; P46; Sokolova, Tatyana...... P52 P77 Strazzulli, Andrea...... P107 P176 Suzuki, Hirokazu...... O25 P115 Tahrioui, Ali...... O27; P157; P158 P129; P115 Takai, Ken...... S5.1 P106 Takano, Kazufumi...... P94 P121 Takashina, Tomonori...... P41 O14; P107 Thomm, Michael...... P58; P156; P162 P161 Tikhonova, Tamara...... P68 P54; P149; P150; P153; P177 Tikunova, Nina...... P20; P21; P22 O22 Tomita, Hiroya...... O6 P14; P24; P80 Tori, Kazuo...... P141 P184 Torres Béjar, Marta...... P158 P183 Tsubasa, Sato...... P155 S2.4 Tuffin, Marla...... S3.1; O9; P43; P44; P51; P69; P172; P173; P178 O27; P42; P47; P157; P158; P186 Tych, Katarzyna...... O11; P113 S1.3 Ueno, Yuko...... P137

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Urusibata, Wataru...... P8 Ushatinskaya, Galina T...... P30 Vaquero, Isabel...... P190 Vargas, Carmen ...... P149; P150; P151; P153; P177 Vazquez, María...... P189 Velasco, Esther...... P62 Ventosa, Antonio...... P2; P3; P7; P10; P13; P16; P23; P37; P50; P121; P166 Waege, Ingrid...... P156 Watanabe, Rei...... P56 Watkin, Elizabeth...... O15 Weber, Hedda.K...... P188 Weissgram, Michaela...... O23; P188 Wierzchos, Jacek...... O3 Willians, Wesley...... P178 Wu, Zhenfang...... P142; P143; P145 Xiang, Hua...... P76; P142; P143; P144; P145 Xue, Yanfen...... P64; P114 Yahiaoui, Houa...... P32; P48 Yamagami, Ryota...... P97; P98 Yoshii, Takahiro...... P27 Zhai, Binyuan...... P160

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P8 P30 P190 P149; P150; P151; P153; P177 P189 P62 P2; P3; P7; P10; P13; P16; P23; P37; P50; P121; P166 P156 P56 O15 P188 O23; P188 O3 P178 P142; P143; P145 P76; P142; P143; P144; P145 P64; P114 P32; P48 P97; P98 P27 P160 list of participants list of participants list

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344 abstracts book EXTREMOphiles_2012

A Cantoblanco, Madrid, 28049 Spain Adams, Michael W. W. [email protected] Dept. of Biochemistry and Molecular Biology University of Georgia Allen, Eric E. Green Street Scripps Institution of Oceanography Athens, GA, 30602 University of California, San Diego USA 9500 Gilman Drive, Mc0202 [email protected] La Jolla, CA, 92037-0202 USA Akita, Hironaga [email protected] Kyushu University Faculty of Agriculture Al-Mailem, Dina Fukuoka, 812-8581 Dept. of Biological Sciences Japan Kuwait University [email protected] Faculty of Science P.O. Box 5969 Alavi, Sana Safat, 13060 Dept. of Microbiology Kuwait University of Tehran [email protected] Extremophiles Lab School of Biology Alsafadi, Diya Tehran, 14155-6455 School of Chemistry and Chemical Biology Iran University College Dublin [email protected] Belfield, Dublin Ireland Albers, Sonja-Verena [email protected] Dept. of Molecular Biology of Archaea Max Planck Institute for Terrestrial Microbiology Amils, Ricardo Karl-von-Frisch-Strasse 10 Dept. of Molecular Biology Marburg, 35043 Universidad Autónoma de Madrid Germany CBMSO (CSIC-INTA) [email protected] Cantoblanco, Madrid, E28049 Spain Albuquerque, Luciana [email protected] Departamento de Ciências da Vida Universidade de Coimbra Amoozegar, Mohammad Ali Laboratório de Microbiologia Dept. of Microbiology Centro de Neurociências e Biologia Celular University of Tehran Coimbra, 3004-517 Extremophiles Lab Portugal School of Biology [email protected] College of Science Enghelab Street Alcaide, María Tehran, 14155-6455 Enzyme Discovery Iran Spanish National Council for Research (CSIC) [email protected] C/ Marie Curie 2

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Anderson, Dominique Argandoña, Montserrat Dept. of Biotechnology Dept. of Microbiology and Parasitology University of the Western Cape University of Sevilla Institute for Microbial Biotechnology and Faculty of Pharmacy Metagenomics C/ Profesor García González 2 Modderdam Road Sevilla, 41012 Bellville, Cape Town, 7535 Spain South Africa [email protected] [email protected] Ascaso, Carmen Andrei, Stefan-Adrian Dept. of Environmental Biology Dept. of Molecular Biology and Biotechnology Museo Nacional de Ciencias Naturales - CSIC Babes-Bolyai University C/ Serrano 115 bis 5-7 Clinicilor Street Madrid, 28006 Cluj-Napoca, 400006 Spain Romania [email protected] [email protected] Atomi, Haruyuki Antón, Josefa Dept. of Synthetic Chemistry and Biological Departamento de Fisiología, Genética y Chemistry Microbiología Kyoto University Universidad de Alicante Graduate School of Engineering Alicante, 3080 Katsura, Nishikyo-ku, Kyoto, 615-8510 Spain Japan [email protected] [email protected]

Antranikian, Garabed Averhoff, Beate Institute of Technical Microbiology Dept. of Molecular Microbiology and Bioenergetics Hamburg University of Technology Goethe University Frankfurt Kasernenstr. 12 Max-von-Laue-Str. 9 Hamburg, 21073 Frankfurt, 60438 Germany Germany [email protected] [email protected]

Antunes, André B Red Sea Research Center 4700 King Abdullah University of Science and Babkin, Igor Technology (KAUST) Institute of Chemical Biology and Fundamental Building 2, office 2227-W3 Medicine SB Thuwal, 23955-6900 Russian Academy of Sciences Saudi Arabia Laboratory of Molecular Microbiology [email protected] Novosibirsk, 630090 Russia Aono, Riku [email protected] Dept. of Synthetic Chemistry and Biological Chemistry Bai, Wenqing Kyoto University Institute of Microbiology 216, A4, A-cluster Chinese Academy of Sciences Katsura, Nishikyo-ku, Kyoto, 615-8510 No.1 Beichen West Road Japan Chaoyang District, Beijing, 100101 [email protected] China [email protected]

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Banciu, Horia Berenguer, Jose Dept. of Molecular Biology and Biotechnology Centro de Biología Molecular Severo Ochoa Babes-Bolyai University Universidad Autónoma de Madrid 5-7 Clinicilor Street C/ Nicolás Cabrera 5 Cluj-Napoca, 400006 Campus UAM Romania Madrid, 28049 Horia.Banciu@ubbcluj,ro Spain [email protected] Matthieu, Barba UCB Lyon 1 Bergquist, Peter Laboratoire de Biométrie et Biologie Evolutive Dept. of Chemistry and Biomolecular Sciences (UMR 5558) Macquarie University UCB Lyon 1 - Bât. Grégor Mendel (2e étage) Herring Road, North Ryde Villeurbanne, 69100 Sydney, NSW, 2109 France Australia [email protected] [email protected]

Baricz, Andreea Berkmen, Mehmet Dept. of Molecular Biology and Biotechnology Gene Expression Babes-Bolyai University New England Biolabs 5-7 Clinicilor Street 240 County Rd. Cluj-Napoca, 400006 Ipswich, MA, 1938 Romania USA [email protected] [email protected]

Bartlett, Douglas Blamey, Jenny Scripps Institution of Oceanography Facultad de Química y Biología University of California, San Diego Universidad de Santiago de Chile 4405 Hubbs Hall Fundación Científica y Cultural Biociencia 8750 Biological Grade José Domingo Cañas 2280 La Jolla, CA, 92037 Ñuñoa, Santiago, 7750132 USA Chile [email protected] [email protected]

Birkeland, Nils-Kåre Blesak, Karol Department of Biology Laboratory of Protein Evolution University of Bergen Institute of Molecular Biology, Slovak Academy of P.O. Box 7803, Sciences Bergen, NO-5020 Dubravska cesta 21 Norway Bratislava, SK-84551 [email protected] Slovakia [email protected] Béjar, Victoria Dept. of Microbiology Bonch-Osmolovskaya, Elizaveta Faculty of Pharmacy Winogradsky Institute of Microbiology University of Granada Russian Academy of Sciences Campus Universitario de Cartuja Prospekt 60-Letiya Oktyabrya 7/2 Granada, 18071 Moscow, 117312 Spain Russia [email protected] [email protected]

abstracts book 347 EXTREMOphiles_2012

Borges, Nuno Calderón-Torres, Marissa Biology Division Unidad de Investigación en Biomedicina (UBIMED) Instituto de Tecnologia Química e Biológica (ITQB) Universidad Nacional Autónoma de México Av. da República, Estação Agronómica Nacional Facultad de Estudios Superiores (Iztacala) Oeiras, 2780-157 Av. de los Barrios 1 Portugal Col. los Reyes Iztacala [email protected] Tlanepantla, 54090 Mexico Bondavalli, Athos [email protected] Universita Degli Studi di Parma Scandiano, Regio Emmilia 42019 Callac, Nolwenn Italy Laboratoire de Microbiologie des Environnements [email protected] Extrêmes Institut Universitaire Européen de la Mer (IUEM) Brockwell, David BP70 Faculty of Biological Sciences Plouzané, 29280 University of Leeds France 3, Cavendisk Road [email protected] Leeds, L529JT UK Cárdenas, Juan Pablo [email protected] Center for Bioformatics and Genome Biology Fundación Ciencia y Vida C Avda. Zañartu 1482 Ñuñoa, Santiago, 7780272 Cabral, Sara Chile Fundação para o Desenvolvimento Sócio- [email protected] Profissional e Cultural de Ribeira Grande Science Center – Azores Microbial Observatory Cario, Anaïs (OMIC) Laboratoire de Géologie de Lyon Rua Nª Sra de Fátima s/n Ecole Normale Supérieure de Lyon Rabo de Peixe, Ribeira Grande 46 allée d’Italie São Miguel, Azores, 9600-998 Lyon, 69007 Portugal France [email protected] [email protected]

Çaðlayan, Pinar Carre-Mlouka, Alyssa Dept. of Biology Molecules of Communication and Adaptation of Marmara University Microorganisms (MCAM-UMR7245) Faculty of Arts and Sciences National Museum of Natural History (MNHN) Göztepe Kampüsü CP54, 63 rue Buffon Kat: 2, C-201 Paris, 75005 Kadýköy, Istanbul, 34100 France Turkey [email protected] [email protected] Casamayor, Emilio O. Cai, Lei Centro de Estudios Avanzados de Blanes (CEAB) Institute of Microbiology Spanish National Council for Research (CSIC) Chinese Academy of Sciences Acc. Cala Sant Francesc 14 No.1 West Beichen Road Blanes, Girona, 17300 Chaoyang District, Beijing, 100101 Spain China [email protected] [email protected]

348 abstracts book EXTREMOphiles_2012

Cavicchioli, Ricardo School of Biotechnology and Biomolecular Sciences Cowan, Don The University of New South Wales (UNSW) Dept. of Genetics Sydney, NSW, 2052 University of Pretoria Australia Pretoria, 0002 [email protected] South Africa [email protected] Charlier, Daniel Dept. of Microbiology Crespo Melgar, Carla Fabiana Vrije Universiteit Brussel Dept. of Biotechnology Pleinlaan 2 Lund University Brussels, B-1050 Getingevägen 60 (Kemicentrum) Belgium Lund, 22241 [email protected] Sweden [email protected] Chernyh, Nikolai Winogradsky Institute of Microbiology Cunha, Sofia Russian Academy of Sciences Centro de Neurociências e Biologia Celular Prospekt 60-Letiya Oktyabrya 7/2 Universidade de Coimbra Moscow, 117312 Largo Marquês de Pombal Russia Coimbra, 3004-517 [email protected] Portugal [email protected] Cockell, Charles School of Physics and Astronomy University of Edinburgh D James Clerk Maxwell Building Edinburgh, EH9 3JZ d’Avó, Filipa UK Centro de Neurociências e Biologia Celular [email protected] Universidade de Coimbra Largo Marquês de Pombal Coman, Cristian Coimbra, 3004-517 Ecology and Taxonomy Portugal Institute of Biological Research [email protected] 48 Republicii Street Cluj-Napoca Da Costa, Milton Romania Dept. of Life Sciences [email protected] University of Coimbra Coimbra, 3301-104 Corcelli, Angela Portugal Dept. of Basic Medical Sciences [email protected] University of Bari «Aldo Moro» Bari, 70124 da Silva Esteves, Ana María Italy Cell Physiology and NMR Group [email protected] New University of Lisbon Instituto de Tecnologia Química e Biológica (ITQB) Corral, Paulina Av. da República, Estação Agronómica Nacional Dept. Microbiology and Parasitology Oeiras, 2780-157 University of Sevilla Portugal Faculty of Phamacy [email protected] C/ Profesor García González 2 Sevilla, 41012 Spain [email protected]

abstracts book 349 EXTREMOphiles_2012

Danson, Michael J. del Pozo-Cevallos, Mercedes Verónica Dept. of Biology and Biochemistry Enzyme Discovery Lab University of Bath Instituto de Catálisis y Petroleoquímica (ICP-CSIC) Centre for Extremophile Research C/ Marie Curie 2 L10 Bath, BA2 7AY Cantoblanco, Madrid, 28049 UK Spain [email protected] [email protected]

Daupan, Socorro Martha Meg-Ay Didari, Maryam Institute of Biology, College of Science Dept. of Microbiology University of the Philippines Diliman, Quezon City University of Tehran 91-J New Lucban Extremophiles Lab Baguio City, 2600 School of Biology Philippines College of Science [email protected] Enghelab Street Tehran, 14155-6455 de la Fuente, Vicenta Iran Dept. of Biology [email protected] Universidad Autónoma de Madrid C/ Nicolás Cabrera 1 Diruggiero, Jocelyne Campus UAM Dept. of Biology Madrid, 28049 Johns Hopkins University Spain 3400 N. Charles Street – Mudd Hall [email protected] Baltimore, MD, 21218 USA de la Haba, Rafael R. [email protected] Dept. of Microbiology and Parasitology University of Sevilla Dougan, Lorna Faculty of Pharmacy Dept. of Physics and Astronomy C/ Profesor García González 2 University of Leeds Sevilla, 41012 Stoner building, Woodhouse Lane Spain Leeds, Ls2 9JT [email protected] UK [email protected] de los Rios, Asuncion Dept. of Environmental Biology Doukyu, Noriyuki Museo Nacional de Ciencias Naturales - CSIC Dept. of Life Science C/ Serrano 115 dpdo Toyo University Madrid, 28006 1-1-1 Izumino Spain Itakura-machi, Ora-gun, Gunma, 374-0193 [email protected] Japan [email protected] del Moral Juarez, Catalina Ecología Molecular de Ambientes Extremos Driessen, Arnold Centro de Biología Molecular «Severo Ochoa» Dept. of Molecular Microbiology C/ Nicolás Cabrera 1 University of Groningen Campus UAM Groningen Biomolecular Sciences and Madrid, 28049 Biotechnology Institute Spain Nijenborgh 7 [email protected] Groningen, 9747AG The Netherlands [email protected]

350 abstracts book EXTREMOphiles_2012

E Fernández-Castillo, Rosario Dept. of Microbiology and Parasitology Elleuche, Skander University of Sevilla Institute of Technical Microbiology Faculty of Pharmacy Hamburg University of Technology C/ Profesor García González 2 Kasernenstr. 12 Sevilla, 41012 Hamburg, D-21073 Spain Germany [email protected] [email protected] Fernández, Ana Beatriz Escudero González, Lorena V. Dept. Microbiology and Parasitology Centro de Biotecnologia University of Sevilla Universidad Católica del Norte Faculty of Pharmacy Centro de Investigación Científica y Tecnológica C/ Profesor García González 2 para la Minería (CICITEM) Sevilla, 41012 Avenida Angamos 0610 Spain Antofagasta, 1240000 [email protected] Chile [email protected] Ferrara, Maria Carmina National Research Council (CNR) Espinosa, Constanza Institute of Protein Biochemistry Dept. of Microbiology and Parasitology Naples, 80131 University of Sevilla Italy Faculty of Pharmacy [email protected] C/ Profesor García González 2 Sevilla, 41012 Flores Moraga, Patricio Alejandro Spain Facultad de Química y Biología [email protected] Universidad Santiago de Chile Fundación Científica y Cultural Biociencia F José Domingo Cañas 2280 Ñuñoa, Santiago, 7750132 Faramarzi, Mohammad Ali Chile Dept. of Pharmaceutical Biotechnology [email protected] Tehran University of Medical Sciences Faculty of Pharmacy Forterre, Patrick P. O. Box 14155-6451 Dept. of Microbiology Tehran, 14174 Institut Pasteur Iran Unit BMGE [email protected] 25 rue du Dr. Roux Paris, 75015 Fazeli, Seyed A. S. France Iranian Biological Resource Center [email protected] No. 66 Nikoughadam St., Sohrevardi Ave. Tehran, 1551813513 Iran Franzetti, Bruno [email protected] Institut de Biologie Structurale National Center for Scientific Research (CNRS) Fedøy, Anita-Elin 41 rue J. Horowitz Dept. of Biology Grenoble, 38240 University of Bergen France Pb 7803 [email protected] Bergen, 5020 Norway [email protected] abstracts book 351 EXTREMOphiles_2012

Fujinami, Shun Gaidamakova, Elena Bio-Nano Electronics Research Centre Dept. of Pathology Toyo University Uniformed Services University of the Health 2100 Kujirai Sciences (USUHS) Kawagoe, Saitama, 350-8585 Henry M. Jackson Foundation (HJF) Japan 4301 Jones Bridge Rd. [email protected] Bethesda, MD, 20814 USA Fujisawa, Makoto [email protected] Faculty of Life Sciences Toyo University Gana, Mohamed Lamine 1-1-1 Izumino Biocorrosion Laboratory Itakura-machi, Oura-gun, Gunma, 374-0193 Centre of Research and Development st Japan Sonatrach, 1 November Avenue [email protected] Boumerdes, 35000 Algeria [email protected] Fujiwara, Shinsuke Dept. of Bioscience García, Mª Teresa Kwansei-Gakuin University Dept. of Microbiology and Parasitology 2-1 Gakuen University of Sevilla Sanda, 669-1337 Faculty of Pharmacy Japan C/ Profesor García González 2 [email protected] Sevilla, 41012 Spain Fukazawa, Risako [email protected] Dept. of Biochemistry and Molecular Biology Nippon Medical School Gavrilov, Sergey 1-1-5 Sendagi Winogradsky Institute of Microbiology Bunkyo-ku, Tokyo, 113-8602 Russian Academy of Sciences Japan Laboratory of Hyperthermophilic Microbial [email protected] Communities Prospekt 60-Letiya Oktyabrya 7/2 Fukuda, Wakao Moscow, 117312 Dept. of Biotechnology, Russia College of Life Sciences [email protected] Ritsumeikan University 1-1-1 Nojihigashi Gerasimenko, Lyudmila Kusatsu, Shiga, 525-8577 Winogradsky Institute of Microbiology Japan Russian Academy of Sciences [email protected] Laboratory of Microbial Communities Prospekt 60-Letiya Oktyabrya 7/2 G Moscow, 117312 Russia [email protected] Gagen, Emma Dept. of Microbiology Ghai, Rohit University of Regensburg Departamento de Producción Vegetal y Universitaetstrasse 31 Microbiología Regensburg, Bayern, 93053 Universidad Miguel Hernández Germany Evolutionary Genomics Group [email protected] San Juan de Alicante, Alicante, 03550 Spain [email protected]

352 abstracts book EXTREMOphiles_2012

Giudici-Orticoni, Marie Therese As Lagoas, Ourense, 32004 Bioenergetic and Protein Engineering Spain National Center for Scientific Research (CNRS) [email protected] 31 chemin Jospeh Aiguier Marseille, 13402 cedex 20 González Grau, Juan Miguel France Institute of Natural Resources and Agrobiology [email protected] Spanish National Council for Research (CSIC) Avda. Reina Mercedes 10 Glaring, Mikkel Andreas Sevilla, 41012 Dept. of Plant and Environmental Sciences Spain University of Copenhagen [email protected] Thorvaldsensvej 40 Frederiksberg C, DK-1871 Gouy, Manolo Denmark Laboratoire de Biométrie et Biologie Évolutive [email protected] Université Lyon 1 National Center for Scientific Research (CNRS) Godfroy, Anne 43 Boulevard du 11 Novembre Laboratoire de Microbiologie des Environnements Villeurbanne, 69622 Extrêmes France Ifremer [email protected] BP 70 Plouzané, 29280 Grant, William D. France Dept. of Infection, Immunity and Inflammation [email protected] University of Leicester Maurice Shock Building Gogliettino, Marta University Road Institute of Protein Biochemistry Leicester, LE1 9HN National Research Council (CNR) UK Via Pietro Castellino 111 [email protected] Naples, 80131 Italy Grote, Ralf [email protected] Institute of Technical Microbiology Hamburg University of Technology Gómez, Felipe Kasernenstr. 12 Dept. of Planetology and Habitability Hamburg 21073 Astrobiology Centre (INTA-CSIC) Germany Ctra. de Torrejón - Ajalvir, km 4 [email protected] Torrejón de Ardoz, Madrid, 28850 Spain Guiliani, Nicolas [email protected] Departamento de Biología Universidad de Chile Gonçalves, Luís G. Las Palmeras, 3425 Instituto de Tecnologia Química e Biológica (ITQB) Ñuñoa, Santiago, 780-0024 Av. da República-EAN, Apartado 127l Chile Oeiras, 2780-157 [email protected] Portugal [email protected] Gulecal, Yasemin Dept. of Freshwater Biology González González, Roberto Istanbul University Dept. of Analytical and Food Chemistry Ordu Cad.No:200 Facultad de Ciencias Fatih, Istanbul, 34470 University of Vigo Turkey [email protected]

abstracts book 353 EXTREMOphiles_2012

Gutiérrez, Mª Carmen Hidalgo, Aurelio Dept. of Microbiology and Parasitology Dept. of Molecular Biology University of Sevilla Universidad Autónoma de Madrid Faculty of Pharmacy C/ Nicolás Cabrera 1 C/ Profesor García González 2 Madrid, 28049 Sevilla, 41012 Spain Spain [email protected] [email protected] Higashibata, Hiroki Graduate School of Life Sciences H Toyo University 1-1-1 Izumino Haenelt, Inga Itakura-machi, Ora-gun, Gunma, 374-0193 Institute of Molecular Biosciences Japan Goethe University Frankfurt [email protected] Molecular Microbiology and Bioenergetics Frankfurt, 60438 Hikmawan, Tyas Germany King Abdullah University of Science and Technology [email protected] (KAUST) Department of Marine Science Hagiuda, Emi Thuwal 23955, Saudi Arabia Dept. of Biochemistry and Molecular Biology [email protected] Nippon Medical School 1-1-5 Sendagi Hirata, Akira Bunkyo-ku, Tokyo, 113-8602 Dept. of Materials Science and Biotechnology Japan Ehime University [email protected] Graduate School of Science and Engineering 3 Bunkyo-cho Hamajima, Yuki Matsuyama, Ehime, 790-8577 Dept. Life Science Japan Rikkyo University [email protected] College of Sciences 3-34-1 Nishi-Ikebukuro Hoffmann, Toni Toshima, Tokyo, 171-8501 School of Physics and Astronomy Japan University of Leeds [email protected] Woodhouse Lane, EC Stoner Building Leeds, West Yorkshire, LS2 9JT Hausner, Winfried UK Lehrstuhl für Mikrobiologie [email protected] University of Regensburg Universitätsstr. 31 Holmes, David Regensburg, 93053 Center for Bioformatics and Genome Biology Germany Fundación Ciencia y Vida [email protected] Avda. Zañartu 1482 Ñuñoa, Santiago, 7780272 Herrera, Laura Chile Dept. of Microbiology and Parasitology [email protected] University of Sevilla Faculty of Pharmacy Horikoshi, Koki C/ Profesor García González 2 Tokyo Institute of Technology Sevilla, 41012 Japan Agency for Marine-Earth Science and Spain Technology (JAMSTEC) [email protected]

354 abstracts book EXTREMOphiles_2012

Tokyo Japan Japan [email protected] [email protected] Iwata, Fumiya Hriscu, Monica Dept. of Chemistry and Biotechnology Dept. of Biochemistry and Biochemical Engineering Graduate School of Engineering Babes-Bolyai University University of Tokyo Faculty of Chemistry and Chemical Engineering Hongo, Bunkyo-ku, Tokyo, 113-8656 Cluj-Napoca, 400028 Japan Romania [email protected] [email protected] J Huang, Li Institute of Microbiology Janecek, Stefan Chinese Academy of Sciences Institute of Molecular Biology No.1 West Beichen Road Slovak Academy of Sciences Chaoyang District, Beijing, 100101 Laboratory of Protein Evolution China Dubravska cesta 21 [email protected] Bratislava, SK-84551 Slovakia I [email protected]

Infante Domínguez, Carmen Jang, Danbie Dept. of Microbiology and Parasitology Dept. of Bio-Food Materials University of Sevilla Silla University Faculty of Pharmacy San 1-1 C/ Profesor García González 2 Gwaebubdong, Sasang, Busan, 617-736 Sevilla, 41012 South Korea Spain [email protected] [email protected] Jebbar, Mohamed Inoue, Akira Institut Universitaire Européen de la Mer (IUEM) Graduate School of Life Sciences Université de Brest Toyo University Rue Dumont d’Urville 1-1-1 Izumino Plouzané, 29280 Itakura-machi, Ora-gun, Gunma, 374-0193 France Japan [email protected] [email protected] Jiang, Kai Institute of Microbiology Ito, Masahiro Chinese Academy of Sciences Faculty of Life Sciences No.1 Beichen West Road Toyo University Chaoyang District, Beijing, 100101 1-1-1 Izumino China Itakura-machi, Ora-gun, Gunma, 374-0193 [email protected] Japan [email protected] Johnsen, Ulrike Institut für Allgemeine Mikrobiologie Iwasaki, Toshio University of Kiel Dept. of Biochmistry and Molecular Biology Am Botanischen Garten 1-9 Nippon Medical School Kiel, 24118 1-1-5 Sendagi Germany Bunkyo-ku, Tokyo, 113-8602 [email protected]

abstracts book 355 EXTREMOphiles_2012

Jones, Brian Silpakorn University Research and Development Faculty of Science Genencor International B.V. Nakhon Pathom, 73000 Archimedesweg 30 Thailand Leiden, 2333CN [email protected] The Netherlands [email protected] Karp, Matti Dept. of Chemistry and Bioengineering Jorge, Carla Tampere University of Technology Biology Division Korkeakoulunkatu 8 New University of Lisbon Tampere, 33720 Instituto de Tecnologia Química e Biológica (ITQB) Finland Av. da República, Estação Agronómica Nacional [email protected] Oeiras, Lisbon, 2780-157 Portugal Kato, Chiaki [email protected] Institute of Biogeosciences Japan Agency for Marine-Earth Science and K Technology (JAMSTEC) 2-15 Natsushima-cho Kaminski, Lina Yokosuka, 237-0061 Ben Gurion University of the Negev Japan Life Scienses, POB 653 [email protected] Beer-Sheva, 84105 Israel Kawamoto, Jun [email protected] Institute for Chemical Research Kyoto University Kanai, Tamotsu Gokasyo, Uji, Kyoto, 611-0011 Graduate School of Engineering Japan Kyoto University [email protected] Katsura, Nishikyo-ku, Kyoto, 615-8510 Japan Kawamura, Takuya [email protected] Dept. of Materials Science and Biotechnology Ehime University Kanao, Tadayoshi 3 Bunkyo Dept. of Biofunctional Chemistry Matsuyama, Ehime, 790-8577 Okayama University Japan 1-1-1, Tsushima-Naka [email protected] Kita-ku, Okayama, 700-8530 Japan Kawarabayasi, Yutaka [email protected] Faculty of Agriculture Kyushu University Kang, Sung Gyun Laboratory for Functional Genomics of Marine Biotechnology Research Center Extremophiles Korea Ocean Research and Development Institute Hakozaki 6-10-1 University of Science and Technology Higashi-ku, Fukuoka, 812-8581 1270 Sa 2-dong Japan Ansan, 426-744 [email protected] South Korea [email protected] Kebbouche-Gana, Salima Laboratory VALCOR Kanoksilapatham, Wirojne University M’Hamed Bougara of Boumerdes st Dept. of Microbiology 1 November Avenue

356 abstracts book EXTREMOphiles_2012

Boumerdes, 35000 Kort, Julia Algeria Biofilm Centre [email protected] University of Duisburg-Essen Molecular Enzyme Technology and Biochemistry Khomarbaghi, Zahra Universitätsstr. 5 Dept. of Microbiology Essen, 45141 University of Tehran Germany Block No.17, Shafiee Avenue [email protected] Ghods Street, Enghelab Square Tehran, 1369665713 Krulwich, Terry Ann Iran Dept. of Pharmacology and Systems Therapeutics [email protected] Mount Sinai School of Medicine 1468 Madison Avenue Kikani, Bhavtosh A. New York, 10029 Department of Biosciences USA Saurashtra University [email protected] Rajkot 360 005 Gujarat, India Kublanov, Ilya [email protected] Winogradsky Institute of Microbiology Russian Academy of Sciences Kim, Yun Jae Laboratory of Hyperthermophilic Microbial Marine Biotechnology Research Center Communities Korea Ocean Research and Development Institute Prospekt 60-Letiya Oktyabrya 7/2 (KORDI) Moscow, 117312 787 Haean-ro Russia Ansan, 426-744 [email protected] South Korea [email protected] Kurata, Atsushi Dept. of Applied Biological Chemistry Kivistö, Anniina Kinki University Dept. of Chemistry and Bioengineering 3327-204 Nakamachi Tampere University of Technology Nara, 631-8505 PL 541 Japan Tampere, 33710 kurata090401@nara,kindai.ac.jp Finland [email protected] Kurosawa, Yasunori Kyokuto Pharmaceutical Industrial Co., Ltd. Kixmüller, Dorthe Product Development Division Dept. of Biology/Chemistry 3333-26, Aza-Asayama, Kamitezuna University of Osnabrück Takahagi-shi, 318-0004 Barbarastraße 11 Ibaraki, Japan Osnabrück, 49076 [email protected] Germany [email protected] Kushida, Takashi Graduate School of Life Sciences Kojima, Mio Toyo University Bioelectronics Research Center 1-1-1 Izumino Toyo University Itakura-machi, Ora-gun, Gunma 374-0193 1-1-1 Izumino Japan Itakura-machi, Ora-gun, Gunma, 374-0193 [email protected] Japan [email protected]

abstracts book 357 EXTREMOphiles_2012

L Lee, Hyun Sook Marine Biotechnology Research Center Labes, Antje Korea Ocean Research and Development Institute Kieler Wirkstoff-Zentrum 787 Haean-ro Helmholtz Center for Ocean Research Sangnok-gu, Ansan, Gyeonggi-do, 426-744 Am Kiel-Kanal 44 South Korea Kiel, 24106 [email protected] Germany [email protected] Lentini, Valeria Dept. of Animal Biology and Marine Ecology Lafraya, Alvaro University of Messina Enzyme Discovery Lab Viale F. Stagno d’Alcontres 31 Instituto de Catálisis y Petroleoquímica (ICP-CSIC) Messina, 98166 Marie Curie 2 L10 Italy Cantoblanco, Madrid, 28049 [email protected] Spain [email protected] León, Mª José Dept. of Microbiology and Parasitology Lage, Claudia University of Sevilla Dept. of Molecular and Structural Biology Faculty of Pharmacy Rio de Janeiro Federal University (UFRJ) C/ Profesor García González 2 Carlos Chagas Filho Institute of Biophysics Sevilla, 41012 373 Carlos Chagas Filho Ave. Spain Centro de Ciências da Saúde, Bldg G [email protected] Rio de Janeiro, 21941-902 Brazil Li, Ming [email protected] Institute of Microbiology Chinese Academy of Sciences Lake, James A. State Key Laboratory of Microbial Resources Dept. of Molecular, Cell, and Developmental Biology No.1 Beichen West Road University of California, Los Angeles (UCLA) Chaoyang District, Beijing, 100101 Molecular Biology Institute China Paul D. Boyer Hall [email protected] 611 Charles E. Young Drive East Los Angeles, CA, 90095-1570 Littlechild, Jennifer USA Exeter Biocatalysis Centre [email protected] University of Exeter Henry Wellcome Building for Biocatalysis Lauro, Federico Biosciences, Stocker Road School of Biotechnology and Biomolecular Sciences Exeter, EX4 4QD University of New South Wales (UNSW) UK Sydney, NSW, 2052 [email protected] Australia [email protected] Liu, Jingfang Institute of Microbiology Lee, Han-Seung Chinese Academy of Sciences Dept. of Bio-Food Materials State Key Laboratory of Microbial Resources Silla University No.1 Beichen West Road San 1-1 Chaoyang District, Beijing, 100101 Gwaebeopdong, Sasang, Busan, 617-736 China South Korea [email protected] [email protected]

358 abstracts book EXTREMOphiles_2012

Llamas Company, Inmaculada Barcelona, 08003 Dept. of Microbiology Spain University of Granada [email protected] Campus Universitario de Cartuja Granada, 18071 Madern, Dominique Spain Institut de Biologie Structurale [email protected] National Center for Scientific Research (CNRS) 41 Rue Jules Horowitz López Hermoso, Clara Grenoble, 38042 Dept. Microbiology and Parasitology France University of Sevilla [email protected] Faculty of Pharmacy C/ Profesor García González 2 Magallanes, Xavier Sevilla, 41012 Laboratory of Molecular Microbiology and Spain Biotechnology [email protected] Universidad de Los Andes (ULA) Av. Universidad, calle Araguaney casa # 25 Lylloff, Jeanette Eva La Victoria, Aragua, 2121 Dept. of Plant and Environmental Sciences Venezuela Copenhagen University [email protected] Copenhagen, 1871 Denmark Marín Palma, Irma [email protected] Dept. of Molecular Biology Universidad Autónoma de Madrid M C/ Nicolás Cabrera 1 Campus UAM Ma, Yanhe Madrid, 28049 Institute of Microbiology Spain Chinese Academy of Sciences [email protected] No.1 Beichen West Road Chaoyang District, Beijing, 100101 Márquez, Mª Carmen China Dept. of Microbiology and Parasitology [email protected] University of Sevilla Faculty of Pharmacy Maciel Paulo, Elinalva C/ Profesor García González 2 Ciências Biológicas Sevilla, 41012 Universidade Estadual de Feira de Santana Spain Feira de Santana, Bahia, 44036900 [email protected] Brasil [email protected] Martínez, Mónica Enzyme Discovery Lab Mackay, Stephen Instituto de Catálisis y Petroleoquímica (ICP-CSIC) Dept. of Biotechnology C/ Marie Curie 2 University of the Western Cape Madrid, 28049 Institute for Microbial Biotechnology and Spain Metagenomics [email protected] Bellville, Cape Town, 7535 South Africa Matsushita, Shinichi [email protected] Dept. of Biochemistry and Molecular Biology Nippon Medical School Mackenzie, Roy 1-1-5 Sendagi Departamento de Biología Marina y Oceanografía Bunkyo-ku, Tokyo, 113-8602 Instituto de Ciencias del Mar Japan Passeig Marítim de la Barceloneta 37-49 [email protected]

abstracts book 359 EXTREMOphiles_2012

Mead, David Lucigen Corporation Merkel, Alexander 2905 Parmenter Street Dept. of Microbial Communities Middleton, WI, 53562 Winogradsky Institute of Microbiology USA Russian Academy of Sciences [email protected] Prospekt 60-Letiya Oktyabrya 7/2 Moscow, 117312 Medeiros, Joana Russia Fundação para o Desenvolvimento Sócio- [email protected] Profissional e Cultural de Ribeira Grande Science Center – Azores Microbial Observatory Miura, Takeshi (OMIC) Faculty of Life Sciences Rua Nª Sra de Fátima s/n Toyo Uuniversity Rabo de Peixe, Ribeira Grande 1-1-1 Izumino São Miguel, Azores, 9600-998 Itakura-machi, Ora-gun, Gunma, 374-0193 Portugal Japan [email protected] [email protected]

Megías, Manuel Mohmmed, Abdolkader A. Dept. of Microbiology and Parasitology Dept. of Molecular Biology and Biotechnology University of Sevilla University of Sheffield and Sebha University Faculty of Pharmacy Sheffield, S3 7NH C/ Profesor García González 2 UK Sevilla, 41012 [email protected] Spain [email protected] Moracci, Marco Institute of Protein Biochemistry Mehrshad, Maliheh National Research Council (CNR) Dept. of Microbiology Via Pietro Castellino 111 University of Tehran Naples, 80131 No.17, Shafiee Street Italy Qods Street, Enghelab Street [email protected] Tehran Iran Moreno, Mª de Lourdes [email protected] Dept. of Microbiology and Parasitology University of Sevilla Mellado, Encarnación Faculty of Pharmacy Dept. of Microbiology and Parasitology C/ Profesor García González 2 University of Sevilla Sevilla, 41012 Faculty of Pharmacy Spain C/ Profesor García González 2 [email protected] Sevilla, 41012 Spain Morgan, Hugh [email protected] Dept. of Biological Sciences University of Waikato Francisco Merchán Private Bag 3105 Dept. of Microbiology and Parasitology Hamilton, 3204 University of Sevilla New Zealand Faculty of Pharmacy [email protected] C/ Profesor García González 2 Sevilla, 41012 Morikawa, Masaaki Spain Faculty of Environmental Earth Science [email protected] Hokkaido University

360 abstracts book EXTREMOphiles_2012

Section of Bioscience Faculty of Pharmacy Kita-ku, N-10 W-5 C/ Profesor García González 2 Sapporo, Hokkaido, 060-0810 Sevilla, 41012 Japan Spain [email protected] [email protected]

Morozova, Vera O Institute of Chemical Biology and Fundamental Medicine SB Oger, Phil Russian Academy of Sciences Laboratoire de Géologie de Lyon Laboratory of Molecular Microbiology Ecole Normale Supérieure de Lyon Novosibirsk, 630090 46 Allée d’Italie Russia Lyon, 69364 [email protected] France [email protected] Müller, Volker Dept. of Molecular Microbiology and Bioenergetics Oggerin, Monike University Frankfurt am Main Dept. of Planetology and Habitability Max-von-Laue Str. 9 Astrobiology Centre (INTA-CSIC) Frankfurt am Main, 60438 Ctra. de Torrejón - Ajalvir, km 4 Germany Torrejón de Ardoz, Madrid, 28850 [email protected] Spain [email protected] N Ohlhoff, Colin Nagaki, Kazunori Dept. of Biotechnology Kyokuto Pharmaceutical Industrial Co., Ltd. University of the Western Cape Product Development Division Institute for Microbial Biotechnology and 3333-26, Aza-Asayama, Kamitezuna Metagenomics Takahagi-shi, 318-0004 Private Bag X17 Ibaraki, Japan Cape Town, 7535 [email protected] South Africa [email protected] Nakasone, Kaoru Faculty of Engineering Onodera, Takefumi Kinki University Graduate School of Life Sciences 1 Umenobe Tokyo University of Pharmacy and Life Science Takaya, Higashihiroshima, 739-2116 1432-1 Horinouchi Japan Hachioji, Tokyo, 192-0392 [email protected] Japan [email protected] Narumi, Issay Quantum Beam Science Directorate Oren, Aharon Japan Atomic Energy Agency Dept. of Plant and Environmental Sciences 1233 Watanuki The Hebrew University of Jerusalem Takasaki, 370-1292 The Institute of Life Sciences Japan Givat Ram [email protected] Jerusalem, 9194 Israel Nieto, Joaquin J. [email protected] Dept. of Microbiology and Parasitology University of Sevilla

abstracts book 361 EXTREMOphiles_2012

Oueriaghli, Nahid Peralta, Rocio Dept. of Microbiology Facultad de Química y Biología University of Granada Universidad de Santiago de Chile Faculty of Pharmacy Fundación Científica y Cultural Biociencia Campus Universitario de Cartuja José Domingo Cañas 2280 Granada, 18017 Ñuñoa, Santiago, 7750132 Spain Chile [email protected] [email protected]

Pereira, Jairo P Facultad de Química y Biología Universidad de Santiago de Chile Palmieri, Gianna Fundación Científica y Cultural Biociencia Institute of Protein Biochemistry José Domingo Cañas 2280 National Research Council (CNR) Ñuñoa, Santiago, 7750132 Via Pietro Castellino 111 Chile Naples, 80131 [email protected] Italy [email protected] Perevalova, Anna Winogradsky Institute of Microbiology Papke, R. Thane Russian Academy of Sciences Dept. of Molecular and Cell Biology Laboratory of Microbial Communities University of Connecticut Prospekt 60-Letiya Oktyabrya 7/2 91 N. Eagleville Rd. Moscow, 117312 Storrs, CT, 6269 Russia USA [email protected] [email protected] Pérez, Dolores Pasic, Lejla Dept. of Microbiology and Parasitology Dept. of Biology University of Sevilla University of Ljubljana Faculty of Pharmacy Biotechnical Faculty C/ Profesor García González 2 Veèna pot 111 Sevilla, 41012 Ljubljana, 1000 Spain Slovenia [email protected] [email protected] Perugino, Giuseppe Pawar, Sudhanshu Institute of Protein Biochemistry Dept. of Chemistry National Research Council (CNR) Lund University, LTH. Via Pietro Castellino 111 Division of Applied Microbiology Naples, 80131 Getingevägen 60 (Kemicentrum), Italy Lund, Skåne, SE-22241 [email protected] Sweden [email protected] Pinto, Leonardo H. Departamento de Bioquimica Médica Pawlowski, Alice Rio de Janeiro Federal University (UFRJ) Dept. of Biological and Evironmental Science 64, 202 Piauí Street University of Jyväskylä Rio de Janeiro, 20770-130 P.O.Box 35 (Survontie 9) Brazil Jyväskylä, 40014 [email protected] Finland [email protected]

362 abstracts book EXTREMOphiles_2012

Piñeiro-Vidal, Maximino Prangishvili, David Institute of Natural Resources and Agrobiology Dept. of Microbiology Spanish National Council for Research (CSIC) Institut Pasteur Avda. Reina Mercedes 10 25 rue du Dr. Roux Sevilla, 41012 Paris, 75015 Spain France [email protected] [email protected]

Piubeli, Francine Dept. of Microbiology and Parasitology Prieto, Patricia University of Sevilla Dept. of Microbiology and Parasitology Faculty of Pharmacy University of Sevilla C/ Prof García González n 2 Faculty of Pharmacy Sevilla, 41012 C/ Prof García González n 2 Spain Sevilla, 41012 [email protected] Spain [email protected] Podar, Mircea Dept. of Microbiology and Biosciences Q University of Tennessee Oak Ridge National Laboratory Quesada Arroquia, Emilia 1 Bethel Valley Road, Bldg. 1505, Room 308 Dept. of Microbiology Oak Ridge, TN, 37831-6038 University of Granada USA Faculty of Pharmacy [email protected] Campus Universitario de Cartuja Granada, 18071 Podosokorskaya, Olga España Winogradsky Institute of Microbiology [email protected] Russian Academy of Sciences Prospekt 60-Letiya Oktyabrya 7/2 Moscow, 117312 R Russia [email protected] Rainey, Fred A. Dept. of Biological Sciences Poklar Ulrih, Natasa University of Alaska Anchorage Dept. of Food Science and Technology 101M CPISB, 3211 Providence Drive University of Ljubljana Anchorage, AK, 99508 Biotechnical Faculty USA Jamnikarjeva 101 [email protected] Ljubljana, 1000 Slovenia Ramírez-Durán, Ninfa [email protected] Laboratorio de Microbiología Médica y Ambiental Universidad Autónoma del Estado de México Pourbabaee, Ahmad Ali Facultad de Medicina Dept. of Soil Science Toluca, 50180 University of Tehran México Biotech Lab [email protected] University College of Agriculture and Natural Resources Ramíred Saad, Hugo Chameran Street, Charah Daneshkadeh Universidad Autónoma Metropolitana-Xochimilco Karaj Depto. de Sistemas Biológicos Iran Calz. del Hueso 1100 [email protected] México, D.F., 04960.

abstracts book 363 EXTREMOphiles_2012

México Spain [email protected] [email protected]

Rauch, Bernadette Rodriguez-Valera, Francisco Biofilm Centre Departamento de Producción Vegetal y University of Duisburg-Essen Microbiología Molecular Enzyme Technology and Biochemistry Universidad Miguel Hernández Universitätsstr. 5 San Juan de Alicante, Alicante, 03550 Essen, 45141 Spain Germany [email protected] [email protected] Rohulya, Olha Reina, Mercedes Dept. of Molecular Microbiology Dept. of Microbiology and Parasitology University of Groningen University of Sevilla Nijenborgh 7 Faculty of Pharmacy Groningen, 9747 AG C/ Profesor García González 2 The Netherlands Sevilla, 41012 [email protected] Spain [email protected] Ron, Emanuel Dept. of Biotechnology Rettberg, Petra Lund University Radiation Biology Department P.O. Box 124 DLR, Institute of Aerospace Medicine Lund, Skåne, 22100 Research Group Astrobiology Skane, Sweden Linder Höhe [email protected] Köln, 51147 Germany Rossi, Mosè [email protected] Institute of Protein Biochemistry National Research Council (CNR) Rivas, Luis A. Via Pietro Castellino 111 Dept. of Molecular Evolution Napoli, 80131 Astrobiology Centre (INTA-CSIC) Italy Ctra. de Torrejón - Ajalvir, km 4 [email protected] Torrejón de Ardoz, Madrid, 28850 Spain Rua, Maria Luisa [email protected] Dept. of Analytical and Food Chemistry University of Vigo Rodrigues, Marta As Lagoas s/n Biology Division Ourense, 32004 New University of Lisbon Spain Instituto de Tecnologia Química e Biológica (ITQB) [email protected] Av. da República, Estação Agronómica Nacional Oeiras, 2780-157 Ruiz Dominguez, María del Carmen Portugal Departamento de Química y CC. de los Materiales [email protected] Universidad de Huelva Parque Dunar s/n Rodriguez, Nuria Matalascañas, Huelva, 21760 Dept. of Planetology and Habitability Spain Astrobiology Centre (INTA-CSIC) [email protected] Ctra. de Torrejón - Ajalvir, km 4 Torrejón de Ardoz, Madrid, 28850

364 abstracts book EXTREMOphiles_2012

S Sánchez-Andrea, Irene Dept. of Molecular Biology Sahm, Kerstin Universidad Autónoma de Madrid Institute of Technical Microbiology Avda. El Ferrol 37, bajo 2 Hamburg University of Technology Madrid, 28029 Kasernenstr. 12 Spain Hamburg, 21071 [email protected] Germany [email protected] Sánchez-Porro Álvarez, Cristina Dept. of Microbiology and Parasitology Saitoh, Yoshimoto University of Sevilla Environmental Science Research Laboratory Faculty of Pharmacy Central Research Institute of Electric Power Supply C/ Profesor García González 2 1646 Abiko Sevilla, 41012 Abiko-shi, Chiba-ken, 270-1194 Spain Japan [email protected] [email protected] Sanchez-Ruiz, Jose Manuel Salvador , Manuel Departamento de Quimica Fisica Dept. of Microbiology and Parasitology Universidad de Granada University of Sevilla Facultad de Ciencias Faculty of Pharmacy Campus Universitario de Fuentenueva C/ Profesor García González 2 Granada, 18071 Sevilla, 41012 Spain Spain [email protected] [email protected] Santos, Helena Salzer, Ralf Instituto de Tecnologia Química e Biológica (ITQB) Dept. of Molecular Microbiology and Bioenergetics New University of Lisbon Goethe University Frankfurt Av. da República, Estação Agronómica Nacional Institute of Molecular Biosciences Oeiras, 2780-157 Max-von-Laue-Str. 9 Portugal Frankfurt, 60438 [email protected] Germany [email protected] Sanz Martín, Jose Luis Dept. of Molecular Biology Samylina, Olga Universidad Autónoma de Madrid Winogradsky Institute of Microbiology C/ Nicolás Cabrera 1 Russian Academy of Sciences Campus UAM Prospekt 60-Letiya Oktyabrya 7/2 Madrid, 28049 Moscow, 117312 Spain Russia [email protected] [email protected] Sarmiento, Felipe San Martín, Patxi Dept. of Microbiology Laboratorio de Extremofilia The University of Georgia Astrobiology Centre (INTA-CSIC) 541 Biological Sciences Building Ctra. de Torrejón - Ajalvir, km 4 Athens, GA, 30602-2605 Torrejón de Ardoz, Madrid, 28850 USA Spain [email protected] [email protected]

abstracts book 365 EXTREMOphiles_2012

Sato, Takaaki Kyungpook National University Dept. of Synthetic Chemistry and Biological 80 Daehakro Chemistry Daegu, 702-701 Kyoto University South Korea Graduate School of Engineering [email protected] A4-building, Room 216 Katsura, Nishikyo-ku, Kyoto, 615-8510 Siristova, Lucie Japan Dept. of Biotechnology [email protected] Institute of Chemical Technology Prague Technicka 5 Satomura, Takenori Prague, 166 28 Dept. of Applied Chemistry and Biotechnology Czech Republic University of Fukui [email protected] 3-9-1 Bnkyo Slobodkin, Alexander Fukui, 910-8507 Winogradsky Institute of Microbiology Japan Russian Academy of Sciences [email protected] Prospekt 60-Letiya Oktyabrya 7/2 Moscow, 117312 Schmidt, Howard Russia Aramco, EXPEC-ARC, Box 8643 [email protected] Dhahran, 31311 Saudi Arabia Slobodkina, Galina [email protected] Winogradsky Institute of Microbiology Russian Academy of Sciences Schönheit, Peter Prospekt 60-Letiya Oktyabrya 7/2 Institut für Allgemeine Mikrobiologie Moscow, 117312 Christian-Albrechts-Universität Kiel Russia Am Botanischen Garten 1-9 [email protected] Kiel, D-24118 Germany Slutskaya, Elvira [email protected] Bakh Institute of Biochemistry Russian Academy of Sciences Servín, Luis Leninsky prospekt 33/2 Ecological Genomics Moscow, 119071 Center for Genomic Sciences Russia National University of Mexico [email protected] Av. Universidad s/n Col. Chamilpa Sokolova, Tatyana Cuernavaca, Morelos, 62210 Winogradsky Institute of Microbiology Mexico Russian Academy of Sciences [email protected] Laboratory of Hyperthermophilic Microbial Communities Shen, Yulong Prospekt 60-Letiya Oktyabrya 7/2 School of Life Sciences Moscow, 117312 Shandong University Russia 27 Shada Nan Rd. [email protected] Jinan, Shandong, 250100 China Sousa, María Helena [email protected] Fundação para o Desenvolvimento Sócio- Profissional e Cultural de Ribeira Grande Shin, Jae-Ho Science Center – Azores Microbial Observatory School of Applied Biosciences (OMIC)

366 abstracts book EXTREMOphiles_2012

Rua Nª Sra de Fátima s/n Takashina, Tomonori Rabo de Peixe, Ribeira Grande Faculty of Life Sciences São Miguel, Azores, 9600-998 Toyo University Portugal 1-1-1 Izumino [email protected] Itakura-machi, Ora-gun, Gunma, 374-0193 Japan Strazzulli, Andrea [email protected] Institute of Protein Biochemistry National Research Council (CNR) Thomm, Michael Via Pietro Castellino 111 Dept. of Microbiology Napoli, 80131 University of Regensbrg Italy Universitaetsstrasse 31 [email protected] Regensburg, D-93053 Germany Suzuki, Hirokazu [email protected] Faculty of Agriculture Kyushu University Tikhonova, Tamara 6-10-1 Hakozaki A.N.Bach Institute of Biochemistry Fukuoka, 812-8581 Russian Academy of Sciences Japan Leninskii prospekt 33 [email protected] Moscow, 119071 Russia T [email protected]

Tahrioui, Ali Tikunova, Nina Dept. of Microbiology Institute of Chemical Biology and Fundamental University of Granada Medicine SB Faculty of Pharmacy Russian Academy of Sciences Campus Universitario de Cartuja Laboratory of Molecular Microbiology Granada, 18071 Novosibirsk, 630090 Spain Russia [email protected] [email protected]

Takai, Ken Tomita, Hiroya Subsurface Geobiology Advanced Research Dept. of Synthetic Chemistry and Biological (SUGAR) Project Chemistry Japan Agency for Marine-Earth Science and Kyoto University Technology Center (JAMSTEC) A4-216 2-15 Natsushima-cho Katsura, Nishikyo-ku, Kyoto, 615-8510 Yokosuka, 237-0061 Japan Japan [email protected] [email protected] Tori, Kazuo Takano, Kazufumi Faculty of Agriculture Dept. of Biomolecular Chemistry Kyushu university Kyoto Prefectural University Laboratory for Functional Genomics of 1-5 Hangi-cho Extremophiles Shimogamo, Sakyo-ku, Kyoto, 606-8522 Hakozaki 6-10-1 Japan Higashiku, Fukuoka, 812-8581 [email protected] Japan [email protected]

abstracts book 367 EXTREMOphiles_2012

Torres Béjar, Marta Urusibata, Wataru Dept. of Microbiology Division of Biosphere Science University of Granada Hokkaido University Faculty of Pharmacy Graduate School of Environmental Science Campus Universitario de Cartuja Kita-ku, N-10 W-5 Granada, 18071 Sapporo, Hokkaido, 060-0810 Spain Japan [email protected] [email protected]

Tropel, David Ushatinskaya, Galina Biovitis Paleontological Institute Saint-Etienne de Chomeil 15400 Russian Academy of Sciences France Profsojuznaya 123 [email protected] Moscow, 117647 Russia Tsubasa, Sato [email protected] Graduate School of Natural Science and Technology Okayama University V 2001-18 Nishifutami Futami-cho, Akashi, Hyogo, 674-0094 Vaquero Calañas, Isabel María Japan Departamento de Química y CC. de los Materiales [email protected] Universidad de Huelva CIECEM, Parque Dunar s/n Tuffin, Marla Matalascañas, Huelva, 21760 Dept. of Biotechnology Spain University of the Western Cape [email protected] Institute for Microbial Biotechnology and Metagenomics Vargas, Carmen Private Bag X17 Dept. of Microbiology and Parasitology Bellville, Cape Town, 7535 University of Sevilla South Africa Faculty of Pharmacy [email protected] C/ Profesor García González 2 Sevilla, 41012 Tych, Katarzyna Spain Molecular and Nanoscale Physics Group [email protected] University of Leeds Woodhouse Lane Vázquez Toscano, María Leeds, West Yorkshire, LS2 9JT Departamento de Química y CC. de los Materiales UK Universidad de Huelva [email protected] Parque Dunar s/n Matalascañas, Huelva, 21760 U Spain [email protected] Ueno, Yuko Dept. of Science Velasco, Esther Rikkyo University Dept. of Petrology College of Life science Vrije Universiteit Amsterdam 3-34-1 Nishi-Ikebukuro De Boelelaan 1085 Toshimaku, Tokyo, 171-8501 Amsterdam, 1082 MA Japan The Netherlands [email protected] [email protected]

368 abstracts book EXTREMOphiles_2012

Ventosa, Antonio Weber, Hedda Dept. of Microbiology and Parasitology Division Wood and Cellulose Chemistry University of Sevilla Kompetenzzentrum Holz GmbH Faculty of Pharmacy Altenbergerstraße 69 C/ Profesor García González 2 Linz, Upper Austria, 4040 Sevilla, 41012 Austria Spain [email protected] [email protected] Weissgram, Michaela Vera, Blanca Division Wood and Cellulose Chemistry Dept. of Microbiology and Parasitology Kompetenzzentrum Holz GmbH University of Sevilla Altenbergerstraße 69 Faculty of Pharmacy Linz, Upper Austria, 4040 C/ Profesor García González 2 Austria Sevilla, 41012 [email protected] Spain [email protected] Wierzchos, Jacek Dept. of Environmental Biology Vera, Joaquín Museo Nacional de Ciencias Naturales - CSIC Dept. of Microbiology and Parasitology C/ Serrano 115 dpdo University of Sevilla Madrid, 28006 Faculty of Pharmacy Spain C/ Profesor García González 2 [email protected] Sevilla, 41012 Spain Williams, Wesley [email protected] Dept. of Biotechnology University of the Western Cape W Institute for Microbial Biotechnology and Metagenomics Bellville, Cape Town, 7535 Waege, Ingrid South Africa Dept. of Microbiology [email protected] University of Regensburg Universitätsstr. 31 Wu, Zhenfang Regensburg, Bavaria, 93053 Institute of Microbiology Germany Chinese Academy of Sciences [email protected] State Key Laboratory of Microbial Resources No.1 Beichen West Road Watanabe, Rei Chaoyang District, Beijing, 100101 Graduate School of Interdisciplinary New Science China Toyo University [email protected] 2100 Kujirai Kawagoe-shi, Saitama, 350-8585 X Japan [email protected] Xiang, Hua Institute of Microbiology Watkin, Elizabeth Chinese Academy of Sciences School of Biomedical Sciences State Key Laboratory of Microbial Resources Curtin University No.1 Beichen West Road GPO Box U1987 Chaoyang District, Beijing, 100101 Perth, Western Australia, 6845 China Australia [email protected] [email protected]

abstracts book 369 EXTREMOphiles_2012

Xing, Xuerong Guangzhou Institute of Biomedicine and Health Chinese Academy of Sciences No.190 Kai Yuan Avenue, Science Park Guangzhou, 510530 China [email protected]

Xue, Yanfen Institute of Microbiology Chinese Academy of Sciences No.1 Beichen West Road Chaoyang District, Beijing, 100101 China [email protected] Y

Yahiaoui, Houa Dept. of Microbiology University Abderrahmane Mira of Bejaia Bejaia, 6000 Algeria [email protected]

Yamagami, Ryota Dept. of Materials Science and Biotechnology Ehime University 4-5-22 Syoenji Matsuyama, Ehime, 790-0904 Japan [email protected]

Yoshii, Takahiro Institute of Environmental Microbiology Kyowa-Kako Co., Ltd. 2-15-5 Tadao Machida, Tokyo, 194-0035 Japan [email protected]

Z

Zhai, Binyuan School of Life Sciences Shandong University 27 Shada Nan Rd. Jinan, Shandong, 250100 China [email protected]

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