DOCSLIB.ORG

THESE Version 18Octobre 09

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
THESE Version 18Octobre 09 Doctoral Thesis under a Cotutelle agreement between l’Université Pierre et Marie Curie and The University of New South Wales French laboratory : Laboratoire d’Océanographie Biologique de Banyuls, Observatoire Océanologique. Université Pierre et Marie Curie. France. Ecole Doctorale B2M – Biochimie et Biologie Moléculaire Spécialité : Génome et Protéines. Australian laboratory: School of Biotechnology and Biomolecular Sciences. Faculty of Science. The University of New South Wales. Sydney, Australia. PHYSIOLOGICAL AND MOLECULAR RESPONSES OF THE MARINE OLIGOTROPHIC ULTRAMICROBACTERIUM SPHINGOPYXIS ALASKENSIS RB2256 TO VISIBLE LIGHT AND ULTRAVIOLET RADIATION Sabine MATALLANA SURGET A thesis submitted in fulfilment of the requirements for the degrees of Doctor of Philosophy awarded by both UPMC and UNSW May, 2009 CERTIFICATE OF ORIGINALITY I hereby declare that this submission is my own work and that, to the best of my knowledge, it contains no material previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. Sabine MATALLANA SURGET I COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed Date : 14-09-09 AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed Date : 14-09-09 II ABSTRACT Ultraviolet radiation reaching the Earth’s surface (UVR, 280-400 nm) may penetrate deep into the clear oligotrophic waters influencing a large part of the euphotic layer. Marine heterotrophic bacteria at the surface of the oceans are especially sensitive to the damaging solar radiation due to their haploid genome with little or no functional redundancy and lack of protective pigmentation. In a context of climate change and ozone depletion, it is clearly important to understand the physiology and underlying molecular UVR responses of abundant marine bacteria species. We chose the marine ultramicrobacterium Sphingopyxis alaskensis as a reference species to study the impact of solar radiation due to its numerical abundance in oligotrophic waters and its photoresistance, previously reported. For this purpose, we focused on the formation of the two major UVB-induced DNA photoproducts (CPDs and 6-4PPs) as well as the differential protein expression under solar radiation. We first demonstrated that the GC content of prokaryotic genome had a major effect on the formation of UVB-induced photoproducts, quantified by HPLC-MS/MS. Due to its high GC content, S. alaskensis presented a favoured formation of highly mutagenic cytosine-containing photoproducts and therefore would be more susceptible to UV- induced mutagenesis. By comparing S. alaskensis to another marine bacterium Photobacterium angustum, we observed for the latter strain a remarkable resistance to high UVB doses associated with a decrease in the rate of formation of CPDs explained by a non-conventional activity of photolyase. We also demonstrated that DNA damage in S. alaskensis was markedly modulated by growth temperature and time spent in stationary phase. In order to assess the effects that environmental UV-R had on regulatory networks and pathways of S. alaskensis, and determine how the cell’s physiology was affected, a quantitative proteomics investigation was performed. Changes in proteome were analyzed, with the recent and powerful mass spectrometry based approach using iTRAQ methodology. Approximately, one third of the proteome of S. alaskensis was identified, with 119 statistically and significantly differentially abundant proteins. Cellular processes, pathways and interaction networks were determined and gave us unique insight into the biology of UV response and adaptation of S. alaskensis. III ACKNOWLEDGEMENTS I am first very grateful to Philippe Lebaron for welcoming me in his lab in Banyuls, when I first started my Master Degree and for encouraging me to do a PhD. Doing my PhD under a cotutelle agreement between France and Australia was a huge and wonderful positive experience. My PhD was supported by fellowship from the French Ministry, a CNRS-INSU through an ATIPE project and by the Australian Research Council. I have had the pleasure of working with and learning from many people. First and foremost, I have constantly felt throughout my PhD, that I was extremely lucky to have not one, but two very supportive and encouraging supervisors. Both Fabien Joux and Rick Cavicchioli have provided invaluable advice on all of the work I did in this thesis. They were always available throughout all stages of my PhD to meet and discuss results and ideas. Rick and Fabien, together your enthusiasm for combining the disciplines of microbial ecology and molecular approaches has created a stimulating environment that enabled our multidisciplinary work to be realised. Your trust in science, belief in me and the freedom you have allowed me have encouraged me to think creatively and take initiative in the lab. I thank you both warmly for a thoroughly enjoyable three and a half years. Thank you very much to Mark Raftery for teaching me mass spectrometry and for his detailed assistance with the computers of BMSF. Thank you also for your patience and availability. I am also very touched that you have accepted our invitation to come in France, to be part of my thesis panel review. I am very grateful for this. Many thanks to Thierry Douki for collaborating on my thesis with the HPLC-MS/MS analysis of the DNA damage. I have learned a lot. It has been such a pleasure working with you and I am very glad to welcome you in Banyuls for my thesis defence. I am very grateful to Wade Jeffrey and Gérard Fonty for having accepted reviewing my manuscript and to be part of my thesis panel review. I would also like to thank Jarah Meador for staying with us in Banyuls for few weeks and working with me on S. alaskensis. I keep excellent memories of working together. It was also very enjoyable working with Maher Abboudi on the optimization of ELISA methodology. Thank you Maher for your calmness. To Laurent Urios, your encouragement and support, especially in my early years as a scientist, was very much appreciated and will always be remembered. Doing a PhD under a cotutelle agreement between two laboratories does not only allow to learn a lot on a scientific point of view but also to meet a lot of very nice people. Because I began my studies in Banyuls, first I would like to thank for their friendship and their help the following students that I met there, namely: Arturo Rodriguez Blanco, Elodie Peyric, Antoine Aze, Antoine Carlier, Olivier Zemb, Mickael Moulager, Raphaël Lami, Benoît Farinas, Marc Auffret, Robin Vuilleumier, Nathalie Parthuisot, Laure Bellec, Sarah Nahon, Charlotte Moritz, Mathieu Chatelain, Mélissa Laghdass and Caroline Sauret. Olivier it was funny to meet you in Banyuls and to become closer friend in Sydney, how small is the world! Thanks for all the good moments, we shared together. I especially thank Arturo, who has been down the long road with me. Thanks for your help and precious encouragement, our wonderful moments sharing together. Thanks for your love, support and the laughs. Thanks for too many things to mention here. To you I am forever grateful! IV I am very grateful to all the members of the “microbes” team, for their invaluable help and friendship during my PhD: namely Laurent Intertaglia, Cécile Vilette, Audrey Calvez, Nicole Batailler, Philippe Catala, Muriel Bourrain, Julia Baudart, Delphine Guillebault, Jeff Ghiglione, Ingrid Obernosterer, Carmem Manes and Sébastien Peuchet. It was always really pleasant to work with enthusiastic people, who eagerly exchange ideas in a specific as well as broader context. Thanks to Gérard Peaucellier for our interesting chats on Proteomics.Thanks to Eliane Rubio and Gilles Vétion that were always on hand to help with the truly enthralling day-to-day activities involved in a PhD. Thanks to Dominic Mooney, Kylie Jones, Claire Biron, Frederique Sanz and Zohra Boumédine for patiently dealing with all the administration that have occurred over the years of my PhD. Thanks also to Owen Parkes for proof-reading my introduction. I would like to acknowledge sincerely all the members of Rick Cavicchioli’s team. It has been rewarding working with people from different scientific and cultural backgrounds and with whom I have shared a lot of laughs.
Load more

Recommended publications
  • A New Insight Into Role of Phosphoketolase Pathway in Synechocystis Sp
    www.nature.com/scientificreports OPEN A new insight into role of phosphoketolase pathway in Synechocystis sp. PCC 6803 Anushree Bachhar & Jiri Jablonsky* Phosphoketolase (PKET) pathway is predominant in cyanobacteria (around 98%) but current opinion is that it is virtually inactive under autotrophic ambient CO2 condition (AC-auto). This creates an evolutionary paradox due to the existence of PKET pathway in obligatory photoautotrophs. We aim to answer the paradox with the aid of bioinformatic analysis along with metabolic, transcriptomic, fuxomic and mutant data integrated into a multi-level kinetic model. We discussed the problems linked to neglected isozyme, pket2 (sll0529) and inconsistencies towards the explanation of residual fux via PKET pathway in the case of silenced pket1 (slr0453) in Synechocystis sp. PCC 6803. Our in silico analysis showed: (1) 17% fux reduction via RuBisCO for Δpket1 under AC-auto, (2) 11.2–14.3% growth decrease for Δpket2 in turbulent AC-auto, and (3) fux via PKET pathway reaching up to 252% of the fux via phosphoglycerate mutase under AC-auto. All results imply that PKET pathway plays a crucial role under AC-auto by mitigating the decarboxylation occurring in OPP pathway and conversion of pyruvate to acetyl CoA linked to EMP glycolysis under the carbon scarce environment. Finally, our model predicted that PKETs have low afnity to S7P as a substrate. Metabolic engineering of cyanobacteria provides many options for producing valuable compounds, e.g., acetone from Synechococcus elongatus PCC 79421 and butanol from Synechocystis sp. strain PCC 68032. However, certain metabolites or overproduction of intermediates can be lethal. Tere is also a possibility that required mutation(s) might be unstable or the target bacterium may even be able to maintain the fux distribution for optimal growth balance due to redundancies in the metabolic network, such as alternative pathways.
    [Show full text]
  • UV-B Induced Stress Responses in Three Rice Cultivars
    BIOLOGIA PLANTARUM 54 (3): 571-574, 2010 BRIEF COMMUNICATION UV-B induced stress responses in three rice cultivars I. FEDINA1*, J. HIDEMA2, M. VELITCHKOVA3, K. GEORGIEVA1 and D. NEDEVA1 Institute of Plant Physiology1 and Institute of Biophysics3, Bulgarian Academy of Sciences, Academic Georgi Bonchev Street, Building 21, Sofia 1113, Bulgaria Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan2 Abstract UV-B responses of three rice (Oryza sativa L.) cultivars (Sasanishiki, Norin 1 and Surjamkhi) with different photolyase activity were investigated. Carbon dioxide assimilation data support that Sasanishiki was less sensitive to UV-B than Norin 1 and Surjamkhi. UV-B radiation sharply decreased the content of Rubisco protein in Surjamkhi and has no effect in Sasanishiki. The photochemical activities of photosystem (PS) 1 and PS 2 was slightly affected by UV-B treatment. The content of H2O2 and the activities of antioxidant enzymes, catalase (CAT), peroxides (POX) and superoxide dismutase (SOD) were enhanced after UV-B treatment. The activities of CAT and POX isoenzymes in Sasanishiki were more enhanced by UV-B radiation than those in Norin 1 and Surjamkhi. 14 Additional key words: catalase, CO2 fixation, hydrogen peroxide, peroxidase, Rubisco, superoxide dismutase. ⎯⎯⎯⎯ UV-B sensitivity of plants is determined by the balance of Furthermore, transgenic rice plants in which the CPD damage incurred and by the efficiency of repair processes photolyase was overexpressed had higher CPD photolyase that can restore the impaired functions. This balance is activity and showed significantly greater resistance to influenced by several factors, including the genetic UV-B than wild plants (Hidema et al.
    [Show full text]
  • Taming the Wild Rubisco: Explorations in Functional Metagenomics
    Taming the Wild RubisCO: Explorations in Functional Metagenomics DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian Hurin Witte, M.S. Graduate Program in Microbiology The Ohio State University 2012 Dissertation Committee : F. Robert Tabita, Advisor Joseph Krzycki Birgit E. Alber Paul Fuerst Copyright by Brian Hurin Witte 2012 Abstract Ribulose bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39) (RubisCO) is the most abundant protein on Earth and the mechanism by which the vast majority of carbon enters the planet’s biosphere. Despite decades of study, many significant questions about this enzyme remain unanswered. As anthropogenic CO2 levels continue to rise, understanding this key component of the carbon cycle is crucial to forecasting feedback circuits, as well as to engineering food and fuel crops to produce more biomass with few inputs of increasingly scarce resources. This study demonstrates three means of investigating the natural diversity of RubisCO. Chapter 1 builds on existing DNA sequence-based techniques of gene discovery and shows that RubisCO from uncultured organisms can be used to complement growth in a RubisCO-deletion strain of autotrophic bacteria. In a few short steps, the time-consuming work of bringing an autotrophic organism in to pure culture can be circumvented. Chapter 2 details a means of entirely bypassing the bias inherent in sequence-based gene discovery by using selection of RubisCO genes from a metagenomic library. Chapter 3 provides a more in-depth study of the RubisCO from the methanogenic archaeon Methanococcoides burtonii.
    [Show full text]
  • Commentary. in the Article “Genetic Code Origins: Experi- Ments Confirm Phylogenetic Predictions and May Explain a Puzzle” B
    5890 Corrections Proc. Natl. Acad. Sci. USA 96 (1999) Commentary. In the article “Genetic code origins: Experi- Neurobiology. In the article “Growth factor-mediated Fyn ments confirm phylogenetic predictions and may explain a signaling regulates a-amino-3-hydroxy-5-methyl-4-isox- puzzle” by Paul Schimmel and Lluis Ribas de Pouplana, which azolepropionic acid (AMPA) receptor expression in rodent appeared in number 2, January 19, 1999 of Proc. Natl. Acad. neocortical neurons” by Mako Narisawa-Saito, Alcino J. Silva, Sci. USA (96, 327–328), the following corrections should be Tsuyoshi Yamaguchi, Takashi Hayashi, Tadashi Yamamoto, noted. The fifth and sixth sentences of the first paragraph on and Hiroyuki Nawa, which appeared in number 5, March 2, page 328 should read as follows (changes are indicated by bold 1999, of Proc. Natl. Acad. Sci. USA (96, 2461–2466), due to a type): “This base pair is found in the spirochetes T. pallidum printer’s error, there were several errors in the author and and B. burgdorferi that contain a class I enzyme. In contrast, affiliations lines. The correct affiliations are as follows: Ibba et al. (1) show that the class II E. coli enzyme cannot MAKO NARISAWA-SAITO*†,ALCINO J. SILVA‡,TSUYOSHI accept G2-U71.” Also, the word “spirocytes” in the sixth YAMAGUCHI*, TAKASHI HAYASHI§,TADASHI YAMAMOTO§, sentence of the second paragraph on page 328 should read AND HIROYUKI NAWA*†‡ spirochetes. Finally, the sixth sentence of the last paragraph on page 328 should read as follows: “So multiple lateral gene *Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan; ‡Cold Spring Harbor Laboratory, Cold transfer from archaebacteria to certain bacteria could account Spring Harbor, NY 11724; and §Institute of Medical Science, University of for the presence of class I LysRS in bacterial organisms such Tokyo, Tokyo 108-8639, Japan as T.
    [Show full text]
  • Microfilmed 199S Information to Users
    UMI MICROFILMED 199S INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with s m a ll overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. A Beil & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313.-761-4700 800.521-0600 Order Number 0517044 Molecular and biochemical studies of RubisCO activation in Anabatna species Li, Lih-Ann, Ph.D. The Ohio State University, 1094 Copyright ©1094 by Li, Llh-Ann.
    [Show full text]
  • The Mechanism of Rubisco Catalyzed Carboxylation Reaction: Chemical Aspects Involving Acid-Base Chemistry and Functioning of the Molecular Machine
    catalysts Review The Mechanism of Rubisco Catalyzed Carboxylation Reaction: Chemical Aspects Involving Acid-Base Chemistry and Functioning of the Molecular Machine Immacolata C. Tommasi Dipartimento di Chimica, Università di Bari Aldo Moro, 70126 Bari, Italy; [email protected] Abstract: In recent years, a great deal of attention has been paid by the scientific community to improving the efficiency of photosynthetic carbon assimilation, plant growth and biomass production in order to achieve a higher crop productivity. Therefore, the primary carboxylase enzyme of the photosynthetic process Rubisco has received considerable attention focused on many aspects of the enzyme function including protein structure, protein engineering and assembly, enzyme activation and kinetics. Based on its fundamental role in carbon assimilation Rubisco is also targeted by the CO2-fertilization effect, which is the increased rate of photosynthesis due to increasing atmospheric CO2-concentration. The aim of this review is to provide a framework, as complete as possible, of the mechanism of the RuBP carboxylation/hydration reaction including description of chemical events occurring at the enzyme “activating” and “catalytic” sites (which involve Broensted acid- base reactions) and the functioning of the complex molecular machine. Important research results achieved over the last few years providing substantial advancement in understanding the enzyme functioning will be discussed. Citation: Tommasi, I.C. The Mechanism of Rubisco Catalyzed Keywords: enzyme carboxylation reactions; enzyme acid-base catalysis; CO2-fixation; enzyme Carboxylation Reaction: Chemical reaction mechanism; potential energy profiles Aspects Involving Acid-Base Chemistry and Functioning of the Molecular Machine. Catalysts 2021, 11, 813. https://doi.org/10.3390/ 1. Introduction catal11070813 The increased amount of anthropogenic CO2 emissions since the beginning of the industrial era (starting around 1750) has significantly affected the natural biogeochemical Academic Editor: Arnaud Travert carbon cycle.
    [Show full text]
  • Overexpression of an Agave Phosphoenolpyruvate Carboxylase Improves Plant Growth and Stress Tolerance
    cells Article Overexpression of an Agave Phosphoenolpyruvate Carboxylase Improves Plant Growth and Stress Tolerance Degao Liu 1,2,†, Rongbin Hu 1,† , Jin Zhang 1,2 , Hao-Bo Guo 3, Hua Cheng 1 , Linling Li 1, Anne M. Borland 1,4 , Hong Qin 3, Jin-Gui Chen 1,2, Wellington Muchero 1,2, Gerald A. Tuskan 1,2 and Xiaohan Yang 1,2,* 1 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA; [email protected] (D.L.); [email protected] (R.H.); [email protected] (J.Z.); [email protected] (H.C.); [email protected] (L.L.); [email protected] (A.M.B.); [email protected] (J.-G.C.); [email protected] (W.M.); [email protected] (G.A.T.) 2 The Center for Bioenergy Innovation (CBI), Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA 3 Department of Computer Science and Engineering, SimCenter, University of Tennessee Chattanooga, Chattanooga, TN 37403, USA; [email protected] (H.-B.G.); [email protected] (H.Q.) 4 School of Natural and Environmental Science, Newcastle University, Newcastle upon Tyne NE1 7RU, UK * Correspondence: [email protected]; Tel.: +1-865-241-6895; Fax: +1-865-576-9939 † These authors contribute equally to this work. Abstract: It has been challenging to simultaneously improve photosynthesis and stress tolerance in plants. Crassulacean acid metabolism (CAM) is a CO2-concentrating mechanism that facilitates plant adaptation to water-limited environments. We hypothesized that the ectopic expression of a CAM- specific phosphoenolpyruvate carboxylase (PEPC), an enzyme that catalyzes primary CO2 fixation in Citation: Liu, D.; Hu, R.; Zhang, J.; CAM plants, would enhance both photosynthesis and abiotic stress tolerance.
    [Show full text]
  • Greater Efficiency of Photosynthetic Carbon Fixation Due to Single Amino
    ARTICLE Received 7 Aug 2012 | Accepted 16 Jan 2013 | Published 26 Feb 2013 DOI: 10.1038/ncomms2504 OPEN Greater efficiency of photosynthetic carbon fixation due to single amino-acid substitution Judith Katharina Paulus1,*, Daniel Schlieper1,* & Georg Groth1 The C4-photosynthetic carbon cycle is an elaborated addition to the classical C3-photo- synthetic pathway, which improves solar conversion efficiency. The key enzyme in this pathway, phosphoenolpyruvate carboxylase, has evolved from an ancestral non- photosynthetic C3 phosphoenolpyruvate carboxylase. During evolution, C4 phosphoe- nolpyruvate carboxylase has increased its kinetic efficiency and reduced its sensitivity towards the feedback inhibitors malate and aspartate. An open question is the molecular basis of the shift in inhibitor tolerance. Here we show that a single-point mutation is sufficient to account for the drastic differences between the inhibitor tolerances of C3 and C4 phosphoenolpyruvate carboxylases. We solved high-resolution X-ray crystal structures of a C3 phosphoenolpyruvate carboxylase and a closely related C4 phosphoenolpyruvate carboxylase. The comparison of both structures revealed that Arg884 supports tight inhibitor binding in the C3-type enzyme. In the C4 phosphoenolpyruvate carboxylase isoform, this arginine is replaced by glycine. The substitution reduces inhibitor affinity and enables the enzyme to participate in the C4 photosynthesis pathway. 1 Cluster of Excellence on Plant Sciences (CEPLAS), Institute of Biochemical Plant Physiology, Heinrich Heine University, Universitaetsstr. 1, 40225 Du¨sseldorf, Germany. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to G.G. (email: [email protected]). NATURE COMMUNICATIONS | 4:1518 | DOI: 10.1038/ncomms2504 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited.
    [Show full text]
  • Metagenomic Analysis of Phosphorus Removing Sludge Communities
    Metagenomic analysis of phosphorus removing sludge communities. Héctor García Martín1, Natalia Ivanova1, Victor Kunin1, Falk Warnecke1, Kerrie Barry1, Alice C. McHardy4, Christine Yeates2, Shaomei He3, Asaf Salamov1, Ernest Szeto1, Eileen Dalin1, Nik Putnam1, Harris J. Shapiro1, Jasmyn L. Pangilinan1, Isidore Rigoutsos4, Nikos C. Kyrpides1, Linda Louise Blackall2, Katherine D. McMahon3, and Philip Hugenholtz1* 1 DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA. 2 Advanced Wastewater Management Centre, University of Queensland, St Lucia, 4072, Queensland, Australia. 3 Civil and Environmental Engineering Department, University of Wisconsin- Madison, 1415 Engineering Drive, Madison, WI 53706, USA. 4 IBM Research Division, Thomas J. Watson Research Center, P.O. Box 218, Yorktown Heights, NY 10598, USA *corresponding author: [email protected] 1 Abstract Enhanced Biological Phosphorus Removal (EBPR) is not well understood at the metabolic level despite being one of the best-studied microbially-mediated industrial processes due to its ecological and economic relevance. Here we present a metagenomic analysis of two lab-scale EBPR sludges dominated by the uncultured bacterium, “Candidatus Accumulibacter phosphatis”. This analysis sheds light on several controversies in EBPR metabolic models and provides hypotheses explaining the dominance of A. phosphatis in this habitat, its lifestyle outside EBPR and probable cultivation requirements. Comparison of the same species from different EBPR sludges highlights recent evolutionary dynamics in the A. phosphatis genome that could be linked to mechanisms for environmental adaptation. In spite of an apparent lack of phylogenetic overlap in the flanking communities of the two sludges studied, common functional themes were found, at least one of them complementary to the inferred metabolism of the dominant organism.
    [Show full text]
  • Rubisco Folding and Oligomeric Assembly: Detailed Analysis of an Assembly Intermediate
    Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Rubisco folding and oligomeric assembly: Detailed analysis of an assembly intermediate Amanda Windhof (geb. Starling) aus Delaware, Ohio, USA 2011 Acknowledgments I especially want to thank Prof. Dr. F. Ulrich Hartl and Dr. Manajit Hayer-Hartl for giving me the opportunity to conduct my PhD research in the department. I have learned so much on this journey which would not have been possible without your intellectual support and scientific enthusiasm. I also want to thank PD Dr. Dr. Winklhofer for being my 2. Gutacherin and of course the other members of my PhD committee: Prof. Dr. Beckmann, Prof. Dr. Vothknecht, Prof. Dr. Gaul, and Prof. Dr. Hopfner. There are also many people I want to thank for within the department and institute. I am very grateful to Dr. Cuimin Liu for her help and guidance when I first came to this department. She was always open to answer any questions I had. Thank you Dr. Andreas Bracher for your support in many ways, in particular with your crystallographic knowledge and various ideas for developing enhanced methods. I also want to thank Dr. Oliver Müller- Cajar for your never ending passion for science, especially for Rubisco, and for your very insightful discussions. I am grateful for the lunch conversations with Dr. Yi-chin Tsai and your mass spectrometry expertise. I would also like to thank Thomas Hauser and Mathias Stotz for your valuable discussions and input. Thank you to Evelyn Frey-Royston, Silke Leuze-Bütün, and Andrea Obermayr-Rauter for their administrative support as well as Emmanuel Burghardt, Bernd Grampp, Romy Lange, Nadine Wischnewski, Verena Marcus, Elisabeth Schreil, Albert Ries, Andreas Scaia, and Kevin Gaughan for their invaluable technical support.
    [Show full text]
  • Directed Evolution of an Improved Rubisco; in Vitro Analyses to Decipher Fact from Fiction
    International Journal of Molecular Sciences Article Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction Yu Zhou and Spencer Whitney * Australian Research Council Center of Excellence for Translational Photosynthesis, Research School of Biology, The Australian National University, 134 Linnaeus Way, Acton, ACT 0200, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-2-6125-5073 Received: 30 August 2019; Accepted: 4 October 2019; Published: 10 October 2019 Abstract: Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains C four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcat , C up 27%), carboxylation efficiency (kcat /Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production.
    [Show full text]
  • Program and Abstracts Book
    16th International Conference on the Cell and Molecular Biology of Chlamydomonas June 8-13, 2014 Asilomar Conference Center, Pacific Grove, CA, USA Program and Abstracts 16th International Conference on the Cell and Molecular Biology of Chlamydomonas June 8-13, 2014 Asilomar Conference Grounds Pacific Grove, California Program and Abstracts Organizers: Kris Niyogi, University of California, Berkeley Winfield Sale, Emory University Marilyn Kobayashi, University of California, Berkeley Advisory Committee: José Luis Crespo, CSIC - Universidad de Sevilla Susan Dutcher, Washington University School of Medicine Arthur Grossman, Carnegie Institution for Science Sabeeha Merchant, University of California, Los Angeles Jun Minagawa, National Institute for Basic Biology David Mitchell, SUNY Upstate Medical University Rachael Morgan-Kiss, Miami University Michael Schroda, University of Kaiserslautern Carolyn Silflow, University of Minnesota James Umen, Donald Danforth Plant Science Center Chia-Lin Wei, DOE Joint Genome Institute William Zerges, Concordia University 1 2 TABLE OF CONTENTS General Information ............................................................................................................................ 4 Exhibitors ............................................................................................................................................. 4 Schedule of Events ............................................................................................................................... 5 Plenary Session Listings
    [Show full text]