PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: PENESYAN First name: ANAHIT Other name/s: Abbreviation for degree as given in the University calendar: PHD School: BIOTECHNOLOGY AND BIOIVIOLECULAR SCIENCES Faculty: SCIENCE Title: PRODUCTION OF ANTIMICROBIAL COMPOUNDS BY MARINE EPIBIOTIC BACTERIA Abstract The aim of this thesis was to obtain antimicrobiai compounds from marine eukaryote-associated bacteria. The unique environment, present on the surfaces of marine eul^aryotes, creates conditions that promote and favour the production of bioactive compounds, such as antimicrobials, by giving the producers a clear advantage in the competition for nutrients. This study has demonstrated the abundance of antimicrobial producing bacteria on two marine algae, Delisea pulchra and Ulva australis. Two antimicrobial compounds, violacein and tropodithietic acid (TDA), have been successfully purified and chemically identified from two different bacterial isolates. IVIoreover, the production of multiple bioactive compounds was observed for both these bacteria. This study also made an attempt to understand the role of the antimicrobial compounds for the producer organisms. Consequently, the effect of violacein on biofilm formation, as well as the possible role of TDA in the defence of both the producer bacterium and the host, have been proposed. The importance of environmental conditions for the expression of bioactive compounds has also been demonstrated, for example, by showing the necessity of high iron concentrations for the production of bioactives in isolates U156 and D245. Notably, despite the absence of a close phylogenetic relationship between these two isolates, they have shown similar trends in terms of production of bioactive compounds. For the first time, this work also described the construction and analysis of a large insert DNA library in E. coli using genomic DNA from a collection of cultured isolates with demonstrated antimicrobial activity. This approach proved to be successful and led to a substantial increase in the positive hit rates in the functional screening of the library, and provided invaluable information concerning the genes, potentially involved not only in the biosynthesis, but also in the processes associated with the production of bioactive compounds, such as transport and resistance. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to mal<e available my thesis or dissertation in whole or in part in the University libraries in all fomns of media, now or here after known, subject to the provisions of the Copyright Act 1968.1 retain all property 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 Abstracts International (this is applicable to doctoral theses only). ' Signature/^? Witness '' ' Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional FOR OFFICE USE ONLY Date of co mpleti on of requirements for Award: to Production of antimicrobial compounds by marine epibiotic bacteria Anahit Penesyan A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia 2010 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 1968.1 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 ..../.....rt^e^ Date 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 I^/.RBJAPIA. ORIGINALITY STATEMENT 'I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials 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.' Anahit Penesyan Date ACKNOWLEDGEMENTS I would like to thank my supervisors, Prof. Staffan Kjelleberg and Dr. Suhelen Egan, for the opportunity to join the research team at the Centre for Marine Bio- Innovation. Dear Staffan, I would like to thank you for all the advice and support during my PhD project, and thank you for believing in me. Dear Su, I can't express all my gratitude for all your help and guidance and for the continuous support and encouragement during my PhD. It was always very comforting to know that you are there, in all ups and downs, and are always ready to help. I would also like to thank Dr Tilmann Harder for his help in the chemistry related part of my project. Dear Tilmann, thank you for enthusiasm, it was always a pleasure working with you and it was a great learning experience. I am also grateful to Dr. Torsten Thomas, Dr. Matt Lee, Dr. Carola Holmstrom, Dr. David Schleheck, Dr. Sharon Longford, Dr. Flavia Evans, Cathy Burke, Maria Yung, Jan Tebben for all their help during various stages of my project. Thank you guys, I wouldn't be able to do all I did during my project without you. Many thanks to Kirsty Collard and Adam Abdool who do a great job and ensure everything runs smoothly within the CMB. Thank you for all your help in sorting out all the various forms and documents I have encountered during my project. I am also grateful to Bill O'Sullivan for proof-reading my thesis. I would also like to thank all the people working in various labs within CMB where I also happen to work, to all the people in labs 323, 304, and 315: Anne, Sacha, Melissa, Jo, Francesco, Adrian, and everyone else whom I did not mention - thank you for all the fun and for the great working environment. At the end I would like to thank my family and my parents for all their love and support. Words will not be able to express all that I owe you; this work is dedicated to you! LIST OF PUBLICATIONS Published: •Penesvan A. Kjelleberg S and Egan S (2010). "Development of novel drugs from marine surface associated microorganisms." Marine Drugs (special issue) 8(3): 438-459. *Penesvan A. Marshall-Jones Z, Holmstrom C, Kjelleberg S and Egan S (2009). "Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs: Research article." FEMS Microbiology Ecology 69(1): 113-124. Thomas T, Evans F F, Schleheck D, Mai-Prochnow A, Burke C, Penesvan A, Dalisay D S, Stelzer-Braid S, Saunders N, Johnson J, Ferriera S, Kjelleberg S and Egan S (2008). "Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface- associated life style in the marine environment." PLoS ONE 3(9): e3252. *Matz C, Webb J S, Schupp P J, Phang S Y, Penesvan A. Egan S, Steinberg P and Kjelleberg S (2008). "Marine biofilm bacteria evade eukaryotic prédation by targeted chemical defense." PLoS ONE 3(7): e2744. Penesvan A R, Antonyan A P, Vardapetyan H R. (2004). "Interaction of purified hypericin preparations with DNA." Scientific Publications of the Yerevan State University i: 80 -85. Penesvan A R (2004). "Content of podophyllotoxin in different plant species grown in Armenia." lAELS Bulletin 9(8): 56 - 60. Penesvan A R, Vardapetyan H R, Tiratsuyan S G, Hovhannisyan A A and Kabasakalyan E E (2004). " Interactions between podophyllotoxin and DNA." lAELS Bulletin 9 (3): 168-171 Vardapetyan H R, Kirakosyan A B, Hovhannisyan A A, Penesvan A R and Alfermann W. (2003) "Effect of various elicitors on lignan biosynthesis in callus cultures of Linum austriacum. Russian Journal of Plant Physiology 50 (3): 297-300. In Preparation: *Penesvan A. Harder T, Tebben J, Lee M, Kjelleberg S and Egan S (2010). "Production of tropodithietic acid and phenol by an algal-associated alpha-proteobacterium D323 and related sponge isolates". *Penesvan A. Kjelleberg S, and Egan S (2010). "Violacein affects formation of biofilms in marine algal-associated bacterium Microbulbifer sp. D250". *Penesvan A. Novakowsky D, Thomas T, Kjelleberg S, and Egan S (2010) "Functional screening of a large DNA insert library created using the genomic DNA from a collection of antimicrobial-producing bacterial isolates, shows a substantial increase in positive hit rates and identifies genes involved in the production of bioactives".