Microbial and Chemical Diversity of Tetrodotoxin-Producing Marine Animals
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Microbial and chemical diversity of tetrodotoxin-producing marine animals Rocky Chau A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science August 2013 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 ……………………………………………........................... 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 ……………………………………………………………. 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.’ Signed ……………………………………………………………. Date ……………………………………………………………. Abstract Marine molluscs are known to employ a variety of defence systems to improve their survivability, such as the production of bioactive molecules. Recently, microbes have been identified as the true producers of these compounds. Similarly, tetrodotoxin (TTX) is hypothesised to have a bacterial origin, however, much controversy still exists. This thesis attempts to shed further light on the biosynthetic origins of TTX with a focus on Hapalochalaena sp. and Pleurobranchaea maculata. This thesis also investigates the potential for biosynthesis of other natural products by bacteria living in association with these marine molluscs. Culture-based studies attempting to isolate TTX-producing bacterium within Hapalochalaena sp. and P. maculata were unable to isolate any such strains. Furthermore, we attempted to replicate the production of TTX by published TTX-producing bacteria, however, TTX was unable to be detected in these strains via spectrometric methods. Nevertheless, culture-independent methods were able to identify four taxa that were strongly correlated to TTX- concentration in P. maculata. Further experiments, however, are required to isolate or characterise these strains. Molecular screening for natural product biosynthesis genes, putatively involved in the biosynthesis of TTX and other natural products, revealed many candidate bacteria. These experiments also identified a bacterium with significant biosynthetic potential, Pseudoalteromonas sp. HM-SA03. Members of Pseudoalteromonas are known to produce many bioactive compounds. Mining of the HM-SA03 genome identified a total of seven novel NRPS and PKS biosynthesis gene clusters. Bioinformatic analysis of these gene clusters revealed putative novel pathways for the assembly of i bromoalterochromide, alteramide and pseudoalterobactin-like compounds. However, production of these compounds in laboratory-cultures of Pseudoalteromonas sp. HM-SA03 could not be confirmed by chemical studies. Nonetheless, chemical studies were able to identify the production of eight diketopiperazines. These bioactive molecules have been observed to function as cell-signalling molecules, and are proposed to function as such in the complex microbial community identified within the Hapalochalaena octopus. Taken together, these results indicate that marine molluscs are a rich source of biosynthetically potent microbes that deserve further attention for the elucidation of new natural products. ii Acknowledgements First and foremost, I would like to thank my supervisor, Professor Brett A. Neilan for his support, motivation and most of all, his patience when things did not go to plan. I am most fortunate to study in his laboratory, under his tutelage. My co-supervisor was Dr. John A. Kalatizis, the chemistry extraordinaire, who was always willing to bear my frustrations and offer me advice when things went wrong. He has tirelessly, and without complaint, proofed many of my manuscripts. The warmest gratitude goes to my colleagues at the Cawthron Institute, Dr Susanna Wood and Paul McNabb. They assisted me with all the work with Pleurobranchaea maculata, including LC-MS analysis. I am grateful for the kind hospitality you showed during my visits to New Zealand. Thanks to Dr. Mark Brown, who first introduced me to the world of bioinformatics and microbial ecology and Dr. Russell Pickford for his assistance with LC-MS. Thank you to all the members of my laboratory, especially Jason and Sarah. Working alongside you all has been a pleasure; you guys keep the lab fun! Thanks to my rock climbing and F1 buddies, Apurv included, who have kept me distracted (and sane) outside of work. Of course, I could not have gone through this without the support of my loving family. The final and most special thanks must go to my beloved Rebecca, who has quietly and patiently supported me wholeheartedly throughout all these years. iii List of publications and presentations Publications: Chau, R., Kalaitzis, J.A., Neilan, B.A., 2014. Tetrodotoxin. In: Dongyou, L. (ed.) Manual of Security Sensitive Microbes and Toxins, GBR, Taylor and Francis. In press. Chau, R., Kalaitzis, J.A., Wood, S., Neilan, B., 2013. Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals. Marine Drugs 11, 2695-2712. Chau, R., Kalaitzis, J.A., Neilan, B.A., 2011. On the origins and biosynthesis of tetrodotoxin. Aquatic Toxicology 104, 61-72. Kalaitzis, J.A., Chau, R., Kohli, G.S., Murray, S.A., Neilan, B.A., 2010. Biosynthesis of toxic naturally-occurring seafood contaminants. Toxicon 56, 244-258. Presentations: Chau, R., Kalaitzis, J.A., Neilan, B.A., 2012. Genome of an octopus-derived Pseudoalteromonas reveals unprecedented natural product gene clusters. International Congress on Natural Products Research, New York. iv Abstract ...................................................................................................................................... i Acknowledgements ............................................................................................................. iii List of publications and presentations .......................................................................... iv List of abbreviations ............................................................................................................ ix List of figures .......................................................................................................................... xi List of tables ......................................................................................................................... xiii Chapter 1: Introduction to the biosynthesis of natural products ...................... 15 1.1. Biosynthesis of secondary metabolites ........................................................................ 17 1.1.1. Polyketide synthases .................................................................................................. 19 1.1.2. Non-ribosomal peptide synthetases ..................................................................... 20 1.1.3. Structural diversity through biosynthetic exchangeability ......................... 21 1.2. Genome mining for biosynthesis gene clusters ........................................................ 21 1.2.1. Molecular screening for biosynthesis genes ...................................................... 23 1.2.2. Identification of biosynthesis gene clusters through genome analysis .. 24 1.3. Biosynthesis of tetrodotoxin ............................................................................................ 26 1.3.1. The guanidinium moiety of tetrodotoxin. ........................................................... 27 1.1.3.1. Amidinotransferase involvement in tetrodotoxin biosynthesis ....... 27 1.1.3.2. Non-ribosomal peptide synthetase involvement in tetrodotoxin biosynthesis ........................................................................................................................... 29 1.3.2. The carbon backbone of tetrodotoxin .................................................................. 30