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Osmoadaptation Mechanisms of Cyanobacteria and Archaea from the Stromatolites of Hamelin Pool, Western Australia

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Osmoadaptation Mechanisms of Cyanobacteria and Archaea from the Stromatolites of Hamelin Pool, Western Australia Osmoadaptation mechanisms of cyanobacteria and archaea from the stromatolites of Hamelin Pool, Western Australia Falicia Qi Yun Goh B. Sc (Hons.) UNSW A Dissertation Submitted in Fulfilment of the Requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences The University of New South Wales Sydney, Australia 2007 Osmoadaptation mechanisms of cyanobacteria and archaea from the stromatolites of Hamelin Pool, Western Australia Falicia Qi Yun Goh B. Sc (Hons.) (UNSW) A Dissertation Submitted in Partial Fulfilment of the Requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences The University of New South Wales Sydney, Australia Supervisors Prof. Brett A. Neilan Dr. Brendan P. Burns School of Biotechnology and Biomolecular Sciences The University of New South Wales Sydney, Australia Certificate of originality Abstract The stromatolites of Shark Bay Western Australia, located in a hypersaline environment, is an ideal biological system for studying survival strategies of cyanobacteria and halophilic archaea to high salt and their metabolic cooperation with other bacteria. To-date, little is known of the mechanisms by which these stromatolite microorganisms adapt to hypersalinity. To understand the formation of these sedimentary structures, detailed analysis of the microbial communities and their physiology for adaptation in this environment are crucial. In this study, microbial communities were investigated using culturing and molecular methods. Phylogenetic analysis of the 16S rRNA gene was carried out to investigate the diversity of microorganisms present. Unique phylotypes from the bacteria, cyanobacteria and archaea clone libraries were identified. Representative cyanobacteria isolates and Halococcus hamelinensis, a halophilic archaea isolated from in this study, were the focus for identifying osmoadaptation mechanisms. The presence of osmolytes in these microorganisms was detected by Nuclear magnetic resonance spectroscopy (NMR). It was found that the cyanobacterial isolates studied utilised different osmolytes. Glucosylglycerol, unique to marine cyanobacteria was not identified; instead various saccharides, glycine betaine and TMAO were the predominant solutes present. Thus cyanobacteria are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. Findings here also indicated that H. hamelinensis accumulates glycine betaine and glutamate instead of potassium ions. DNA molecular methods were employed to identify candidate genes for the uptake of osmoprotectants. Three putative glycine betaine transporters from Halococcus hamelinensis were identified. Functionality of one of these glycine betaine transporters was determined by complementation studies. For the first time, an archaeal glycine betaine transporter was shown to be successfully complemented in a glycine betaine transport deficient mutant (E. coli MKH13). This study has increased our understanding of how microorganisms co-exist in fluctuating environments in response to solubilisation/precipitation or dilution/evaporation processes, resulting in a hypersaline environment. It also provides an excellent platform for the identification of any novel osmolytes/compatible solutes that might have been produced by these microorganisms that have been isolated for the first time from stromatolites. Acknowledgments Time and tide wait for no man. Solid years of perseverance, hardwork and sheer luck have finally seen to the completion of this dissertation. However it would not be possible without comrades that braved through the failure of experiments, the late nights, the caffeine shoots and the heartbreaking moments throughout the whole time span of this project as well as the joy of success and time spent together. This journey would not be possible without Brett Neilan and Brendan Burns who enticed me to start this PhD and giving me the opportunity to work with them. They have provided endless support and training. It would be so hard to find supervisors like you. Hope this bring tears to your eyes. Long story short, you have made me into what I m and who I m today. No pun intended =P. Another person who leaded me to this path is Professor Sim Tiow Suan, without her mentoring and good advices, I probably would have lacked the courage to settle back into Science for the passion rather than the money. Not to forget Kevin Barrow, Masahiro Kamekura, Tomo Kawaguchi, Yung-Pin Chen for their help and sharing their ideas and experiences with me. To friends, people who come and go in my life & people who have helped me. Especially girls in BAN Lab, Lenora, Rosy, Anne, Hannah, Alex, Kristen, Michelle G., and Michelle A. for all the coffee breaks, lunch breaks, retail therapies, dinners and surprises. It is sad that the time have come when we are all going separate ways but we will meet up again someday, somewhere out there. Last but not the least to my parents and brother: for being there when I need them. Thank you! “Never tire to study and to teach others” Confucius List of Publications Articles in journals Allen, M.A., Goh, F., Leuko, S., Echigo, A., Mizuki, T., Usami, R., Kamekura, M., Neilan, B.A. & Burns, B.P. Haloferax elongans sp. nov. and Haloferax mucosum sp. nov., isolated from microbial mats from Hamelin Pool, Shark Bay. Int J Syst Evol Microbiol (in press). Leuko, S., Goh, F., Ibáñez-Peral,R., Burns, B. P., Walter, M., & Neilan, B. A. Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment. Extremophiles (in press). Leuko, S., Goh, F., Allen, M. A., Burns, B. P., Walter, M., & Neilan, B. A. (2007) Analysis of intergenic spacer region length polymorphisms to investigate the halophilic archaean diversity of stromatolites and microbial mats. Extremophiles 11, 203-210. Burns, B. P., Pomati, F., Goh, F., Yasar, S. A. & Neilan, B. A. (2006) Stromatolites as a resource for novel natural products. Orig Life Evol Biosph. 36(5-6), 623-624. Goh, F., Leuko, S., Allen, M. A., Bowman, J. P., Kamekura, M., Neilan, B. A. & Burns, B. P. (2006). Halococcus hamelinensis sp. nov., a novel halophilic archaeon isolated from stromatolites in Shark Bay, Australia. Int J Syst Evol Microbiol 56, 1323 - 1329. Burns, B. P., Seifert, A., Goh, F., Pomati, F., Jungblut, A., Serhat, A. & Neilan, B. A. (2005). Genetic potential for secondary metabolite production in stromatolite communities. FEMS Microbiology Letters 243, 293-301. Burns, B. P., Goh, F. & Neilan, B. A. (2004). Dynamics of cyanobacterial communities in living stromatolites. Current Trends in Microbiology 1, 103-106. Burns, B. P., Goh, F., Allen, M. A. & B. A. Neilan (2004). Microbial diversity of extant stromatolites in the hypersaline marine environment of Shark Bay, Australia. Environmental Microbiology 6 (10), 1096-1101. Burns, B. P., Goh, F., Pomati, F. & Neilan, B. A. (2004). Big Genes, Small Molecules- Molecular Basis for Toxicity and Biosynthesis of Natural Products in Cyanobacteria. Marine Biotechnology 6, S1-S5 Contributions to academic conferences Burns, B. P., Goh, F., Leuko, S., Marshall, C., Neilan, B. A., Walter, M.R. Stromatolites: resources for understanding early life and the potential for life elsewhere, in 7th Australian Space Science Conference, Sydney, Australia (2007). Goh, F., Jeon, Y-J., Barrow, K. D., Neilan, B. A. & Burns, B. P. Role of compatible solutes in the functioning of microbial communities in the stromatolites from Hamelin Pool, Shark Bay, Western Australia, in Extremophiles, Brest, France (2006) Goh, F., Jeon, Y-J., Barrow, K. D., Neilan, B. A. & Burns, B. P. Mechanisms of osmoadaptation in stromatolites of Shark Bay, Australia, in 4th Astrobiology Science Conference, Washington DC, USA (2006). Goh, F., Jeon, Y-J., Barrow, K. D., Neilan, B. A. & Burns, B. P. Osmoadaptation of a halophilic archaea and cyanobacteria from the stromatolites, Shark Bay, Western Australia, in International Symposium of Extremophiles and its Applications (ISEA), Tokyo, Japan (2005). Burns, B. P., Goh, F., Pomati, F. & Neilan, B. A. Biodiversity and bioprospecting in stromatolite cyanobacterial communities, in 6th European Workshop on the Molecular Biology of Cyanobacteria, Gdansk, Poland (2005). Yagoutyfam, N., Goh, F., Ting, L., Neilan, B.A. & Burns, B.P. Insights into salt adaptation by Halococcus hamelinii, in 4th Annual Biotechnology and Biomolecular Sciences Symposium, The University of New South Wales, (ISBN NO. 0 7334 2295 0), Sydney, Australia (2005). Burns, B.P., Goh, F., Pomati, F. & Neilan, B.A. Genetic potential for secondary metabolite production in stromatolite communities, in National Workshop on Astrobiology, Capri, Italy (2005). Goh, F., Leuko, S., Burns, B. P. & Neilan, B. A. Stromatonome-directed isolation of halophilic archaea, in Australian Society for Microbiology conference, Sydney, Australia (2004). Goh, F., Burns, B. P. & Neilan, B. A. Unexpected microbial diversity of the living stromatolites of Shark Bay, Western Australia, in AbSciCon, Nasa Ames Research Centre,USA (2004). Goh, F., Burns, B. P., & Neilan, B. A. Analysis of microbial communities in the living stromatolites of Shark Bay, Western Australia, in Biotechnology and Biomolecular Sciences Symposium, The University of New South Wales, (ISBN 0-7334-1581-4), Sydney, Australia (2003). Goh, F., Allen, M. A., Burns, B. P. & Neilan, B. A. Analysis of microbial communities in stromatolites, in Australian Society for Microbiology conference, Melbourne, Australia (2002). Book
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