COASTAL BACTERIAL COMMUNITIES: THEIR POTENTIAL ROLES IN DIMETHYLSULPHIDE (DMS) PRODUCTION AND CORAL DEFENCE By FELICITY KUEK WEN IK A thesis submitted in partial fulfilment of the requirements for the degree of Masters of Science (by Research) Faculty of Engineering, Computing and Science Swinburne University of Technology (Sarawak campus) 2014 P a g e | ii P a g e | iii Abstract Little is known about the microbial communities in the South China Sea, especially the eastern region and this study aims to expand our knowledge on the diversity of culturable bacterial communities in this area. The Talang-Satang region is situated off the coast of Sematan and is especially important as it is one of the most diverse ecosystems found off Sarawak. Complex microbial communities are known to have significant influence over coral reef ecosystems. Through isolation and identification (16S rDNA) of native microbes from the open ocean, coral surface mucus layer (SML), as well as the surrounding sediment and waters, we were able to determine the species composition and abundance of the culturable bacteria in the South China Sea (Kuching and Kota Kinabalu), the Celebes Sea (Semporna) and the coral reef ecosystem (Talang-talang reef). Comparisons were made with regards to physico-chemical parameters and bacterial communities. The diversity of bacterial communities in these marine environments were analysed through isolation and identification (16S rDNA) of culturable bacteria, as well as preparation of clone libraries and subsequent restriction fragment length polymorphism (RFLP). It was observed that although the majority of bacteria in Kuching, Kota Kinabalu and Semporna are members of the Proteobacteria group, the composition of bacterial communities in these three areas did vary significantly, and the changes were also mirrored in physico-chemical differences. There is also a clear distinction between the different species found in the different parts of the reef system. Isolates found attached to the coral were mostly related to Vibrio spp., presumably attached to the mucus from the water column and surrounding sediment. Cultures that were isolated from the SML are found to be closely related to antibiotic producers with tolerance towards elevated temperatures and heavy metal contamination. This specialized microbiota may be important for protecting the corals from pathogens by occupying entry niches and/or through the production of secondary metabolites (i.e. antibiotics). The role of the mucus- associated bacteria for the defence of the coral was highlighted by the fact that isolates related to pathogenic Vibrio spp. and Bacillus spp. were dominant amongst P a g e | iv the samples from the water column and sediment, and isolates with closest matches to the known coral pathogens Vibrio coralliilyticus and Vibrio shiloi were obtained from the SML and sediment samples respectively. The ability of isolates living in the SML (associated) to inhibit isolates loosely attached to the SML (attached) and vice versa was assessed at varying temperatures. All isolates were also screened (using specific sets of primers) for the presence of type I modular polyketides synthase (PKS) genes responsible for macrolide polyketides production and non-ribosomal peptide synthetase (NRPS) genes with the ability to produce immunosuppressants and other antibiotics. Our results indicate that the mucus-associated bacteria display maximum efficacy to ward off other bacteria at 28 °C, however the inhibitory abilities of mucus-associated bacteria became less effective as temperatures increased. One major and globally important role of surface bacteria is their involvement in the breakdown or osmoregulation of dimethylsulphoniopropionate (DMSP) to dimethylsulfide (DMS) or methanethiol (MeSH). Using genomic-based studies, enzymes responsible for DMSP degradation within the microbial community can be identified and over 200 culturable bacteria were screened for the existence of two key genes (dmdA, dddP) which are involved in competing, enzymatically mediated DMSP degradation pathways. Roseobacter spp. which are mainly responsible for the degradation of DMSP – a major source of oceans’ organic sulphur – into MeSH were also successfully isolated from the SML. Bacterial DMSP degraders may also contribute significantly to DMS production when temperatures are elevated. This is to our knowledge the first comprehensive study looking at culturable bacteria in the eastern South China Sea and their potential roles in coral defence and the DMS(P) cycle. P a g e | v Acknowledgements For since the creation of the world God’s invisible qualities – his eternal power and divine nature – have been clearly seen, being understood from what has been made, so that people are without excuse. (Romans 1:20) Foremost, I would like to express my sincere gratitude to my principal coordinating supervisor, Dr. Moritz Müller for his continuous support of my MSc study and research, for his patience, motivation, enthusiasm, and immense knowledge. Thank you for giving me the chance to explore this field, allowing me freedom and space to make mistakes and for believing in me. I would also like to extend my appreciation to my co-supervisors: Dr. Aazani Mujahid, Assoc. Prof. Dr. Lim Po Teen, and Dr. Leaw Chui Pin, for their encouragements, insightful comments, hard questions, as well as access to laboratories and facilities in Universiti Malaysia Sarawak (UNIMAS). Heartfelt thanks also to the Biotechnology laboratory officers and technicians: Chua Jia Ni, Dyg. Rafika Atiqah and Nurul Arina, for allowing me to use the labs past office hours and weekends, and for loaning me apparatus and experiment materials when I needed them. Without your help, this project may not have been completed on time. A big thank you to my fellow lab mates and student helpers: Onn May Ling, Jessica Fong, Lim Li Fang, and Ngu Lin Hui, for the stimulating discussions, the company during long hours in the lab, the support during various existential crises and for all the fun we have had in the last two years. Last but not least, I would like to thank my family, especially my mother, for encouraging me to take up this M.Sc. opportunity and for having my back throughout every circumstance in the past two years. I am grateful to the Sarawak Foundation for providing me with funding via the Tunku Abdul Rahman Scholarship which enabled me to pursue this postgraduate study. P a g e | vi Declaration I hereby declare that this research entitled “Coastal Bacterial Communities: Their Potential Roles in Dimethylsulphide (DMS) Production and Coral Defence” is original and contains no material which has been accepted for the award to the candidate of any other degree or diploma, except where due reference is made in the text of the examinable outcome; to the best of my knowledge contains no material previously published or written by another person except where due reference is made in the text of the examinable outcome; and where work is based on joint research or publications, discloses the relative contributions of the respective workers or authors. (FELICITY KUEK WEN IK) Date: 9th September 2014 In my capacity as the Principal Coordinating Supervisor of the candidate’s thesis, I certify that the above statements are true to the best of my knowledge. (MORITZ MÜLLER) Date: 9th September 2014. P a g e | vii Publications Arising from this Thesis The work described in this thesis has been submitted as described in the following: Kuek F.W.I., Mujahid A., Lim P.T., Leaw C.P. & Müller M. ‘Diversity and DMS(P)- related genes in culturable bacterial communities in Malaysian coastal waters’. Systematic and Applied Microbiology (Manuscript ID:). Kuek F.W.I., Lim L.F., Ngu L.H., Mujahid A., Lim P.T., Leaw C.P. & Müller M. ‘The potential roles of bacterial communities in coral defence: a case study at Talang-talang reef’. Ocean Science Journal (Manuscript ID: OSJO-D-14- 00062). Early work has been presented in the following conferences and contributed to the content presented in Chapters 3 and 4 of this thesis: Müller M., Kuek F.W.I., Song J.X.P. & Mujahid A. ‘Potential role of microbes in the local sulphur and nitrogen cycles in Kuching waters’ IOC/WESTPAC 9th International Scientific Symposium, 22-25 April 2014, Nha Trang, Khanh Hoa, Vietnam. (Oral presentation) Kuek F.W.I., Lim L.F., Ngu L.H., Ng C.T., Mujahid A., Lim P.T., Leaw C.P. & Müller M. ‘The potential role of bacterial communities: a case study at Talang-talang reef’ IOC/WESTPAC 9th International Scientific Symposium, 22-25 April 2014, Nha Trang, Khanh Hoa, Vietnam. (Poster presentation) Kuek F.W.I., Lim L.F., Ngu L.H., Ng C.T., Mujahid A., Lim P.T., Leaw C.P. & Müller M. ‘Coral mucus bacterial communities of Talang-talang reef and their potential role in coral defence’ International conference on sustainable development of tropical coastal zones, 5-6 September 2013, Port Dickson, Malaysia. (Poster presentation) Klaus Pfeilsticker and the SHIVA consortium ‘Findings of the SHIVA field campaign in the South China Sea in Nov.-Dec. 2011’ Geophysical Research Abstracts Vol. 15, EGU2013-1702, 2013. European Geosciences Union General Assembly, 7-12 April 2013. Vienna, Austria. (Oral presentation) P a g e | viii Mujahid A., Müller M., Ngu E.S.L., Lee S.T.H., Lew Y.L., Kuek F.W.I., Lim H.C., Teng S.T., Leaw C.P., & Lim P.T. ‘SHIVA local boat deployment in Kuching, and major findings from Sarawak’ SONNE status seminar, 13-15 February 2013, Kiel, Germany. (Poster presentation) Kuek F.W.I., Ngu E.S.L., Lee S.T.H., Mujahid A., Lim P.T., Leaw C.P. & Müller M. ‘Microbial communities of the eastern South China Sea and their possible role in the DMS(P) cycle’ SONNE status seminar, 13-15 February 2013, Kiel, Germany. (Poster presentation) Klaus Pfeilsticker and the SHIVA consortium ‘SHIVA consortium: Overview on the SHIVA activities and results’ South China Sea Conference, 21-24 October 2012, Kuala Lumpur, Malaysia.