Coastal Bacterial Communities: Their Potential Roles in Dimethylsulphide (Dms) Production and Coral Defence

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

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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.

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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.

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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. (Oral presentation) Kuek F.W.I., Mujahid A., Lim P.T., Leaw C.P. & Müller M. ‘Diversity of culturable bacteria from Talang-talang reef and its surrounding waters’ South China Sea Conference, 21-24 October 2012, Kuala Lumpur, Malaysia. (Poster presentation)

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Table of Contents

Page List of Figures xii List of Tables xviii 1 Introduction 1 1.1 Microbes in the ocean 1 1.2 South China Sea and the Celebes Sea 2 1.3 Surface microbes and their roles in the DMS(P) cycle 4 1.4 Coral reefs 8 1.4.1 Coral reefs of Malaysia 10 1.5 Coral bleaching 10 1.6 Coral Surface Mucus Layer (SML) and associated microbes 11 1.7 Coral diseases and the role of microbes in the SML 13 1.8 Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) 14 1.9 Significance and aims of the present study and dissertation outline 18 2 Methodology 19 2.1 Field sampling 19 2.1.1 Reef samples 20 2.2 Laboratory procedures 22 2.2.1 Isolation of bacteria 22 2.2.2 Molecular characterisation 23 2.2.3 Clone libraries from water samples 24 2.2.3.1 Extraction of genomic DNA from sea water 24 2.2.3.2 DNA cloning and plasmid extraction 25 2.2.3.3 Bacterial communities based in the construction of clone libraries 26 2.2.4 PCR amplification of bacterial DMSP cleavage (dddP) and demethylation (dmdA) genes 28 2.2.5 PCR amplification of bacterial antimicrobial PKS and NRPS genes 30 P a g e | x

2.2.6 Antimicrobial tests 31 2.2.6.1 Extraction of bioactive compounds 31 2.2.6.2 Inhibitory interaction tests (well diffusion assay) 32 3 Bacterial Communities from the eastern South China Sea and the Celebes Sea, and Their Potential Role in the DMS(P) Cycle 34 3.1 Introduction 35 3.2 Materials and Methods 38 3.2.1 Study site and sample collection 38 3.2.2 DNA extraction and purification of cultured bacteria 40 3.2.3 PCR amplification of bacterial 16S rRNA genes 41 3.2.4 Sequencing and phylogenetic analysis 41 3.2.5 Nucleotide sequence accession numbers 41 3.2.6 PCR amplification of bacterial DMSP cleavage (dddP) and demethylation (dmdA) genes 42 3.3 Results and Discussion 42 3.3.1 Physico-chemical parameters 42 3.3.2 Diversity of culturable bacterial communities 44 3.3.3 Variations in the bacterial communities in Kuching, Kota Kinabalu and Semporna waters 59 3.3.4 Bacterial strains with potential to metabolise DMS and/or demethylate DMSP 60 3.4 Conclusion 66 3.5 Acknowledgement 67 4 Bacterial Communities from Talang-Talang Reef and Their Potential Role in Coral Defence and the DMS(P) Cycle 68 4.1 Introduction 69 4.2 Materials and Methods 71 4.2.1 Study site and sample collection 71 4.2.2 DNA extraction and purification of cultured bacteria 72 4.2.3 PCR amplification of bacterial 16S rRNA genes 72 4.2.4 Sequencing and phylogenetic analysis 72 4.2.5 Nucleotide sequence accession numbers 73 P a g e | xi

4.2.6 PCR amplification of bacterial DMSP cleavage (dddP) and demethylation (dmdA) genes 73 4.2.7 PCR amplification of bacterial polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes 73 4.2.8 Extraction of bioactive compounds 74 4.2.9 Well diffusion assay 74 4.3 Results and Discussion 74 4.3.1 Diversity of culturable bacterial communities 74 4.3.2 Bacterial strains with PKS and NRPS genes 81 4.3.3 Role of mucus-associated bacteria in coral defence 83 4.3.4 Bacterial strains with potential to metabolise DMS and/or demethylate DMSP 88 4.4 Conclusion 90 4.5 Acknowledgement 91 5 Summary and Future Work 92 5.1 Future Research 93 References 95 Appendix 121

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List of Figures

Figure Page 1.1 Map of the South China Sea (source: Morton & Blackmore 2001). 3 1.2 Overview of DMSP catabolic pathways in marine bacteria and the fates of carbon and sulphur (taken from Reisch, Moran & Whitman 2011). 5 1.3 Biochemical pathways of DMSP demethylation. [1] DMSP demethylase (DmdA); [2] 5,10-methylene-THF reductase (MetF, E.C.1.5.1.20); [3] methylene-THF dehydrogenase (FolD, E.C.1.5.1.5); [4] methenyl-THF-cyclohydrolase (E.C.3.5.4.9); [5] methionine synthase (MetH, E.C. 2.1.1.13); [6] methionine salvage pathway (multiple enzymes); [7] MMPA-CoA ligase (DmdB); [8] MMPA-CoA dehydrogenase(DmdC); [9] methylthioacryloyl-CoA hydratase (DmdD); [10] acetaldehyde dehydrogenase (E.C.1.2.1.10) (source: Reisch, Moran & Whitman 2011). 6 1.4 Dimethylsulfoniopropionate cleavage pathways leading to central carbon metabolism. Reactions 5 and 6 may be coenzyme-A mediated and would therefore bypass reaction 8. [1] DMSP-cleavage enzyme (DddD); [2] DMSP lyase (DddL, DddP, DddQ, DddY, DddW, E.C. 4.4.1.3); [3] acrylate hydratase; [4] 3-hydroxypropionate dehydrogenase; [5] 3-hydroxypropionate reductase; [6] acrylate reductase (1.3.99.3); [7] malonate semialdehyde dehydrogenase/decarboxylase (E.C.1.2.1.18); [8] propionate-CoA ligase (PrpE, E.C.6.2.1.17) (source: Reisch, Moran & Whitman 2011). 7 1.5 Distribution of coral reefs in the East Asian Seas (source: http://www.ncdc.noaa.gov/paleo/outreach/coral/sor/sor_asia.html). 9 1.6 Basic steps during PKS. Each PKS module consists of three core domains: an acyltransferase (AT) domain, which selects the appropriate extender unit (usually malonyl-CoA or methylmalonyl- CoA) and transfers it to the ACP domain where a thioester bond is formed, and a ketosynthase (KS) domain, responsible for decarboxylative condensation between the extender unit present on P a g e | xiii

the ACP domain of the same module and the polyketide intermediate bound to the ACP domain of the preceding module. All elongating modules present these core domains, while the loading module lacks a functional KS domain and the last module contains an additional TE domain, for release of the finished polyketide from the PKS. Most PKS modules contain additional domains for processing the newly formed b-keto: the b-ketoreductase (KR), the dehydratase (DH) and the enoylreductase (ER) domains carry out the reactions (source: Donadio, Monciardini & Sosio 2007). 15 1.7 Basic steps during NRPS. Each NRPS module consists of three core domains: an adenylation (A) domain, which selects the cognate amino acid, activates it as an amino acyl adenylate and transfers it to the T domain (also known as peptidyl carrier protein, or PCP) where a thioester bond is formed, a condensation (C) domain, responsible for peptide bond formation between the amino acid present on the T domain of the same module and the peptidyl intermediate bound to the T domain of the preceding module, and the T domain itself. Usually, all elongation modules present these core domains. A dedicated loading module (carrying just A and T domains) and a termination module, containing a thioesterase (TE) domain, usually complete the NRPS assembly line (source: Donadio, Monciardini & Sosio 2007). 16 2.1 Overview of the SHIVA cruise (source: http://shiva.iup.uni- heidelberg.de/a_activities.html). 19 2.2 Overview of the Talang-talang Islands just off the shores of Kuching, Sarawak (source: Yahya, Hassan & Husaini 2012) 21 2.3 Collection of fresh coral mucus. 22 2.4 16S rRNA bands of bacterial isolates. Impure bands can be seen at BSD 16-5, 16-7, 16-11. These isolates were later reisolated to ensure pure cultures. 24 2.5 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in clone libraries from Kuching and Kota Kinabalu. The phylogenetic tree was generated with distance methods, and P a g e | xiv

sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated. 27 2.6 PCR-based screening of dmdA genes. Bands highlighted in this figure indicate presence of the genes. 29 2.7 PCR-based screening of dddP genes. Bands highlighted in this figure indicate presence of the genes. 30 2.8 PCR-based screening of PKS genes. Bands highlighted in this figure indicate presence of the genes. 31 2.9 PCR-based screening of NRPS genes. Bands highlighted in this figure indicate presence of the genes. 31 2.10 Inhibition zones from bioactive compoinds with antimicrobial properties. 33 3.1 The RV Sonne ship track leading from Singapore to Manila between November 15-29, 2011 during the SHIVA SO 218 cruise. 38 3.2 Locations of sampling stations in Kuching, Sarawak. 39 3.3 Locations of sampling stations in Kota Kinabalu, Sabah. 40 3.4 Locations of sampling stations in Semporna, Sabah. 40 3.5 Pie charts illustrating the diversity of bacterial groups based on partial 16S rRNA gene sequences from bacteria isolated from (a) Kuching, (b) Kota Kinabalu and (c) Semporna. 45 3.6 Phylogenetic groups of isolates from the waters of Kuching, Kota Kinabalu and Semporna at depths of 1, 5 and 10 m. 45 3.7 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kuching 1611. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated. 47 3.8 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kuching 1911. The phylogenetic tree was P a g e | xv

generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.1 substitution per site. Accession numbers for the reference sequences are indicated. 48 3.9 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kota Kinabalu. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated. 49 3.10 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Semporna. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.1 substitution per site. Accession numbers for the reference sequences are indicated. 51 3.11 Relative abundance of dmdA and dddP genes in cultured bacterial communities from the waters of (a) Kuching, (b) Kota Kinabalu and (c) Semporna. 61 3.12 Presence of dmdA and/or dddP genes in bacterial isolates from the waters of Kuching, Kota Kinabalu and Semporna. 62 3.13 Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Kuching. 63 3.14 Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Kota Kinabalu. 63 3.15 Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Semporna. 64 4.1 Overview of the Talang-talang Islands just off the shores of Kuching, Sarawak. Enlarged map indicates sampling area. 71 4.2 Pie charts illustrating the diversity of bacterial groups based on partial 16S rRNA gene sequences from bacteria isolated from (a) P a g e | xvi

coral mucus, (b) water column and (c) sediment. 75 4.3 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in coral mucus The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.1 substitution per site. Accession numbers for the reference sequences are indicated. 76 4.4 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in water column. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated. 77 4.5 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in reef sediment. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated. 78 4.6 Percentage of Vibrio isolates in mucus attached and mucus associated communities. 79 4.7 Relative abundance of PKS and NRPS genes in cultured bacterial communities from coral mucus. 82 4.8 Presence of PKS and/or NRPS genes in bacterial isolates from coral mucus. 82 4.9 Total inhibitions of mucus attached bacteria at temperatures of 28, 30 and 32 °C. 83 4.10 Total inhibitions of mucus associated bacteria at temperatures of 28, 30 and 32 °C. 83 4.11 Average zone of inhibitions (cm) of mucus attached bacteria at P a g e | xvii

temperatures of 28, 30 and 32 °C. 84 4.12 Average zone of inhibitions (cm) of mucus associated bacteria at temperatures of 28, 30 and 32 °C. 84 4.13 Relative abundance of dmdA and dddP genes in cultured bacterial communities from coral mucus. 88 4.14 Presence of dmdA and/or dddP genes in bacterial isolates from coral mucus. 89

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List of Tables

Table Page 1.1 Regional distribution of coral reefs (source: Veron & Stafford-Smith 2000). 9 3.1 Locations of sampling stations at Kuching, Kota Kinabalu and Semporna. 39 3.2 Physico-chemical parameters measured from Kuching and Kota Kinabalu at depths of 1 and 5 m. 42 3.3 Indices used to quantify the diversity of bacterial communities at Kuching, Kota Kinabalu and Semporna. 46 A.1 16S rRNA gene sequence analysis of bacterial cultures from Kuching 1611, based on BLAST analysis. 126 A.2 16S rRNA gene sequence analysis of bacterial cultures from Kuching 1911, based on BLAST analysis. 130 A.3 16S rRNA gene sequence analysis of bacterial cultures from Kota Kinabalu, based on BLAST analysis. 133 A.4 16S rRNA gene sequence analysis of bacterial cultures from Semporna, based on BLAST analysis. 136 A.5 16S rRNA gene sequence analysis of bacterial cultures from Talang- talang reef and its surrounding waters, based on BLAST analysis. 139 A.6 Screening of antimicrobial (PKS/NRPS) and DMSP-degrading (dmdA/dddP) genes in coral mucus isolates. 145 A.7 Total inhibition and inhibition zones of mucus attached isolates at 28, 30 and 32 °C. 146 A.8 Total inhibition and inhibition zones of mucus associated isolates at 28, 30 and 32 °C. 147

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CHAPTER 1

Introduction

1.1 Microbes in the oceans The oceans are made up of a web of different marine organisms that form an interdependent community. Microbes, a major component of this community, have been studied by scientists for years in attempts to establish a better understanding of their diversity, distribution and nature. An estimated total of 3.6×1029 microbial cells reside in the oceans (Singh 2010). These marine microorganisms have experienced billions of years’ worth of evolution, forming vast and complex communities of bacteria, archaea, protists and fungi, within what is said to be the dominant biome of the Earth (DeLong 2009). The actual number of microbes that exist in the ocean, however, is thought to surpass published estimates; indicating that while many have been and are in the process of being identified, an equally great percentage still remains undiscovered (Karl 2002; Sogin et al. 2006).

These microbes play vital roles in the marine ecosystem by mediating the geochemical cycles in the ocean (Arrigo 2005) and allowing for rapid nutrient recycling in an environment that is poor in essential nutrients (Mayer & Wild 2010). Consequently, they are responsible for around 98% of overall primary production in the ocean, providing short-term sustainability to the marine ecosystem while a longer term supply of nutrients comes from external sources (Karl 2002; Sogin et al. 2006). As a result of dominating an ecosystem that constitutes approximately 40% of the Earth’s surface, these microbes and their involvement in biogeochemical processes are significant on a global scale (Karl 2002).

For decades, microbiologists have aimed to unravel the mysteries of the microbial world through culture-based studies. This approach allowed them to discover new species, as well as to study their biochemical properties. Today, the advances in molecular biology have brought ecological studies in microbiology to even greater P a g e | 2

heights. Physiological and biochemical studies, previously hindered by obstacles in culturing the ‘unculturable’, can now be carried out to establish the identities, phylogenetic relationships and metabolic processes of both cultured and uncultured microbial populations via DNA or RNA based methods (Jørgensen 2006).

Characterization of microbes by genera and species, which previously could not be achieved through biochemical methods alone, can now be executed with relative ease with the help of sequence-classifier algorithms (Petrosino et al. 2009). Sequencing studies are conventionally carried out using the Sanger method (Sanger, Nicklen & Coulson 1977) which is widely used in microbial population studies. Each metabolic function is encoded by a specific set of genes, thus scientists today rely on molecular-based protocols for a more rapid and effective study of the nature of these bacteria (Rappé & Giovannoni 2003). Sequencing will provide us with an indication of whether these specific genes are present in a sample, signalling that these special groups of bacteria inhabit the population of study (Rajendhran & Gunasekaran 2011); a far easier alternative to culture-based protocols which can eventually lead to more in-depth studies on microbial metabolism.

1.2 South China Sea and the Celebes Sea The South China Sea is a marginal sea that is part of the Pacific Ocean, encompassing an area from the Singapore and Malacca Straits to the Strait of Taiwan (Morton & Blackmore 2001; see Figure 1.1 for a map of the South China Sea). The Celebes Sea is connected to the South China Sea through the Sulu Sea (Yoshida, Nishimura & Kogure 2007).

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Figure 1.1: Map of the South China Sea (source: Morton & Blackmore 2001).

Not much is known about the diversity and function of the microbial communities in the South China Sea, especially regarding the eastern region (Kuching and Kota Kinabalu) and the Celebes Sea. To our knowledge, there is no study on regional scale or large-scale distribution patterns of microbes in the Malaysian area of the South China Sea. Most studies about bacterial communities focused on regions near China, such as those carried out by Li et al. (2006), Jiang et al. (2007) and Tao, Peng & Pinxian (2008) and a brief mention of communities in the Celebes Sea by Yoshida, Nishimura & Kogure (2007). All the studies mentioned used culture- independent techniques to reveal the community structure and diversity of the predominant bacteria at the sampling environment. No studies on culturable communities in the region have been made at this time.

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1.3 Surface microbes and their roles in the DMS(P) cycle Through recent studies, the biogeochemical processes of marine microbes have been discovered not only to regulate marine ecosystems but to potentially have an indirect influence on the Earth’s climate as well. In 1983 it was first hypothesized that the sulphur gases released from biotic ecosystems, upon their oxidation in the atmosphere, would also affect the Earth’s climate (Shaw 1983) and marine planktonic algae have since been found to contribute largely to the exchange of sulphur gases across the ocean-atmosphere boundary. They are known to produce dimethylsufoniopropionate (DMSP), a metabolite precursor to dimethylsulfide (DMS) which is the primary source of sulphur released into the atmosphere (Strom 2008; Reisch, Moran & Whitman 2011). Once in the atmosphere, DMS is oxidized into sulphate aerosols that will act as cloud condensation nuclei (CCN) which will promote cloud formation (Charlson et al. 1987). The protective layer of clouds formed will serve to reduce the amount of solar radiation that reaches the Earth’s surface (Welsh 2000; Vallina & Simó 2007) thus, possibly reducing the Earth’s overall temperature by several degrees. Certain species of marine microbes have been found to be mediators to the reaction by producing the enzymes necessary to cleave DMSP into the desired gas product (Vila-Costa et al. 2010) and are therefore being put under extensive study as a model for climate change studies.

In research today, scientists use modern molecular technology to locate the genes responsible for the production of these enzymes that will determine the fate of DMSP. Two possible pathways exist for DMSP (see Figure 1.2 for an overview), catabolism into DMS or demethylation into methanethiol (MeSH), the former of which – while quantitatively less important than demethylation (Kiene 1996a) – is the largest biogenic source of sulphur to the atmosphere (Andreae & Raemdonck 1983). The cleavage pathway is important in mediating organic sulphur emission into the atmosphere by splitting of the DMSP molecule into acrylate and DMS (Reisch, Moran & Whitman 2011). The demethylation pathway involves the removal of a methyl group from DMSP to produce 3-methiolpropionate, which is then cleaved to methanethiol and probably acrylate or propionate (González, Kiene & Moran 1999). The potential of bacterial strains to use more than one DMSP P a g e | 5

catabolic pathway has been previously noted, especially among Roseobacter spp. by González et al. (1999).

Figure 1.2: Overview of DMSP catabolic pathways in marine bacteria and the fates of carbon and sulphur (taken from Reisch, Moran & Whitman 2011).

To date, six different enzymes responsible for DMSP cleavage have been identified, each encoded by different genes and known to catalyse different reactions that ultimately lead down different pathways. Using genomic-based studies, specific metabolic processes within a microbial population can be identified more easily which will contribute to studies on the different biochemical pathways and regulatory factors involved in DMSP metabolism – something that still remains very poorly understood (Reisch, Moran & Whitman 2011). Vila-Costa et al. (2010) carried out a transcriptomic analysis on the marine microbial population in the Sargasso Sea to study gene expression of the microbes in the presence of low amounts of DMSP. They were able to identify several genes known to be directly involved in DMSP degradation and could classify them according to the taxonomic groups.

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Figure 1.3: Biochemical pathways of DMSP demethylation. [1] DMSP demethylase (DmdA); [2] 5,10-methylene-THF reductase (MetF, E.C.1.5.1.20); [3] methylene- THF dehydrogenase (FolD, E.C.1.5.1.5); [4] methenyl-THF-cyclohydrolase (E.C.3.5.4.9); [5] methionine synthase (MetH, E.C. 2.1.1.13); [6] methionine salvage pathway (multiple enzymes); [7] MMPA-CoA ligase (DmdB); [8] MMPA-CoA dehydrogenase(DmdC); [9] methylthioacryloyl-CoA hydratase (DmdD); [10] acetaldehyde dehydrogenase (E.C.1.2.1.10) (source: Reisch, Moran & Whitman 2011). P a g e | 7

Figure 1.4: Dimethylsulfoniopropionate cleavage pathways leading to central carbon metabolism. Reactions 5 and 6 may be coenzyme-A mediated and would therefore bypass reaction 8. [1] DMSP-cleavage enzyme (DddD); [2] DMSP lyase (DddL, DddP, DddQ, DddY, DddW, E.C. 4.4.1.3); [3] acrylate hydratase; [4] 3- hydroxypropionate dehydrogenase; [5] 3-hydroxypropionate reductase; [6] acrylate reductase (1.3.99.3); [7] malonate semialdehyde dehydrogenase/decarboxylase (E.C.1.2.1.18); [8] propionate-CoA ligase (PrpE, E.C.6.2.1.17) (source: Reisch, Moran & Whitman 2011).

The identification of the DMSP-demethylase gene (dmdA), which encodes the first step in the demethylation pathway (see Figure 1.3 for a complete overview of the demethylation pathway), has enabled quantification of the gene in marine metagenomic surveys and revealed it to be taxonomically diverse and highly abundant (del Valle, Kiene & Karl 2012). In comparison to dmdA, the genes involved in DMS production (dddD, dddL, dddP dddQ, dddY, and dddW, all of which mediate the same step of DMSP cleavage; see Figure 1.4) are present in less than 10% of bacteria based on marine metagenomic surveys (Todd et al. 2007; P a g e | 8

Curson et al. 2008; Howard et al. 2008; Todd et al. 2009; Curson, Sullivan, et al. 2011; Todd et al. 2011; Todd, Curson, et al. 2012). The most abundant ddd genes in bacterial taxa are dddP and dddQ, occurring in genomes of some Roseobacter spp. (Howard et al. 2008; Todd et al. 2009, 2011) and SAR116.

As can be seen from the lack of studies mentioned above, there is very limited information available on bacteria in the South China Sea (SCS) and even less on their potential role in the DMS(P) cycle. One aim of this thesis is to provide data on both. Besides surface waters, bacteria also play major roles in other oceanic ecosystems for example coral reefs. In the following, coral reefs will be introduced as well as the role that bacteria play in them

1.4 Coral reefs Coral reefs are among the most diverse and productive ecosystems on this planet. Millions of people rely on harvests derived from coral reefs as their major source of protein and income (Wilkinson & Buddemeier 1994). In addition, revenue earned from tourism, recreation, education and research are of major importance to our national economy (Wilson et al. 2012). Coral reefs also act as a natural protection between the open seas and coastlines by acting as wave breaks, thus effectively preventing coastal erosion (Buddemeier, Kleypas & Aronson 2004; McLeod et al. 2010; Eghtesadi-Araghi 2011). They perform a vital role in protecting coastal areas from the consequences of rising sea levels such as storm flooding (Wilkinson 1999). There is also increasing evidence of the potential of reefs to act as bio-indicators for climate change, as they are sensitive to rising sea levels and increasing sea temperature (Awang, Moshidi & Muda 2003). In addition, reefs are good indicators of coastal pollution, as they are sensitive to changes in their ambient environment (Moberg & Folke 1999). Coral reefs in the South Pacific cover the highest amount of space (116,200 km2; see Table 1.1), closely followed by Southeast Asia (87,760 km2, see Table 1.1.), indicating their important role for the local communities.

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Table 1.1: Regional distribution of coral reefs (source: Veron & Stafford-Smith 2000).

Region Reef area (km2) South Pacific 116,200 Southeast Asia 87,760 Indian Ocean 31,930 Middle East 21,450 Caribbean 20,360 Western Atlantic 2.820

Reefs are widely distributed in the area (see Figure 1.5 for an overview of reef distribution in the East Asian Seas) and Southeast Asia’s coral reefs have the highest biodiversity of all the world’s reefs (Veron & Stafford-Smith 2000). This region contains more than 600 of the nearly 800 reef building coral species found worldwide (Veron & Stafford-Smith 2000).

Figure 1.5: Distribution of coral reefs in the East Asian Seas (source: http://www.ncdc.noaa.gov/paleo/outreach/coral/sor/sor_asia.html).

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1.4.1 Coral reefs of Malaysia The wide geographic range that Malaysia covers means that coral reefs can be found in varied conditions across the country. Malaysia is included in the Coral Triangle, a global centre of marine biodiversity (Lee & Mohamed 2009). The waters of the Coral Triangle hold the highest diversity of iridescent corals, fish, crustaceans, mollusks and marine plant species in the world (Veron et al. 2009). The area sustains over 120 million people and garners more than $12 billion a year from nature-based tourism (Cabral et al. 2013).

Malaysia has an estimated 4,000 km2 of coral reefs (Yaman n.d.). Little reef development occurs along the west coast of Peninsular Malaysia, but the east coast has some fringing reefs along the coast and many reefs around the offshore islands (Harborne et al., 2000; see Figure 1.6). In East Malaysia, reef development around Sarawak is limited due to high sedimentation (Pilcher & Cabanban 2000a) (Yaman n.d.; see Figure 1.5). However, Sabah contains more than 75% of all Malaysian reefs and has high levels of coral diversity (Pilcher & Cabanban 2000b). Overall, more than 350 coral species have been recorded in Peninsular Malaysia and over 500 in East Malaysia (Praveena, Siraj & Aris 2012). Unfortunately, there are several threats to coral reefs such as sedimentation, eutrophication, temperature rise and ocean acidification (Praveena, Siraj & Aris 2012) and Malaysia is no exception to the rule. Corals that are under stress for long time will eventually die, mainly due to a process called "coral bleaching".

1.5 Coral bleaching Bleaching is defined as the disruption of the symbiosis between the coral host and its endosymbiotic zooxanthellae, resulting in the loss of the algal symbiont and/or of the algal pigments, thus making the coral tissue transparent and exposing the underlying white calcium carbonate skeleton (Rosenberg et al. 2009). If symbiont populations are not restored within weeks or months of a bleaching event, then whole or partial coral mortality is likely (Hoegh-Guldberg 2004). Coral bleaching has increased in frequency, intensity and geographical extent over the last few decades (Huppert & Stone 1998) and has been correlated with increased seawater temperatures as well as high levels of solar irradiance (Jokiel & Brown 2004). P a g e | 11

Predicted ocean warming in the current century is expected to result in higher frequency of mass coral bleaching events and associated mortality (Hoegh- Guldberg 1999).

Temperature-related bleaching is the most widely reported cause of coral reef stress (Buddemeier, Kleypas & Aronson 2004). The range of temperatures tolerated by reef-building corals worldwide is relatively narrow, and studies have shown that a temperature of only 1-2 °C above the normal threshold temperature for a few weeks is sufficient to cause a bleaching event (Fitt et al. 2001). Sea- surface temperatures have now moved so close to coral thermal limits that the fluctuations of temperatures within natural climatic events such as the El Niño Southern Oscillation (ENSO) can cause massive coral bleaching (Praveena, Siraj & Aris 2012). The most severe ENSO event since statistics have been recorded occurred in 1997-1998. Although the effects from the 1997-1998 event were most severe in the central Indian Ocean, major bleaching was also reported across Southeast Asia, where an estimated 18% of reefs were damaged (Praveena, Siraj & Aris 2012).

Two mechanisms for causing bleaching have been discovered: photoinhibition leading to the damage of photosystem II (Jones et al. 1998); and infection by a pathogen that targets the zooxanthellae (Rosenberg 2004). As discussed in the following, bacteria play a significant role in the latter and other coral diseases.

1.6 Coral Surface Mucus Layer (SML) and associated microbes All corals have a surface mucus layer (SML) that is generated by secretion of a polysaccharide-protein complex by mucocytes (Sharon & Rosenberg 2008). The SML serves as an ecological niche rich in nutrients and diverse in bacterial populations (Shnit-Orland & Kushmaro 2008). It plays an important role in structuring microbial communities on the coral surface by providing a hostile environment for some bacteria and a nurturing environment for others (Ritchie 2006). Various functions have been ascribed to the SML including defence against disease-causing pathogens, desiccation resistance, shedding of sediments and protection against radiation (Sharon & Rosenberg 2008). On average, 20-30 % of P a g e | 12

bacterial isolates originating from coral SML possess antibacterial properties (Ritchie 2006) that may assist the coral holobiont as a first line of defence against pathogens and fouling organisms (Shnit-Orland & Kushmaro 2009). It has been suggested that these antimicrobial compounds are temperature sensitive (Ritchie 2006). Antibacterial activity was found to be optimal at 26 °C and slightly decreased at 30 °C, with partial inactivation occurring at 60 °C and complete loss of activity occurring at 80 °C (Shnit-Orland & Kushmaro 2009).

High temperatures act on the coral microorganisms as well as on the coral host, causing a change in microbial community (Rosenberg et al. 2009). There is evidence that a community shift in the coral SML from beneficial bacteria to Vibrio- dominance occurs prior to zooxanthellae loss (Ritchie 2006). Studies have shown that Vibrio may be normal constituents of the coral microbial assemblages and can opportunistically proliferate if holobiont health is compromised (Bourne & Munn 2005). Previous studies have implicated Vibrio spp. as the principal causative agent in seasonal and species-specific episodes of coral bleaching (Kushmaro et al. 1996, 1997; Ben-Haim & Rosenberg 2002; Ben-Haim et al. 2003). It was speculated that the endosymbiotic zooxanthellae (Symbiodinium spp.) play a significant role in restricting Vibrio growth in the coral SML by producing free radicals (Sharon & Rosenberg 2008). However, three separate studies (Ritchie et al. 1994; Kushmaro et al. 1996; Ben-Haim, Zicherman-Keren & Rosenberg 2003) showed that the number of Vibrio in coral SML did increase with increasing temperatures. In elevated temperatures, Vibrio spp. will produce a photosynthesis inhibitor (Ben- Haim et al. 1999), thereby allowing them to multiply, leading to overgrowth and in turn, causing the loss of antibiotic properties of the SML inhabiting microorganisms (Ritchie 2006). During bleaching, coral mucus production changes in quality and can decrease in quantity (Ritchie 2006). Elevated sea water temperatures can also induce pathogens to produce adhesions that allow it to adhere to the coral surface and subsequently establish infections in the pathogenic systems of the coral (Banin, Ben-Haim, et al. 2000). The production of toxins and lytic enzymes which cause bleaching and lysis of zooxanthellae were also found to be temperature-regulated (Banin, Ben-Haim, et al. 2000). Although temperature may affect the metabolism and diversity of the microbial community, the loss of P a g e | 13

zooxanthellae is potentially the fundamental driving parameter changing the microbiota of the corals (Bourne et al. 2008).

1.7 Coral diseases and the role of microbes in the SML Emerging diseases have been responsible for the death of about 30% of corals worldwide in the last few decades and it is predicted that by 2050, most of the world’s coral reefs will be destroyed (Reshef et al. 2006). Many disease outbreaks involve opportunistic infections by endemic microbes following periods of stress (Lesser et al. 2007; Rosenberg et al. 2009; Bourne et al. 2009). Bleached corals are additionally vulnerable because the loss of algae reduces the concentration of oxygen and the resulting radicals that protect the coral animal (Banin et al. 2003).

Disease susceptibility is positively correlated with a change in coral SML composition, loss of antibiotic activity and an increase in pathogenic microbes (Reshef et al. 2006). The bacterial communities of diseased corals are different from healthy ones, both qualitatively and quantitatively (Reshef et al. 2006). The bacterial population of apparently healthy corals undergo changes within a period of a few months, probably as a result of temperature changes (Koren & Rosenberg 2006). Previous studies have shown a sudden shift to pathogen dominance occurring in the coral SML prior to a bleaching event (Lipp, Huq & Colwell 2002; Rosenberg & Ben-Haim 2002; Ritchie 2006) and it has been demonstrated that antibiotic activity and antibiotic-producing bacteria in the SML decline in times of increased water temperature when bleaching is most likely to occur (Ritchie 2006). One possible explanation for an increased incidence of coral diseases is stress-induced susceptibility to opportunistic microbes trapped in the coral SML (Ritchie 2006). Indigenous bacteria may help prevent infection by pathogens by producing antibacterial materials (Koh 1997).

Vibrio shiloi is a known bacterial pathogen to the coral Oculina patagonica found in the Mediterranean sea (Kushmaro et al. 1996, 1997, 2001). It induces bleaching by reducing the amount of viable zooxanthellae available for symbiosis with the coral. This is achieved by the secretion of a toxin (a proline-rich, 12 amino acid peptide) (Banin, Israely, et al. 2000) that inhibits photosynthesis, and bleaches and lyses P a g e | 14

zooxanthellae (Ben-Haim et al. 1999). Vibrio shiloi is only actively pathogenic at temperatures of 20-32°C and displays maximum efficacy around 29-30°C (Kushmaro et al. 2001).

A more recently discovered temperature-dependent agent of bleaching is Vibrio coralliilyticus which infects the coral Pocillopora damicornis (Ben-Haim et al. 2003). A patchy pattern of bleaching of Pocillopora damicornis has been observed at 24 °C, suggesting that bacterial bleaching results from an attack on the zooxanthellae, followed by bacterium-induced coral lysis and death caused by bacterial extracellular proteases which were produced at temperatures of 24 to 28 °C (Ben-Haim, Zicherman-Keren & Rosenberg 2003; Rosenberg 2004).

There has been only one published report about coral reefs in Sarawak (Awang, Moshidi & Muda 2003) and this report does not contain any information about coral-associated microbial communities. Since associated bacteria play a major role as a first line of defence against pathogens (Shnit-Orland & Kushmaro 2009), and are of significance to the survival of coral reefs in the area, the second aim of the thesis was therefore to take a closer look at the bacteria in a local reef and their potential role in coral defence as well as their response to changing temperatures.

1.8 Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) Polyketides and non-ribosomal peptides comprise two large families of secondary metabolites and numerous natural products belonging to these groups are widely used as pharmaceuticals, industrial agents or agrochemicals (Silakowski, Kunze & Müller 2001). Both types are biosynthesized by extremely large polyfunctional enzyme systems within the protein. The responsible biosynthetic proteins are known as polyketide synthases (PKS) and nonribosomal polypeptide sythetases (NRPS) (Cane 1997).

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Figure 1.6: Basic steps during PKS. Each PKS module consists of three core domains: an acyltransferase (AT) domain, which selects the appropriate extender unit (usually malonyl-CoA or methylmalonyl-CoA) and transfers it to the ACP domain where a thioester bond is formed, and a ketosynthase (KS) domain, responsible for decarboxylative condensation between the extender unit present on the ACP domain of the same module and the polyketide intermediate bound to theACP domain of the preceding module. All elongating modules present these core domains, while the loading module lacks a functional KS domain and the last module contains an additional TE domain, for release of the finished polyketide from the PKS. Most PKS modules contain additional domains for processing the newly formed b-keto: the b-ketoreductase (KR), the dehydratase (DH) and the enoylreductase (ER) domains carry out the reactions (source: Donadio, Monciardini & Sosio 2007).

PKS is known from both the systems of eukaryotes and prokaryotes. This enzyme catalyses the fusion of carbon chains into long polymers via Claisen condensation reaction (Heath & Rock 2002). PKS is related to fatty acid synthase structurally and functionally as both of the enzymes catalyse the condensation of activated primary metabolites to produce β-ketoacetyl polymers attached to the enzyme via thioester bonds (Donadio, Monciardini & Sosio 2007). In synthesis of polyketides, these reduction steps are eliminated partly or completely in a controlled way and thus results in polyketides chain with respect to the production of β-hydroxyl, β-ketone and alkyl groups (Fujii et al. 2001; see Figure 1.6 for an overview of PKS). P a g e | 16

PKS has been characterized in terms of their subunits number and the synthesis mode, such as type I modular PKS, type I iterative PKS, type II PKS and type III PKS (Ansari et al. 2004). Type I modular PKS that can be found in bacteria is the best categorized class, but the functional information derived from these generally apply to other categories (Watanabe & Ebizuka 2004). Type I PKS are categorized by being multi enzymes, carrying out enzymatic domains that are needed for polyketides formation, in particular, clinical and economical macrolide polyketides production, for instance rifamycin and erythromycin A (Ansari et al. 2004). For type II PKS, the catalytic domains are located on individual proteins which interact to produce a functional PKS enzyme complex (Ansari et al. 2004). The type III PKS is different from the types I and II as it does not rely on acyl carrier protein domains (Meier & Burkart 2009).

Figure 1.7: Basic steps during NRPS. Each NRPS module consists of three core domains: an adenylation (A) domain, which selects the cognate amino acid, activates it as an amino acyl adenylate and transfers it to the T domain (also known as peptidyl carrier protein, or PCP) where a thioester bond is formed, a condensation (C) domain, responsible for peptide bond formation between the amino acid present on the T domain of the same module and the peptidyl intermediate bound to the T domain of the preceding module, and the T domain itself. Usually, all elongation modules present these core domains. A dedicated loading module (carrying just A and T domains) and a termination module, P a g e | 17

containing a thioesterase (TE) domain, usually complete the NRPS assembly line (source: Donadio, Monciardini & Sosio 2007).

NRPS is a group of enzymes that typically found in most of the bacteria and fungi which synthesizes non-ribosomal peptides, a family of complex natural products synthesized from amino acid monomers (see Figure 1.7 for an overview of NRPS). NRPS is achieved by the thiotemplate function of modular enzyme complexes known collectively as peptide synthetases (Radjasa & Sabdono 2003). It has been established that the specific combination of modules and various functional domains within the peptide synthetase determines the structure and the activity of peptide product (Neilan et al. 1999). Most non-ribosomal peptides from microorganisms are classified as secondary metabolites, rarely having a role in primary metabolism, growth or reproduction, but instead having evolved to benefiting the producing organisms (Neilan et al. 1999). The products of microbial NRPS include the immunosuppressant cyclosporine and antibiotics such as erythromycin, gramicin S, lovastatin, rapamycin, surfactins, and tyrocin A (Kleinkauf & Von Döhren 1996; Du, Sánchez & Shen 2001). NRPS usually works in conjunction with PKS to give hybrid products which are significant pharmaceutical products (Ansari et al. 2004).

NRPS is organized based on modules, where each of the modules is responsible to catalyse a single cycle of product length elongation and modification of that functional group. The minimum set of domains necessary for a single elongation cycle consists of a module with Thiolation (T), Adenylation (A) or Peptidyl Carrier Protein (PCP) and a Condensation (C) domain. The structural variation of the peptide product is determined by the number and order of the module as well as the type of domains present in a module of NRPS (Ansari et al. 2004). Thus, with advanced techniques such as polymerase chain reaction (PCR), the screening for the presence of PKS and NRPS genes is possible by using specific primers of PKS and NRPS.

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1.9 Significance and aims of the present study and dissertation outline To our knowledge, microbial communities in the eastern South China Sea and the Celebes Sea are practically unknown and we are therefore missing vital data to understand these ecosystems. Understanding will help to predict their reaction to changes in the global climate and other factors such as anthropogenic pollution. One major and globally important role of surface bacteria is their involvement in the breakdown or osmoregulation of DMSP to DMS or MeSH and this will be the first comprehensive study looking at culturable bacteria in the eastern South China Sea and their potential roles in the DMS(P) cycle.

In chapter 3, we look at samples from different regions to: (a) distinguish differences in species distributions and (b) discuss their potential involvement in the DMS(P) cycle.

Another globally important role of bacteria is their involvement in the coral defence. The biodiversity and natural diversity of coral reefs in our region are under threat from various anthropogenic and natural impacts, causing major changes in their structure and function. Current research suggests that coral reefs support an unknown number of organisms that may prove to be of major benefit in the treatment of critical human diseases. In chapter 4, we have isolated bacteria in a local reef and looked at their potential involvement in coral defence. The isolation of native microbes from the coral SML allowed us to determine the species composition and abundance of various bacteria in the SML. Results from this study will update our current understanding of the basic ecology of coral- associated microbial communities. This will help improve monitoring and management efforts and aid in related issues of coral health.

The objectives of this study are: i. Isolation and identification of native microbes in the South China Sea and the coral SML. ii. Testing of bacterial isolates for potential DMSP-degrading and/or antibiotic properties. iii. Determination of the effects of elevated temperatures on the antibiotic properties of bacterial isolates. P a g e | 19

CHAPTER 2

Methodology

2.1 Field sampling In November 2011, a core field campaign took place in the South China Sea, and along the coastline of Peninsula Malaysia and Borneo using the Sonne Research Vessel, the DLR Falcon aircraft, satellites, and land-based investigation teams (see Figure 2.1 for a schematic overview of activities and cruise track). The project was supported by the 7th Framework programme of the European Union (call ENV.2008.1.1.2.1) and is called Stratospheric Ozone: Halogen Impacts in a Varying Atmosphere (SHIVA). By combining measurements from land, ship, aircraft, and space-based platforms, with sophisticated numerical models, SHIVA aims to better predict the rate, timing and climate-sensitivity of ozone-layer recovery, and identify potential risks to that recovery.

Figure 2.1: Overview of the SHIVA cruise (source: http://shiva.iup.uni- heidelberg.de/a_activities.html). P a g e | 20

The sampling of the RV Sonne was supported by local cruises to provide complimentary data from the coast. As part of the complementary three-part series of local boat deployments, two transects were organised in Kuching on the 16th and 19th November 2011, one in Kota Kinabalu on the 23rd November 2011, and one in Semporna on the 26th November 2011, respectively. Air and water samples were collected at (at least) 5 stations with intervals of 5 km, along transects at 1 to 20 km off the coasts.

The main aims of the local boat deployments were two-fold: i. To obtain coastal samples to complement open ocean samples made by RV Sonne, Falcon aircraft and satellites; ii. To enable the exchange of samples collected at the coasts (for example VSLS and nutrients) to be taken onboard RV Sonne for further analyses, and those collected onboard RV Sonne (sensitive biological samples) to be taken back for storage and further analyses.

The rendezvous stations were at the RV Sonne diurnal stations on the 19th (Kuching) and 23rd (Kota Kinabalu) November 2011. Samples used in this thesis were collected from 10 stations in Kuching (16th and 19th November 2011), five stations in Kota Kinabalu (23rd November 2011) and eight stations in Semporna (26th November 2011).

The water samples were collected using a Ruttner water sampler up to 10 m depth and stored in sterile water bottles placed in cooling boxes to be transported back to the laboratory for further analysis.

2.1.1 Reef samples Samples of coral mucus from corals of different colonies, sediment and water samples (up to 5 m depth) were collected from the reefs of Talang-talang (see Figure 2.2) and its surrounding waters in July 2011.

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Figure 2.2: Overview of the Talang-talang Islands just off the shores of Kuching, Sarawak (source: Yahya, Hassan & Husaini 2012).

Loose coral fragments were collected and brought to the surface. The corals were held upside down, allowing excess water to drip off and fresh mucus to form at the surface of the coral. Coral mucus were dripped into sterile falcon tubes (see Figure 2.3) and stored in in cooling boxes maintained at 4 °C to be transported back to the laboratory for further analysis.

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Figure 2.3: Collection of fresh coral mucus.

Sediment samples were collected at areas adjacent to coral colonies in sterile falcon tubes. Water samples were collected in sterile bottles at the surface and 5 m depth while diving. These samples were also stored in cooling boxes maintained at 4 °C to be transported back to the laboratory for further analysis.

2.2 Laboratory procedures 2.2.1 Isolation of bacteria Most marine bacteria face an oligotrophic environment with diverse needs for growth (Schut, Prins & Gottschal 1997) so to avoid a ‘nutrient shock’, the sea water samples and coral mucus were streaked on marine agar at half strength. Bacterial colonies were isolated based on their morphological differences. Colonies were picked and purified by repeated streaking on plates. Pure cultures were preserved as a glycerol suspension (20%, w/v) at -70 °C (Feltham et al. 1978).

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2.2.2 Molecular characterisation Small subunit ribosomal RNA (16S rRNA) has been proven to be most useful for establishing evolutionary relationships because of their high information content, conservative nature, and universal distribution (Lane et al. 1985). The 16S sequence analysis is used in two major applications: (a) identification and classification of isolated pure cultures and, (b) estimation of bacterial diversity in environmental samples without culturing through metagenomic approaches. New bacterial isolates are identified based on the 16S sequence homology analysis with existing sequences in the databases (Rajendhran & Gunasekaran 2011).

Bacterial isolates were grown overnight in half strength marine broth at 30 °C and pelleted by centrifugation at 13,000 x g for 5 min. The pellet was resuspended in 50 µl of TE buffer (pH 8.0). Three cycles of freezing in a -80 °C freezer and thawing in a 85°C water bath were conducted to release DNA from the microbial cells.

The bacterial DNA were amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of bacterial 16S rRNA genes was performed with broad-specificity primers 8F (Eden et al. 1991) and 519R (Lane et al. 1985).

Ampliﬁcation was performed by using REDTaq® ReadyMix™ PCR Reaction Mix (Sigma Aldrich) using instructions provided by the Sigma Aldrich with the following cycling conditions: . Initial denaturation at 96 °C for 4 min. . 40 cycles of: - 96 °C for 1 min. - 55 °C for 2 min. - 72 °C for 3 min. . Final elongation at 72 °C for 4 min.

Samples of extracted DNA were analyzed on a 1% agarose gel containing 1 µg of ethidium bromide per ml (see Figure 2.4). P a g e | 24

Figure 2.4: 16S rRNA bands of bacterial isolates. Impure bands can be seen at BSD 16-5, 16-7, 16-11. These isolates were later reisolated to ensure pure cultures.

Nucleotide sequences were determined by the dideoxynucleotide method by cycle sequencing of the puriﬁed PCR products. An ABI Prism BigDye Terminator Cycle Sequencing Kit was used in combination with an ABI Prism 877 Integrated Thermal Cycler and ABI Prism 377 DNA Sequencer (Perkin Elmer Applied Biosystems).

Sequences (typically 500 bp) were analysed against the NCBI (USA) database (Zhang et al. 2000) using BLAST program packages and matched to known 16S rRNA gene sequences. Gene sequences were corrected manually. Results are shown in the Appendix. Sequences were aligned and phylogenetic trees were created with MEGA 5 (Tamura et al. 2011) using the neighbor-joining method.

2.2.3 Clone libraries from water samples 2.2.3.1 Extraction of genomic DNA from sea water Total DNA from a few selected water samples from the local SHIVA cruises were cloned (see chapter 3 for further information). Seawater from different depths (1, 5 and 10 m) was collected using a Ruttner water sampler, filtered onto a 0.22 µm membrane filter (Milipore). The filters were immersed in saline ethanol (70% EtOH, 0.9% NaCl) and kept at -22 °C until further processed in the laboratory.

The filtrate samples were sonicated for 20 seconds to dislodge bacterial cells from the filter and a total of 10 ml of each sample centrifuged at 10,000 rpm for 10 min P a g e | 25

to concentrate the samples. The I-genomic BYF DNA extraction mini kit and the I- genomic CTB DNA extraction mini kit (iNTRON Biotechnology, Korea) were used on the Kuching samples, while three freeze and thaw cycles followed by ethanol washing were carried out on the Kota Kinabalu samples. No samples were processed for Semporna.

Prior to the freeze and thaw cycles, 10 ml of each sample were pelleted by centrifugation at 5,000 rpm and 4 °C for 40 min. The pellets were resuspended in 50 µL of TE buffer (10 mM Tris-HC pH 8.0, 1 mM EDTA). Three cycles of freezing in a -80 °C freezer for 3 min and thawing in a 85 °C water bath for 3 min were conducted to release DNA from the microbial cells.

The bacterial DNA were amplified by polymerase chain reaction (PCR) using broad-specificity primers 8F (Eden et al. 1991) and 519R (Lane et al. 1985). Amplification was performed by using REDTaq® ReadyMix™ PCR Reaction Mix (Sigma Aldrich) using instructions provided by the Sigma Aldrich with the following cycling conditions: . Initial denaturation at 96 °C for 4 min. . 40 cycles of: - 96 °C for 1 min. - 55 °C for 2 min. - 72 °C for 3 min. . Final elongation at 72 °C for 4 min.

2.2.3.2 DNA cloning and plasmid extraction The replicated DNA was inserted into vectors using the p-GEMT Easy Vector Systems (Promega, USA) cloning kit and cloned with Escherichia coli JM109 competent cells as the host cell. The white colonies on the cloning agar plate which contain species DNA were selected.

Plasmid extraction by alkaline Lysis method (Birnboim & Doly 1979) was carried out on the selected white colonies. Each colony was cultured in 5 ml Luria Broth (Conda Laboratories, Spain) and incubated overnight in an incubator shaker (37 P a g e | 26

°C, 250 rpm). After incubation, an Eppendorf tube (1.5 ml) was filled with bacterial culture and centrifuged at 13,000 rpm for 1 min. The supernatant was removed and more bacterial culture was added and centrifuged to obtain more plasmid. To resuspend the pellets, 200 µl of Solution 1 (50 mM glucose, 25 mM Tris-HCL pH 8.0 and 10 mM EDTA pH 8.0) were added before adding 400 µl of Solution 2 (1% Sodium Dodecyl Sulphate, 0.2 N NaOH). The tubes were then inverted 5 times and incubated at room temperature for 5 min. 300 µl of Solution 3 (3 M K+, 5 M acetate) were added and the tubes were inverted five times. After inverting the tubes, they were placed on ice for 10 minutes. After the incubation, the tubes were centrifuged for 5 min at 13,000 rpm. The supernatant was transferred into a new tube and filled with isopropanol before incubating the tubes at room temperature for 2 min. The tubes were centrifuged again at 13,000 rpm for 5 min, the supernatant was removed and 1 ml of ice-cold 70% ethanol was added. Lastly, the tubes were subjected to quick spin (1 min) and the supernatant was removed. The tubes were dried and 50 µl of TE buffer (10 mM Tris-HC pH 8.0, 1 mM EDTA) was added before storage.

2.2.3.3 Bacterial communities based in the construction of clone libraries The following is a brief discussion of the clone libraries obtained in this study. The data available for the bacterial communities based on clone libraries is not significant as the sample size is too small; therefore the results were not included in chapter 3.

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Figure 2.5: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in clone libraries from Kuching and Kota Kinabalu. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated.

The phylogenetic tree (see Figure 2.5) shows the evolutionary relationships between the 12 bacterial clones from the samples with nine species obtained from the NCBI BLAST program, based on similarities in the DNA sequences. The tree showed only the Proteobacteria were eligible for comparison with the sample species because of the repeat of the species in a majority of BLAST results, proving that the bacteria species extracted from the samples are distantly related to this group. The BLAST results also showed the highest query coverage of less than 75%, raising the possibility that the clones may be novel, yet-to-be-described species. The species selected from the BLAST results showed a diversity of bacteria from various parts of the world; ranging from a fish pathogen causing fish pasteurellosis (Juíz-Río et al. 2005), to a bacteria found in the North Atlantic Ocean (Muhling et al. 2008) and a bacteria found in the hot springs of Tunisia (Sayeh et al. 2010). The Kota Kinabalu species were grouped on a separate branch from the Kuching species indicating that the two locations clearly contain two separate bacterial communities. However, the identities of the sampled sequences could not be identified due to limited overlap in sequences. P a g e | 28

The species composition of the clone libraries differed dramatically from that of the cultured bacterial community. It is important to note that the samples used were collected at the same time from the same stations. This finding supports the idea that a majority of microorganisms are not easily cultured using standard microbiological techniques (Rappé & Giovannoni 2003).

The influence of riverine input with regards to species composition was further supported by KCH (1) 1 metre which was obtained from Kuching 1911 Station 1 at 1 m depth, and which was grouped away from the other samples. The lower pH and salinity of the surface water at the station allowed what may be a different community to thrive. The other Kuching 1911 bacterium which was obtained from the same station but at 10 m depth is grouped together with a bacterium from Kota Kinabalu which is representative of ocean waters, showing that the denser sea water at the location provides a slightly different environment, influencing the community at that depth. It can also be observed that a most of our samples are grouped together on a separate branch, raising the possibility that the bacteria obtained are undescribed novel species.

2.2.4 PCR amplification of bacterial DMSP cleavage (dddP) and demethylation (dmdA) genes dmdA is a key gene in the catabolism of DMSP which involves demethylation where DMSP is degraded to MeSH (Curson et al. 2008) while dddP is the one of the most common gene which mediates DMSP cleavage in DMS production (Varaljay et al. 2010; Curson, Fowler, et al. 2011). Based on gene abundance, we can measure the distribution of DMSP degrading genes and hypothesize the involvement of members of the bacterial community in the local sulphur cycle (Varaljay et al. 2012). The presence of DMSP degrading genes in our cultured bacterial isolates were screened using degenerate dddP primers and universal dmdA primers.

Bacterial DNA was amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of dddP genes was performed with degenerate dddP primers dddP_874F and dddP_971R P a g e | 29

(Levine et al. 2012) while amplification of dmdA genes was performed with universal dmdA primers dmdAUF160 and dmdAUR697 (Varaljay et al. 2010).

Ampliﬁcation was performed by using REDTaq® ReadyMix™ PCR Reaction Mix (Sigma Aldrich) using instructions provided by the Sigma Aldrich with the following cycling conditions: . Initial denaturation at 95 °C for 30 s. . 40 cycles of: - 95 °C for 30 s. - 41 ° C for 30 s. - 72 °C for 30 s. . Final elongation at 72 °C for 4 min.

Samples of extracted DNA were analyzed on a 1% agarose gel containing 1 µg of ethidium bromide per ml (see Figures 2.6 and 2.7).

Figure 2.6: PCR-based screening of dmdA genes. Bands highlighted in this figure indicate presence of the genes.

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Figure 2.7: PCR-based screening of dddP genes. Bands highlighted in this figure indicate presence of the genes.

2.2.5 PCR amplification of bacterial antimicrobial PKS and NRPS genes Small subunit ribosomal DNA (16S rDNA) based approach has been very successful in the search of secondary metabolites particularly by using specific degenerated primers (Radjasa & Sabdono 2003). The application of PCR-based approach on screening of secondary metabolites-producing parts is used to identify PKS and NRPS genes within our cultured bacterial isolates.

Bacterial DNA was amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of PKS genes was performed with PKS degenerated primers KSDPQQF and KSHGTGR (Piel 2002) while amplification of NRPS genes was performed with NRPS degenerated primers A2gamF and A3gamR (Marahiel, Stachelhaus & Mootz 1997).

Ampliﬁcation was performed by using REDTaq® ReadyMix™ PCR Reaction Mix (Sigma Aldrich) using instructions provided by the Sigma Aldrich with the following cycling conditions: . Initial denaturation at 94 °C for 2 min. . 45 cycles of: - 94 °C for 1 min. - 55 °C for 1 min. - 72 °C for 2 min. . Final elongation at 72 °C for 4 min. P a g e | 31

Samples of extracted DNA were analyzed on a 1% agarose gel containing 1 µg of ethidium bromide per ml (see Figures 2.8 and 2.9).

Figure 2.8: PCR-based screening of PKS genes. Bands highlighted in this figure indicate presence of the genes.

Figure 2.9: PCR-based screening of NRPS genes. Bands highlighted in this figure indicate presence of the genes.

2.2.6 Antimicrobial tests 2.2.6.1 Extraction of bioactive compounds Crude extract from each mucus associated isolate was extracted using ethyl acetate solvent. This extraction method is particularly useful for extraction of both extracellular (excreted by bacteria into the medium) and intracellular bioactive compounds. P a g e | 32

The bacterial isolates were grown in half strength marine broth at 28, 30 and 32 °C for three days. 20 ml of ethyl acetate was added into 20 ml of bacterial broth and shaken on a rotary shaker overnight. The mixtures were poured into separating funnels and the broth layer was discarded while the layer containing the ethyl acetate phase was collected in pre-weighed beakers. Another 20 ml of ethyl acetate were added into the funnel and the extraction was repeated to rinse out any residue extract. The ethyl acetate extract was then dried in the fume hood to give a solid and oily residue. The dried extract was kept at -20 °C until further use.

2.2.6.2 Inhibitory interaction tests (well diffusion assay) Modified well diffusion assays were used to detect the antimicrobial activities of samples (Ndyetabura, Lyantagaye & Mshandete 2010). The well diffusion assay is an alternative approach to the Kirby-Bauer disk diffusion method (Boyle, Fancher & Ross 1973) for testing antimicrobial activities of the isolate microbes.

The dried extract was weighed and the extracted metabolite was diluted to 500 ppm using dimethyl sulfoxide (DMSO) (Matu et al. 2012). Mucus associated bacterial isolates (test organisms) were grown overnight in half strength marine broth at 28, 30 and 32 °C. Wells with a diameter of 5 mm were punched into half strength marine agar and the test organisms were swabbed onto the agar plates. 50 μl of extract from each bacterial culture were loaded each well. Chloramphenicol and DMSO adjusted to concentrations of 500 ppm were used as positive and negative controls. Chloramphenicol is a broad-spectrum antibiotic and is effective against a wide variety of Gram-positive and Gram-negative bacteria (Neu & Gootz 1996).

The agar plates were incubated at 28, 30 and 32 °C for three days. The agar plates were observed for any zone of inhibitions and recorded (see Figure 2.10).

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Figure 2.10: Inhibition zones from bioactive compounds with antimicrobial properties.

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CHAPTER 3

Bacterial Communities from the eastern South China Sea and the Celebes Sea, and Their Potential Role in the DMS(P) Cycle

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 Malaysian waters which include parts of the eastern South China Sea (Kuching and Kota Kinabalu) and the Celebes Sea (Semporna). Comparisons were made with regards to physico-chemical parameters and bacterial communities. The diversity of bacterial communities in these waters were analysed through isolation and identification (16S rDNA) of culturable bacteria. 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. Riverine input was the highest in Kuching which was mirrored by dominance of potentially pathogenic Vibrio, whereas the Kota Kinabalu community is more indicative of an open ocean environment. Isolates obtained from Kota Kinabalu and Semporna show that the communities in these areas have potential roles in bioremediation, nitrogen fixing and sulphate reduction. One major and globally important role of surface bacteria is their involvement in the breakdown or osmoregulation of DMSP to DMS or MeSH. The cultured bacteria were screened for the existence of two key genes (dmdA, dddP) which are involved in competing, enzymatically mediated DMSP degradation pathways. This is to our knowledge the P a g e | 35

first study looking at the diversity of culturable bacteria in coastal waters of Malaysia and also their potential roles in the DMS(P) cycle.

Keywords: culturable bacterial communities, diversity, dimethylsulphide, dimethylsulphoniopropionate

3.1 INTRODUCTION The South China Sea is a marginal sea that is part of the Pacific Ocean, encompassing an area from the Singapore and Malacca Straits to the Strait of Taiwan (Morton & Blackmore 2001). The Celebes Sea is connected to the South China Sea through the Sulu Sea (Yoshida, Nishimura & Kogure 2007). While the bacterial community structure in these two regions have been previously reported to display some similarities when compared (Yoshida, Nishimura & Kogure 2007) not much is known about the diversity and function of the microbial communities in the South China Sea, especially regarding the eastern region (Kuching and Kota Kinabalu) and the Celebes Sea and no studies on culturable communities in the region have been made at this time.

Studies indicate that Alphaproteobacteria, together with SAR 11 and SAR 86 phylotypes, dominate bacterial communities of the surface ocean waters (up to 50% of rDNA analyses; (González et al. 2000). Members of the Gammaproteobacteria, and Bacteroidetes also often make up a significant portion of marine microbial communities (Abell et al., 2005; Alonso et al., 2007). Other common bacteria found in marine sediment surfaces include Gammaproteobacteria, Bacteroidetes and sulphate reducing bacteria (Stevens & Brinkhoff 2005). However, information about the diversity and function of the microbial communities in the South China Sea is still sparse; especially regarding the eastern region (Kuching and Kota Kinabalu) and the Celebes Sea. To our knowledge, there is no study on regional scale or large-scale distribution patterns of microbes in the Malaysian area of the South China Sea. Most studies about bacterial communities focused on regions near China, such as those carried out by Li et al. (2006), Jiang et al. (2007) and Tao, Peng & Pinxian (2008). All three studies used culture-independent techniques to reveal the community structure and P a g e | 36

diversity of the predominant bacteria at the sampling environment. Their results showed no significant difference in community structure with depth at each location, but significant difference between geographical sites and hosts. The latter two studies revealed that most lineages within the Proteobacteria represented uncultured microorganisms, suggesting that a vast amount of microbial resource in the South China Sea is unknown and unexplored.

Studies of microbial communities in rivers and estuarine communities have shown that the Alpha-, Beta- and Gammaproteobacteria, and gram-positive bacteria are the dominant groups. This is true for different regions of the world such as the Columbia River (Crump, Armbrust & Baross 1999) and Changjiang River in China (Sekiguchi et al. 2002).

In an interesting study, Nakase et al. (2007) found that a predominance of Alphaproteobacteria and the Cytophaga–Flavobacterium cluster appear to be beneficial for successful fish larval production, whereas Gammaproteobacteria seem to increase their mortality. A comprehensive study of the bacterial communities in the Malaysian area of the South China Sea could therefore potentially be beneficial to fisheries as well.

The ocean is the major source of sulphur (Andreae 1986) and microorganisms residing in the ocean have the ability to metabolise organic and inorganic sulphur (Sievert, Kiene & Schultz-Vogt 2007). Dimethylsulphoniopropionate (DMSP) does represent a major carrier for sulphur transfer through microbial food webs and organic sulphur cycling in the pelagic ocean as it is an abundant component in many phytoplankton taxa and is very prone to microbial degradation, making it very appetizing for bacteria and grazers (Kiene, Linn & Bruton 2000). The Roseobacter which are part of the Alphaproteobacteria lineage are mainly responsible for the degradation of DMSP – a major source of oceans’ organic sulphur – into methanethiol (MeSH) (González, Kiene & Moran 1999; González et al. 2000). Hence, these bacteria play an important role in organic sulphur cycling and are found in different regions ranging from the Sargasso Sea to the Black Sea (González, Kiene & Moran 1999). P a g e | 37

A competing metabolic pathway results in the production of dimethylsulfide (DMS) from DMSP (González, Kiene & Moran 1999; Johnston et al. 2008). Due to the highly efficient bacterial DMSP demethylation and DMS consumption processes, only a small percentage (1-2 %) of DMSP produced by marine phytoplankton is ventilated to the atmosphere as DMS (Levine et al. 2012). Despite the low percentage, DMS does, however, represent a major source of biogenic sulphur to the atmosphere, where oxidation products form cloud condensation nuclei and ultimately influence radiative backscatter (Lovelock, Maggs & Rasmussen 1972; Andreae & Crutzen 1997; Simó 2001).

Using genomic-based studies, enzymes responsible for DMSP degradation within the microbial community can be identified more easily, which will contribute to studies on the different biochemical pathways and regulatory factors involved in DMSP metabolism. The DMSP demethylase gene (dmdA), encodes the first step in the demethylation pathway. It has been revealed to be taxonomically diverse and highly abundant (present in >50 % of marine bacterioplankton) (Howard et al. 2008). In comparison to dmdA, the genes involved in DMS production (dddD, dddL, dddP dddQ, dddY, and dddW; all of which mediate the same step of DMSP cleavage) are present in less than 10% of bacteria based on marine metagenomic surveys (Todd et al. 2007; Curson et al. 2008; Howard et al. 2008; Curson, Sullivan, et al. 2011; Todd et al. 2011; Todd, Kirkwood, et al. 2012). We focus on dddP as past studies indicate that this gene, is one of most abundant occurring ddd genes (Todd et al. 2009; Varaljay et al. 2012; Levine et al. 2012).

The present study tries to expand our knowledge on microbial communities in the South China Sea by assessing the surface bacterial communities along the eastern region of the South China Sea as well as the Celebes Sea. Isolates obtained were also screened for the existence of key genes involved in the competing, enzymatically mediated DMSP degradation pathways (dmdA, dddP) to identify potential key players in the local DMS(P) cycle.

3.2 MATERIALS AND METHODS 3.2.1 Study site and sample collection P a g e | 38

In conjunction with European and Malaysian research partners, the SHIVA (Stratospheric ozone: Halogen Impacts in a Varying Atmosphere, EU call ENV.2008.1.1.2.1) Western Pacific field campaign was performed in the fall of 2011. The core field campaign took place in the South China Sea and along the coastline of Peninsula Malaysia and Borneo using the German Research Vessel (RV) Sonne during a cruise leading from Singapore to Manila, Philippines (see Figure 3.1 for cruise track).

Figure 3.1: The RV Sonne ship track leading from Singapore to Manila between November 15-29, 2011 during the SHIVA SO 218 cruise.

Local cruises took place in Kuching on both November 16 and 19, 2011 (see Figure 3.2 for sampling stations), Kota Kinabalu (November 23, 2011; see Figure 3.3 for sampling stations) and Semporna (November 26, 2011; see Figure 3.4 for sampling stations) to provide additional data for coastal input. Samples for this study were collected during the local cruises. Table 3.1 provides an overview of the sampling stations and their respective GPS coordinates.

Raw sea water samples were streaked on marine agar at half strength and bacterial colonies were isolated based on their morphological differences. Colonies P a g e | 39

were picked and purified by repeated streaking on plates. Pure cultures were preserved as a glycerol suspension (20%, w/v) at -70 °C.

Table 3.1: Locations of sampling stations at Kuching, Kota Kinabalu and Semporna.

Sampling GPS coordinates stations Kuching (1611) Kuching (1911) Kota Kinabalu Semporna 1°38'36.24"N, 1°39'28.81"N, 6° 3'4.56"N, 116° 4°35'15.96"N, Station 1 110°30'5.28"E 110°31'24.42"E 5'54.60"E 118°32'58.14"E 1°39'44.82"N, 1°42'44.24"N, 6° 3'5.82"N, 116° 4°38'37.86"N, Station 2 110°32'7.26"E 110°33'23.46"E 4'1.45"E 118°20'25.44"E 1°42'2.80"N, 1°45'32.93"N, 6° 3'4.02"N, 116° 4°42'31.68"N, Station 3 110°37'12.36"E 110°35'16.86"E 0'2.77"E 118°23'19.38"E 1°42'46.62"N, 1°48'2.16"N, 6° 2'49.85"N, 4°40'42.48"N, Station 4 110°39'17.40"E 110°37'51.53"E 115°57'38.26"E 118°32'11.34"E 1°45'49.07"N, 1°50'54.15"N, 6° 4'23.64"N, 4°37'31.26"N, Station 5 110°41'27.77"E 110°40'11.26"E 115°54'36.42"E 118°41'5.99"E 4°35'56.76"N, Station 6 – – – 118°43'19.14"E 4°35'30.66"N, Station 7 – – – 118°42'17.10"E 4°33'17.83"N, Station 8 – – – 118°39'22.57"E *1611 and 1911 denotes November 16 and 19, 2011 respectively.

Figure 3.2: Locations of sampling stations in Kuching, Sarawak.

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Figure 3.3: Locations of sampling stations in Kota Kinabalu, Sabah.

Figure 3.4: Locations of sampling stations in Semporna, Sabah.

3.2.2 DNA extraction and purification of cultured bacteria The isolates were grown in marine broth at half strength and pelleted by centrifugation at 13,000 rpm for 5 min. The pellets were then suspended in 50 µl of TE buffer (10 mM Tris-HC pH 8.0, 1 mM EDTA). Three cycles of freezing in a -80 °C P a g e | 41

freezer for 3 min and thawing in an 85 °C water bath for 3 min were conducted to release DNA from the microbial cells.

3.2.3 PCR amplification of bacterial 16S rRNA genes The bacterial DNA were amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of bacterial 16S rRNA genes was performed with broad-specificity primers 8F (Eden et al. 1991) and 519R (Lane et al. 1985). Amplification was performed by using RedTaqMix (Sigma Aldrich) using instructions provided by the Sigma Aldrich with the following cycling conditions: initial denaturation at 96 °C for 4 min, 40 cycles of 96 °C for 1 min, 55 °C for 1 min, extension at 72 °C for 2 min, and then a final elongation at 72 °C for 4 min. Samples of extracted DNA were analysed on a 1% agarose gel containing 1 µg of ethidium bromide per mL.

3.2.4 Sequencing and phylogenetic analysis Sequences were analysed against the NCBI (USA) database (Zhang et al. 2000) using BLAST program packages and matched to known 16S rRNA gene sequences. Gene sequences were corrected manually. Results are shown in Appendix (see Tables A.1 to A.4). Sequences were aligned and phylogenetic trees created with MEGA 5 (Tamura et al. 2011) using the maximum likelihood method, and are presented in Figures 3.7, 3.8, 3.9 and 3.10.

3.2.5 Nucleotide sequence accession numbers The nucleotide sequences obtained in the present study have been deposited in GenBank database (http://www.ncbi.nlm.nih.gov) under accession numbers KF373266 to KF373440.

3.2.6 PCR amplification of bacterial DMSP cleavage (dddP) and demethylation (dmdA) genes The bacterial DNA were amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of dddP P a g e | 42

genes was performed with degenerate dddP primers dddP_874F and dddP_971R (Levine et al. 2012) while amplification of dmdA genes was performed with universal dmdA primers dmdAUF160 and dmdAUR697 (Varaljay et al. 2010). Ampliﬁcation was performed by using RedTaqMix (Sigma Aldrich) with the following cycling conditions: initial denaturation at 95 °C for 5 mins, 40 cycles of 95 °C for 30 s, 41 °C for 30 s, extension at 72 °C for 30 s, and then a final denaturation and annealing for 1 min each. Samples of extracted DNA were analyzed on a 1% agarose gel containing 1 µg of ethidium bromide per mL.

3.3 RESULTS AND DISCUSSION 3.3.1 Physico-chemical parameters Basic physico-chemical parameters were recorded during sampling in Kuching (November 19, 2011) and Kota Kinabalu (see Table 3.2). Values for Kuching (November 16, 2011) and Semporna are not reported as the measuring instruments were inconsistent and not functioning properly at the time of sampling.

Table 3.2: Physico-chemical parameters measured from Kuching and Kota Kinabalu at depths of 1 and 5 m.

Depth Temp Salinity Nitrate Phosphate Nitrite Silicate Station pH (m) (°C) (ppt) (ppm) (ppm) (ppm) (ppm) 1 29.06 7.90 28.48 9.13 0.60 1.46 23.47 KCH-1 5 29.34 8.10 30.59 BD BD BD BD 1 28.98 8.25 30.65 2.02 0.33 0.60 7.48 KCH-2 5 29.11 8.25 30.89 BD BD BD BD 1 29.05 8.33 31.18 0.85 0.15 0.03 2.97 KCH-3 5 29.16 8.30 30.53 BD BD BD BD 1 29.00 8.33 31.07 0.49 0.10 0.00 4.52 KCH-4 5 29.10 8.29 30.52 BD BD BD BD 1 29.27 8.31 31.61 0.15 0.06 0.00 5.86 KCH-5 5 29.40 8.29 31.85 0.15 0.05 0.00 2.21 KCH 29.15 8.24 30.74 2.13 0.22 0.35 7.75 mean 1 29.80 8.44 31.85 1.04 0.15 BD 3.47 KK-1 5 29.90 8.37 32.04 BD BD BD BD 1 29.73 8.36 31.44 0.25 0.17 BD 3.21 KK-2 5 29.78 8.33 31.95 BD BD BD BD 1 29.55 8.34 31.88 0.23 0.11 BD 2.67 KK-3 5 29.54 8.33 31.87 BD BD BD BD 1 29.52 8.36 31.93 BD BD BD BD KK-4 5 29.45 8.34 31.91 BD BD BD BD 1 29.68 8.38 32.03 0.13 0.03 BD 2.74 KK-5 5 29.50 8.37 31.92 0.15 0.02 BD 2.79 P a g e | 43

KK 29.65 8.36 31.88 0.36 0.10 BD 2.98 mean *KCH denotes Kuching; KK denotes Kota Kinabalu. **BD denotes values that are below detection limit.

Sampling stations at Kota Kinabalu stretched further away from the coastline and displayed average values of salinity at 31.88 ppt, pH of 8.36 and temperature of 29.65 °C (see Table 3.2), all indicative of a typical ocean environment (Raven et al. 2005). The first sampling station at Kuching (KCH-1) was closer to the river mouth of the Sarawak river and displayed a visible influence by riverine water with its surface water displaying a salinity of 28.48 ppt and pH of 7.90 (see Table 3.2). The salt and minerals in sea water made it denser than fresh water thus at the mouth of the river where it meets the sea, the fresh water flows downstream across the surface and the sea flows upstream at the bottom, resulting in an increasing trend from low to high salinity as it goes deeper (salinity at depth is 2 units higher; see Table 3.2). The pH values at KCH-1 also showed an increase from pH 7.90 at 1 m depth to pH 8.10 at 5 m depth. However, the pH is still lower than the average pH of seawater, indicating mixing of fresh- and marine water and/or supporting the theory that not only the salt content of the sea water contributed to its pH but other compounds present in sea water as well (Millero, Lee & Roche 1998). Subsequent stations were however more representative of ocean waters with pH around 8.3 and salinity around 31ppt (see Table 3.2). Temperatures at Kuching and Kota Kinabalu are consistent at both 1 and 5 m depth (mean temperature of 29.15 and 29.65 °C respectively; see Table 3.2). The majority of the South China Sea is in the tropical region where warm temperatures are constant and deviations in temperature are not significant (McKnight & Hess 2000).

The riverine input at Kuching was also visible with significantly higher nitrate, phosphate, nitrite, and silicate values closer to the river mouth (KCH-1 and KCH-2; see Table 3.2). Nutrient levels in Kuching were also generally higher than in Kota Kinabalu. To assess differences in distribution in the upper surface layers, samples were also taken from 5 m depth (KCH-5 and KK-5; see Table 3.2). Interestingly, the samples for Kota Kinabalu show consistent values, however, for Kuching, the P a g e | 44

silicate drops from 5.86 ppm to 2.21 ppm within the first 5 metres (see Table 3.2), indicative of an active biological pump (Dugdale, Wilkerson & Minas 1995).

3.3.2 Diversity of culturable bacterial communities The present study provides what we believe to be the first analysis of cultured bacterial communities from the eastern South China Sea and the Celebes Sea. The bacterial communities from the sampling sites in Kuching, Kota Kinabalu and Semporna were found to be diverse with representatives from several groups. The total bacterial assemblage had representatives within the Alpha-, Beta- and Gammaproteobacteria, as well as Firmicutes (see Figure 3.5). The general similarity in groups can be explained by the use of a singular isolation media (marine agar at half strength). However, the total number of bacterial isolates obtained and assemblages at the three sampling sites were different as discussed in the following.

From Kuching waters, 89 isolates were obtained over two sampling periods (November 16 and 19, 2011). The diversity of bacterial phylotypes is presented in Figures 3.7 and 3.8, and Tables 3.3 and 3.4. Overall, 89% of the cultured bacteria were clustered within the Gammaproteobacteria, 8% within the Alphaproteobacteria and 3% within the Firmicutes (see Figure 3.5). In Kota Kinabalu waters, 39 isolates were obtained and the majority (72% of the cultured bacteria) were clustered within the Gammaproteobacteria. The remaining isolates were members of the Firmicutes (18%) and Alphaproteobacteria (10%) (see Figures 3.5, 3.9 and Table 3.5). In Semporna waters, 48 isolates were obtained from four phylogenetic groups. In total, 88% of the cultured bacteria were members of the Gammaproteobacteria, 6% of the Firmicutes, 4% of the Alphaproteobacteria and 2% Betaproteobacteria (see Figures 3.5, 3.10 and Table 3.6).

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Figure 3.5: Pie charts illustrating the diversity of bacterial groups based on partial 16S rRNA gene sequences from bacteria isolated from (a) Kuching, (b) Kota Kinabalu and (c) Semporna.

35 30 25 20 15 10 Number Number of isolates 5 0 1m 5m ≥10m 1m 5m ≥10m 1m 5m ≥10m Kuching Kota Kinabalu Semporna

α-proteobacteria β-proteobacteria γ-proteobacteria Firmicutes

Figure 3.6: Phylogenetic groups of isolates from the waters of Kuching, Kota Kinabalu and Semporna at depths of 1, 5 and 10 m.

Several ecological diversity indices frequently applied to microbial community profile data were used in order to compare diversity among microbial communities, enabling us to quantify diversity within the communities and describe their numerical structure (see Table 3.3). Taxonomic classification up to genus was used as some BLAST results could only relate the isolates to strains which have been identified up to genus level.

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Table 3.3: Indices used to quantify the diversity of bacterial communities at Kuching, Kota Kinabalu and Semporna.

Genus Kuching Kota Kinabalu Semporna Total isolates (N) 89 39 48 Total genus (S) 14 15 14 Margalef index (DMg) 2.90 3.82 3.36 Shannon index (H’) 1.60 2.42 2.18 Shannon evenness (J’) 0.61 0.89 0.83 Smith and Wilson evenness (Evar) 0.49 0.69 0.59 *Formulae of diversity indices are from Margalef (1958), Shannon & Weaver (1963) and Smith & Wilson (1996).

The Margalef index (DMg) measures species richness and is highly sensitive to sample size (Magurran 2004). DMg is a more accurate index when it comes to sample richness as it utilises absolute numbers compared to a density data matrix (Gamito 2010). The commonly used Shanon index (H’) considers proportions, ensuring no differences when using either data set (Gamito 2010). However, calculated H’ values can be underestimations due to incomplete coverage as it gives more weight to rare than to common species (S), making it more sensitive to absolute (but not relative) changes in their abundance (Hill et al. 2003). Values for both indices indicate that the bacterial community in Kota Kinabalu is the most diverse with a greater number of genuses within the community, followed by the communities in Semporna and Kuching.

The Shannon evenness index (J’) is derived from H’ which therefore makes it sensitive to changes in evenness of rare species, thereby possibly overestimating its true value (Hill et al. 2003). The Smith and Wilson evenness index (Evar), however, is known to show greater resolution in reflecting true values (Blackwood et al. 2007). The evenness values from both J’ and Evar show that not only does the bacterial community in Kota Kinabalu have a greater amount of genuses present, but the individuals in the community are distributed most equitably among these genuses, and this corelation is replicated in the results from Semporna and Kuching.

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Figure 3.7: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kuching 1611. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour- joining method. Bootstrap values ≥50 are shown and the scale bar represents a P a g e | 48

difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated.

Figure 3.8: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kuching 1911. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour- joining method. Bootstrap values ≥50 are shown and the scale bar represents a P a g e | 49

difference of 0.1 substitution per site. Accession numbers for the reference sequences are indicated.

Figure 3.9: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Kota Kinabalu. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour- P a g e | 50

joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated.

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Figure 3.10: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in Semporna. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining P a g e | 52

method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.1 substitution per site. Accession numbers for the reference sequences are indicated.

Gammaproteobacteria are the dominant phylogenetic group at all three locations and at all sampling depths, followed by Alphaproteobacteria (see Figure 3.5). Betaproteobacteria were only found at Semporna at 1m depth (see Figure 3.6). These results correlate with existing records of microbial communities found in coastal and open-ocean environments (Bernard et al. 2000) although samples from Kuching have some riverine influence. The percentage of bacterial culturability is 2% (Button et al. 1993), thus, giving the possibility that although some groups may be present in low numbers in cultures, they may still occupy a significant portion of the bacterial community. However, to better understand their physiology and ecology, the isolation of bacteria in pure culture remains an essential step in microbial ecology (Bernard et al. 2000).

In the following, we discuss some highlights of the diversity found within the major bacterial groups and also try and establish differences between the three different sampling sites.

The cultured Alphaproteobacteria group consisted of representatives from the Caulobacteraceae, Phyllobacteriaceae, Rhodobacteraceae and Rhodospirillaceae. Caulobacteraceae were only found in Kota Kinabalu at 1 m depth with two isolates related to Brevundimonas diminuta (see Figure 3.9). They are aerobic, non- photosynthetic organisms which are widespread in natural bodies of water (Stove Poindexter & Cohen-Bazire 1964). The closest related strains were Brevundimonas diminuta strain c138 (GenBank accession number FJ950570; 99% similarity) and Brevundimonas diminuta strain KSC_AK3a (GenBank accession number EF191247; 100% similarity), both of which have shown antibiotic resistance under extreme conditions (La Duc et al. 2007; Li et al. 2010). Members of the family Phyllobacteriaceae are part of a large variety of bacteria able to reduce nitrate to nitrite or to molecular nitrogen (Zumft 1997; Labbé, Parent & Villemur 2004). Isolates related to this family were grouped with Nitratireductor spp. and were P a g e | 53

found in Kota Kinabalu and Semporna, at depths of 5 and 10 m (see Figures 3.9 and 3.10). Isolates belonging to Rhodobacteraceae were related to Roseovarius pacificus strain 81-2 (GenBank accession number NR_043564) and Rhodobacter capsulatus strain PSB-06 (GenBank accession number FJ866784), with overlaps across Kuching and Semporna at 1 and 5 m depth (see Figures 3.7, 3.8 and 3.10). The property to reduce nitric oxide is not restricted to denitrifiers within Phyllobacteriaceae as strains of Rhodobacter capsulatus have been shown to be able to transform nitric oxide to nitrous oxide at a significant rate (Bell, Richardson & Ferguson 1992) and are also able to convert nitrous oxide to nitrogen through the involvement of cytochrome bc1 complex (Itoh, Matsuura & Satoh 1989; Richardson et al. 1989). Roseovarius pacificus was previously isolated from deep- sea sediment of the Western Pacific Ocean and displayed the ability to degrade polycyclic aromatic hydrocarbons (Wang, Tan & Shao 2009). Rhodospirillaceae are typically purple non-sulphur photosynthetic bacteria, possessing the adaptive capacity to grow photosynthetically and by oxidative phosphorylation (Saunders 1978). Cultures from this family were related to Thalassospira spp. which generally form opaque, unpigmented or slightly yellow colonies on agar (López-López et al. 2002) and are potential bioremediation agents as they have the ability to degrade polycyclic aromatic hydrocarbons and diesel fuel (Liu et al. 2007; Kim & Kwon 2010; Lai & Shao 2012). Isolates related to the Alphaproteobacteria at all three areas seem to be involved in the nitrogen cycle and possibly in the degradation of hydrocarbons.

The sole Betaproteobacteria that was cultured was related to Alcaligenes faecalis (GenBank accession number JF264463; 88% similarity) which was previously isolated from a coastal aquaculture environment. The isolate was obtained from Semporna (see Figure 3.10), an area that is surrounded with aquaculture and seaweed farms. Alcaligenes faecalis have also been found in salt marsh and estuarine waters (Ansede, Friedman & Yoch 2001). It has the potential to degrade DMSP to DMS via acrylate metabolism through the induction of β- hydroxypropionate (Yoch, Ansede & Rabinowitz 1997; Ansede, Pellechia & Yoch 1999). It is easily recognizable by its diffusable yellow pigment on agar plates, a characteristic not produced by the non-DMS-producing terrestrial Alcaligenes P a g e | 54

faecalis strains (Ansede, Friedman & Yoch 2001) and our isolate displayed yellow pigmentation. Other non-DMSP degrading strains of Alcaligenes faecalis have been found to contribute towards coral defence by exhibiting anti-nematode activity (Kooperman et al. 2007).

Within the Gammaproteobacteria group, isolates from Aeromonadaceae, Pseudoalteromonadaceae, Shewanellaceae, Pseudomonadaceae and Vibronaceae can be found across all three sampling sites (see Figures 3.7, 3.8, 3.9 and 3.10). Uncommon groups of bacteria from Gammaproteobacteria that are related to isolates in this study include Burzelia and Stenotrophomonas from Kuching (see Figure 3.7); and Bowmanella, Idiomarina and Allomonas from Semporna (see Figure 3.10).

Aeromonadaceae are primarily found in freshwater and associated with aquatic animals and sewage, with the ability to reduce nitrate (Colwell, Macdonell & De Ley 1986). An isolate related to Aeromonas enteropelogenes strain KADR14 (GenBank accession number JX136699; 99% similarity) was successfully cultured from surface waters of Kota Kinabalu (see Figure 3.9). Aeromonas enteropelogenes was previously found to be synonymous with Aeromonas trota on the basis of 16S rRNA sequences (Collins, Martinez-Murcia & Cai 1993), and is a clinically relevant species (Figueras, Guarro & Martínez-Murcia 2000) . Oceanimonas spp. were isolated from all three sampling locations (see Figures 3.7, 3.9 and 3.10). Isolates were closely matched with Oceanimonas smirnovii strain 31-13 (GenBank accession number NR_042963), which was previously isolated from the Black Sea (Ivanova et al. 2005), had overlaps across Kota Kinabalu and Semporna. Oceanimonas spp. are one of the earliest Gammaproteobacteria to have been studied biochemically for multiple DMSP degrading genes including dddP (Yoch 2002). Studies have indicated that Oceanimonas spp. have multiple DMSP degrading genes, allowing them to play a role in the sulphur cycle (Curson, Fowler, et al. 2011). The availability of different ddd genes in Oceanimonas spp. implies that DMSP may be a key substrate for this bacteria genus, enabling them to produce DMS from DMSP (Ledyard, DeLong & Dacey 1993). They also have a cytoplasmic DMSP lyase (de Souza & Yoch 1995; Yoch, Ansede & Rabinowitz 1997) P a g e | 55

resembling the periplasmic dddY of Alcaligenes faecalis (de Souza, Yoch & Souza 1996). Our results displayed a slightly more diverse picture with isolates related to Oceanimonas spp. from Kuching possessing dddP genes, whereas isolates from KK and Semporna possessed dmdA genes (see Figures 3.14, 3.15, 3.16).

Members of the Pseudoalteromonadaceae have been previously reported to be found in marine environments, invertebrates, fish and algae, generally without the ability to denitrify (Ivanova, Flavier & Christen 2004). Isolates from Kuching collected on November 16 and 19, 2011 were closely related to Pseudoalteromonas maricaloris strain KMM636 (GenBank accession number NR_025009; 100% similarity) and Pseudoalteromonas ganghwensis (GenBank accession number DQ011614; 99% similarity) respectively, from surface water (1 m depth). Pseudoalteromonas maricaloris strain KMM636 was isolated from an Australian sponge (Fascaplysinopsis reticulate) which was collected at 10 m depth from the Coral Sea, and exhibits antibacterial properties which possibly play a role in the chemical defence of the sponge (Ivanova et al. 2002). Our cultures displayed similar growth characteristics as Pseudoalteromonas maricaloris strain KMM636, such as, translucent colonies which turn lemon yellow after 48 h of incubation (Ivanova et al. 2002). Different strains of Pseudoalteromonas ganghwensis have been shown to possess the ability to form biofilms and contribute in part to the removal of excess proteineous matters from the sediment sludge of fish farms (Iijima et al. 2009). Kota Kinabalu had isolates that were closely related to Pseudoalteromonas lipolytica strain ZR064 (GenBank accession number JX173567). Pseudoalteromonas lipolytica has only been recently characterised (Xu et al. 2010) and has the ability to hydrolyse lipids and reduce nitrate to nitrite.

Members of the Shewanellaceae family generally have the ability to reduce nitrate to nitrite and can be isolated from marine invertebrates and marine environments (Ivanova, Flavier & Christen 2004). They are also known to be opportunistic pathogens (Aguirre et al. 1994; Brink, van Straten & van Rensburg 1995). An isolate from Kuching (collected on November 16, 2011) was related to Shewanella chilikensis strain JC5 (GenBank accession number HM016088; 99% similarity). Not much is known about Shewanella chilikensis as it has only been recently described P a g e | 56

by Sucharita et al. in 2009. They were previously isolated from lagoon sediment and do not share the family’s ability to reduce nitrate (Sucharita et al. 2009). Our isolate was obtained near the river mouth at 10 m depth (Station 1; see Figure 3.7). Isolates related to various Shewanella haliotis strains were cultured from the waters of Kuching and Kota Kinabalu (see Figures 3.7 and 3.9) at depths of 1 and 5 m. Shewanella haliotis has been described to be sensitive to antibiotics (Kim et al. 2007). In Kota Kinabalu, an isolate related to Shewanella putrefaciens strain R1418 (GenBank accession number AB208055; 99% similarity) was found at 1 m depth. S. putrefaciens is a hydrogen sulphide producing bacteria (Satomi et al. 2006) frequently isolated from marine water and spoiling fish (Ziemke et al. 1998) and in rare cases can be a human pathogen (Brink, van Straten & van Rensburg 1995).

The family Pseudomonadaceae is an extremely diverse group of bacteria. Pseudomonas spp. are found at all three sites and all three depths. Isolates obtained from Kuching were related to Pseudomonas aeruginosa strain 11.2 (GenBank accession number JX286673; 100% similarity) and Pseudomonas oleovorans strain HNS030 (GenBank accession number JN128264; 99% similarity). Both were isolated from 1 and 7 m respectively at stations near the river mouth. Pseudomonas aeruginosa is a clinically relevant opportunistic pathogen, ubiquitous in the environment due to its resistance to the antibiotics and disinfectants, and environmental adaptability (Stover et al. 2000). Pseudomonas oleovorans was first isolated from oil-water emulsions used as lubricants and cooling agents in the cutting and grinding of metals (Lee & Chandler 1941). The species is classified part of the Pseudomonas aeruginosa group (Anzai et al. 2000). An isolate from Kota Kinabalu is also found to be closely related to the same Pseudomonas oleovorans strain mentioned (99% similarity) at 1 m depth, indicating an overlap between the locations. Other Pseudomonas spp. related isolates from Kota Kinabalu include Pseudomonas plecoglossicida strain AIMST Aie20 (GenBank accession number JQ312025; 100% similarity) and Pseudomonas stutzeri strain UP-1 (GenBank accession number AY364327; 99% similarity) at 5 and 1 m depth respectively. Pseudomonas stutzeri is distributed widely in the environment with denitrifying abilities (Lalucat et al. 2006). It has also been isolated as an opportunistic pathogen from humans although infections are rare (Noble & Overman 1994), and are P a g e | 57

involved in the degradation of biogenic and xenobiotic compounds (oil derivatives – aromatic and nonaromatic hydrocarbons – and biocides) (Lalucat et al. 2006). Pseudomonas plecoglossicida was first isolated by Nishimori, Kita-Tsukamoto & Wakabayashi (2000) from ayu fish (Plecoglossus altivelis). It is well known as a fish pathogen causing bacterial haemorrhagic ascites and has the ability to reduce nitrate to nitrite (Nishimori, Kita-Tsukamoto & Wakabayashi 2000). This species is also related to an isolate from Semporna, although of a different strain (Pseudomonas plecoglossicida strain R8-591-1; GenBank accession number JQ659971; 100% similarity) at 10 m depth. The occurence of these bacteria at Kota Kinabalu and Semporna is possibly due to the numerous aquaculture farms observed near or surrounding the two locations. Isolates closely related to Pseudomonas pseudoalcaligenes strain K29411 (GenBank accession number DQ298030) were also obtained from the surface waters (1 m depth) of Semporna. This species was found to be synonymous with Pseudomonas oleovorans (Saha et al. 2010) as they share the same morphological, biochemical, and molecular characteristics. An isolate related to Pseudomonas fulva strain SMA24 (GenBank accession number JQ618288; 100% similarity) was also cultured from the waters of Semporna at 1 m depth. Pseudomonas fulva was originally isolated from Japanese rice paddy and are commonly associated with rice plants, grains and paddy fields (Uchino et al. 2001). Our study indicates that they can also be in other environments, potentially involved in hydrocarbon degradation (Ni et al. 2010).

Members of Vibrionaceae are common in the marine environment, with species found in hydrothermal vents, deep sea, open water, estuaries, and marine sediments (Lee & Ruby 1994; Raguénès et al. 1997; Eilers et al. 2000; Maruyama et al. 2000) and is the most heavily represented within the group, with several overlaps between the sites displaying the same GenBank hits (e.g. Vibrio splendidus strain AP625, GenBank ascension number GQ254509). Studies have suggested that Vibrios degrade some ecologically hazardous compounds, such as polycyclic aromatic hydrocarbons (Ramaiah et al. 2000), and are major decomposers of chitin in the ocean (Nagasawa & Terazaki 1987; Hedlund & Staley 2001). Members of Vibrionaceae have on the other hand also been shown to cause potentially lethal diseases in humans and fish (Kusuda & Kawai 1998; McCarter 1999). More P a g e | 58

recently, studies have shown Vibrio shiloi to be a coral pathogen, producing toxins that inhibit photosynthesis and lyse zooxanthellae resulting in bleaching (Banin, Ben-Haim, et al. 2000; Banin, Israely, et al. 2000). Species such as Vibrio parahaemolyticus and Vibrio vulnificus have been shown to express virulence- related properties such as production of the toxR gene (Lin et al. 1993; Okuda et al. 2001) and production of phenolate siderophore (Stelma et al. 1992). Vibrio harveyi and Photobacterium spp. are luminous bacteria which often cause disease in aquaculture (Baticados et al. 1990; Prayitno & Latchford 1995). While most Vibrio spp. isolated from Kuching appear to be related to pathogenic strains, many of the isolates from Kota Kinabalu and Semporna have potential roles in bioremediation, nitrogen fixing and sulphate reduction.

Members of the cultured Firmicutes group consisted of members of the Bacillaceae, Bacillaceae Family XII. incertae sedis and Paenibacillaceae. Isolates from Bacillaceae were mostly related to Bacillus spp. and Lysinibacillus spp. with no overlaps across the sampling sites. Bacillaceae are able to form endospores that allow them to survive for extended periods under adverse environmental conditions and have been shown to fix nitrogen (Jordan, McNicol & Marshall 1978), synthesise antifungal peptides (Kajimura 1995) and plant growth promoting substances, including gibberellin and indoleacetic acid (Broadbent, Baker & Waterworth 1977; Turner & Backman 1991). As such, members of this group have been used for agricultural crop enhancement (Wipat & Harwood 1999). Related strains were obtained from agricultural soil and compost with the exception of Bacillus sphaericus isolate BS11 (GenBank accession number AM269451; 100% similarity) which was isolated from the East China Sea. Isolates from the Bacillaceae Family XII. incertae sedis were matched with Exiguobacterium spp., which have previously been isolated from, or molecularly detected in, a wide range of habitats including cold and hot environments with temperature range from -12 to 55 °C (Vishnivetskaya, Kathariou & Tiedje 2009). Interestingly, members of this family were only isolated from Kota Kinabalu and Semporna where recent temperature spikes resulted in mass coral bleaching in the region (Tan & Heron 2011) and of the three sampling sites, Sarawak was the only area with no reported bleaching events (Tun et al. 2010). The different strains of Exiguobacterium spp. P a g e | 59

did not overlap between sites (see Figures 3.9 and 3.10). The only Paenibacillaceae isolated (from Kota Kinabalu at 1 m depth) was related to Brevibacillus laterosporus strain GZUB11 (GenBank accession number FJ434663; 100% similarity). Brevibacillus laterosporus are aerobic spore-forming bacteria that have demonstrated pathogenicity towards insects and nematodes, with a potential to be used as a biological control agent (Zahner et al. 1999; de Oliveira et al. 2004; Tian et al. 2007). It is also reported to have the ability to produce lignin peroxidase which can be used to degrade sulfonated azo dyes (Gomare, Jadhav & Govindwar 2008).

3.3.3 Variations in the bacterial communities in Kuching, Kota Kinabalu and Semporna waters The bacterial communities in the waters of Kuching, Kota Kinabalu and Semporna are almost entirely unknown and have not been sampled by either culture or culture-independent techniques. Previous studies have shown that microbial community composition is influenced by physico-chemical variables such as salinity, pH and temperature among others (Lamberti & Resh 1983; Nold & Zwart 1998; Arnon et al. 2005; Fierer & Jackson 2006). Our isolates are also mostly related to species that are highly adaptable environmentally, indicating that the communities in these waters employ various mechanisms that regulate the activity of cells in natural communities (Bernard et al. 2000).

It is not surprising that cultured bacterial communities differ from clone libraries which lacks culturable species (Ward, Weller & Bateson 1990; Bidle & Fletcher 1995; Suzuki et al. 1997; Bernard et al. 2000). Only 2% of bacteria grow in culture (Button et al. 1993) as they can be affected by nutrients in growth mediums (Schut et al. 1993), viral infection (Rehnstam et al. 1993) or lack of bacterial commensalism (Saville Waid 1999; Grover 2000). Our cultures were isolated from diluted marine agar, so these results may differ if the growth medium were used at full strength.

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3.3.4 Bacterial strains with potential to metabolise DMS and/or demethylate DMSP Since there are no published reports on the microbial biodiversity in the eastern region of the South China Sea, their role in local biogeochemical cycles is also unclear. To date, there are no available reports on the sulphur cycle in the region, or of DMSP catabolism from bacterial communities of Kuching, Kota Kinabalu and Semporna; neither are any bioinformatics data available on the prevalence of dmdA and dddP genes in bacteria from these regions. As part of our effort to understand the importance of bacteria in the region for the local sulphur cycle, we screened our isolates for the existence of dmdA and dddP genes. Since our isolates have been cultured in a very general way using a method that does not involve selection for DMSP utilisation, any presence of these genes in our isolates is most likely a fundamental trait of these bacteria.

Previously reported bacteria with the ability to demethylate DMSP and/or metabolise DMS which we also managed to isolate and culture include Rhodobacter and Roseovarius within the Alphaproteobacteria (González et al. 2003; Moran et al. 2007; Curson et al. 2008; Johnston et al. 2008; Todd et al. 2009; Kirkwood et al. 2010); the aforementioned Alcaligenes faecalis within the Betaproteobacteria; Oceanimonas, Pseudomonas, Shewanella and Vibro within the Gammaproteobacteria (de Souza & Yoch 1995; Yoch, Ansede & Rabinowitz 1997; Ansede, Pellechia & Yoch 1999; Yoch 2002; Moran et al. 2007; Sievert, Kiene & Schultz-Vogt 2007; Johnston et al. 2008; Raina et al. 2009, 2010); and Bacillus within the Firmicutes (Todd et al. 2009).

DMSP lyase enzymes are present in diverse bacteria (Taylor 1993). Past studies have revealed that DMS is a relatively minor product of DMSP metabolism under normal circumstances in the marine water column (Kiene 1996b; Ledyard, Dacey & Dacey 1996; van Duyl et al. 1998). Past studies found that the demethylation pathway is the major fate of DMSP in seawater (Kiene 1996a). There are two schools of thought regarding the regulation of the two competing pathways: Kiene, Linn & Bruton (2000) and Simó (2001) hypothesized that the ‘bacterial switch’ is influenced by bacterial carbon and sulphur demands and by DMSP availability; P a g e | 61

while Slezak & Brugger (2001), Sunda et al. (2002), Toole et al. (2006), Archer et al. (2010) and Levine et al. (2012) suggest that phytoplankton DMS production is enhanced by UV-A radiation while bacterial DMSP consumption may be UV-A intolerant.

Figure 3.11: Relative abundance of dmdA and dddP genes in cultured bacterial communities from the waters of (a) Kuching, (b) Kota Kinabalu and (c) Semporna.

Bacteria isolated from Kuching displayed the highest abundance of both DMSP degrading genes (36%) compared to communities isolated from Kota Kinabalu and Semporna with 13 and 19 %, respectively. The bacterial community in Kota Kinabalu has the highest percentage of dmdA gene occurrence (28%) while the dddP gene responsible for DMS production appears to be most abundant (29%) within the bacterial community Semporna (see Figure 3.11).

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100% 90% 80%

70% 60% 50% 40% 30% Percentage Percentage (%) 20% 10% 0%

Firmicutes Firmicutes Firmicutes proteobacteria proteobacteria proteobacteria proteobacteria proteobacteria proteobacteria proteobacteria proteobacteria proteobacteria ------γ γ γ β β β α α α Kuching Kota Kinabalu Semporna

Only dmdA Only dddP Both None

Figure 3.12: Presence of dmdA and/or dddP genes in bacterial isolates from the waters of Kuching, Kota Kinabalu and Semporna.

The Gammaproteobacteria group is the largest identified fraction within the communities at all three sampling sites with the potential for DMSP-assimilation. Interestingly, the composition of the DMSP-assimilating community generally mirrored the composition of the total bacterial community at each sampling site (see Figures 3.6 and 3.12). This is unlike previous studies at the Gulf of Maine and the Sargasso sea where the dominating group are the Alphaproteobacteria (Malmstrom, Kiene & Kirchman 2004). Our findings indicate that the community structure of Gammaproteobacteria in the area could be tightly linked to the local sulphur and also possibly the nitrogen cycle.

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100% 90% Vibrio sp. 80% 70% Stenotrophomonas sp. 60% Shewanella sp. 50% Pseudomonas sp. 40% Pseudoalteromonas sp. 30% Oceanimonas sp. 20% Citrobacter sp 10% Burzellia sp. 0% dmdA dddP Both

Figure 3.13: Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Kuching.

100% 90% 80%

70% Vibrio sp. 60% Shewanella sp. 50% Pseudomonas sp. 40% Oceanimonas sp. 30% Enterobacter sp. 20% 10% 0% dmdA dddP Both

Figure 3.14: Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Kota Kinabalu.

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100% 90% 80% Vibrio sp. 70% Shewanella sp. 60% Pseudomonas sp. 50% Photobacterium sp. 40% Oceanimonas sp. 30% Bowmanella sp. 20% Allomonas sp 10% 0% dmdA dddP Both

Figure 3.15: Relative abundance of dmdA and dddP genes in isolated Gammaproteobacteria from Semporna.

Vibrio appear to be the dominant group within Gammaproteobacteria with dmdA and dddP genes at all three stations. At Kuching, they appear well adapted with the potential ability to undergo both competing pathways as a majority of them (88%; see Figure 3.13) have both DMSP degrading genes; in Kota Kinabalu, Vibrio are the only genus possessing dddP genes (see Figure 3.14); and in Semporna, Vibrio appear to have even number of isolates with either one or both genes (see Figure 3.15).

It was previously hypothesized that DMSP production is an overflow mechanism for when growth is unbalanced by lack of nutrients and the need to release excess energy and excess reduced sulphur (Stefels 2000). These carbon-energy overflow substances might evolve through natural selection to be useful in the cell (e.g. through auxiliary structures or defence mechanisms) (Hill, White & Cottrell 1998). Based on our findings, it seems likely that at low nutrient conditions, the distribution of dmdA and dddP genes within the bacterial community become more specific (e.g. more dmdA in KK and more dddP in Semporna; see Figure 3.11) to adapt to a preferred pathway to degrade DMSP. This is discussed in the following.

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The sampling locations at Kuching and Kota Kinabalu were observed to have heavy shipping traffic which may influence the sulphur concentration in the area. Ship plumes emit large amounts of anthropogenic nitrogen and sulphur into the atmosphere, particularly within potential transport distance of land regions (Corbett, Fischbeck & Pandis 1999) which may influence the algal production of DMSP (Malin & Erst 1997).

The waters of Kota Kinabalu are known for having seasonal phytoplankton blooms (Adam et al. 2011). The relative production of DMSP was suggested to depend on nitrogen availability (Andreae 1986). Small haptophytes (e.g. coccolithophorids) and many small dinoflagellates are typical of more nitrogen-deficient conditions, so they have evolved to produce more DMSP, implying the probability of finding higher levels of DMSP is greater under conditions of nitrogen depletion during phytoplankton blooms (Simó 2001).

Nitrate and nitrite concentrations at Kota Kinabalu are low (0.36 ppm and not detectable respectively; see Table 3.2), especially in comparison with Kuching, indicating a low nutrient environment and suggesting the likelihood of high concentration of DMSP in the area especially in the event of phytoplankton blooms. The bacterial community in the area have possibly evolved to adapt to these conditions and preferred the demethylation pathway as the occurrence of dmdA genes in the community is the highest (see Figure 3.11). Due to riverine input, the waters of Kuching have significantly higher in nutrients compared to Kota Kinabalu (see Table 3.2). It is possible that the high nutrient environment at Kuching forces the bacterial community in the area to be more flexible, hence the diverse occurrence of dmdA and/or dddP genes in the community (see Figure 3.11) which allows them to use different pathways in DMSP degradation.

Sampling stations at Semporna were surrounded by seaweed farms. Micro- and macroalgae and halophytic plants are abundant sources of DMSP in the marine environment (Yoch 2002) and past studies (Scarratt et al. 2000) suggested that bacteria growing near algal cells might be exposed to high local levels of DMSP, which would lead to DMS yields that are higher than those inferred from bulk P a g e | 66

seawater measurements. Our results support this as the dddP gene which is responsible for DMS production is most abundant in the bacterial community at Semporna.

Most studies show that bacteria are a major sink for DMS. Therefore, because bacterioplankton are involved in both DMSP and DMS utilization, factors controlling bacterial activity (e.g. UV radiation, temperature, nutrients and dissolved organic matter) (Kirchmann 2000) ultimately play a role in controlling DMS concentration.

Based on our preliminary observations, we believe that these isolates have the ability to undergo both DMSP-degradation processes depending on current environmental conditions. Considering the observed conditions of the sampling sites, our data supports the hypothesis of a ‘bacterial switch’. However, UV radiation measurements at the sampling locations were not taken at this time and may play a role in the local sulphur cycle.

3.4 CONCLUSION The bacterial communities that could be cultured from water samples taken in Kuching, Kota Kinabalu and Semporna vary significantly. Differences between the three areas can partly be explained by differences in physico-chemical parameters. The Kuching community is influenced by higher nutrients and riverine input, and is dominated by potentially pathogenic Vibrio spp., while the Kota Kinabalu community is more indicative of an open ocean environment. The bacterial community in Kota Kinabalu were found to be the most diverse, followed by communities in Semporna and Kuching. This correlates with community evenness from each site. Isolates obtained from Kota Kinabalu and Semporna show that the communities in these areas have potential roles in bioremediation, nitrogen fixing and sulphate reduction.

The preliminary study on the potential role of the bacterial communities in the local sulphur cycle indicates a major role for Gammaproteobacteria, and in particular Vibrio spp.. Occurrence of dddP, dmdA in Gammaproteobacteria mirrors P a g e | 67

total Gammaproteobacteria community structure at all three sampling sites. Kota Kinabalu and Semporna are dmdA- and dddP-dominant respectively, indicating DMSP degrading pathway preferences of the communities in these areas. The majority of isolates from Kuching have almost equal abundance both genes, showing high adaptability in DMS(P) utilisation. Considering how the majority of isolates within the three communities have DMSP-degrading genes, we believe that they are influenced by bacterial carbon and sulphur demands and by DMSP availability, giving some of them the ability to ‘switch’ pathways according to necessity.

Further in-depth characterization of these communities through a combination of physical, chemical and molecular biological studies will improve our understanding of the role of bacteria in DMS(P) cycling in the eastern South China Sea and the Celebes Sea and their impacts on climate change.

3.5 ACKNOWLEDGEMENTS The authors would like to thank N.M. Levine for her assistance with the identification of dddP and dmdA genes. We also thank the Sarawak Biodiversity Centre for their kind permission to conduct research in Sarawak waters (Permit No. SBC-RA-0094-MM). F.W.I. Kuek is funded by the Sarawak Foundation’s Tunku Abdul Rahman scholarship. The research leading to these results has received funding from the European Union's Seventh Framework Programme FP7/2007- 2013 under grant agreement no. 226224 - SHIVA.

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CHAPTER 4

Bacterial Communities from Talang- Talang Reef and Their Potential Role in Coral Defence and the DMS(P) Cycle

ABSTRACT 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, including a healthy coral reef. Complex microbial communities are known to have signiﬁcant inﬂuence over coral reef ecosystems. Through isolation and identification (16S rDNA) of native microbes from corals, their surface mucus layer (SML), as well as the surrounding sediment and waters, we were able to determine the species composition and abundance of culturable bacteria in the coral reef ecosystem. There was a clear distinction between the 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 were 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 the samples from the water column and sediment. 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 P a g e | 69

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 bacterial microbes 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. Roseobacter spp. which are mainly responsible for the degradation of dimethylsulphoniopropionate (DMSP) – a major source of oceans’ organic sulphur – into methanethiol (MeSH) were also successfully isolated from the SML. Bacterial DMSP degraders may also contribute significantly to dimethylsulfide (DMS) production when temperatures are elevated.

Keywords: culturable bacterial communities; coral mucus; antimicrobial; increasing temperatures; coral reefs

4.1 INTRODUCTION Coral reefs are a rare feature in Sarawak due to its shallow sea shelf extending a long way into the ocean. The reefs of Sarawak are limited to the areas off the shores of Bintulu, Miri and offshore islands including the Talang-Satang region in Kuching. 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.

Reef-building corals have a narrow range of thermal tolerance, making them extremely susceptible to temperature stress and outbreaks of coral diseases, whereby the immunity of corals decrease (Baker, Glynn & Riegl 2008). This makes the corals more vulnerable towards pathogens that are more virulent, especially at higher temperatures (Goreau & Hayes 2008). The coral surface mucus layer (SML) contains a complex microbial community that respond to such changes in the environment (Ritchie & Smith 2004). The normal microbial flora within the SML can protect the coral against pathogen invasion and disturbances which may have led to coral diseases (Sutherland, Porter & Torres 2004). On average, 20-30 % of bacterial isolates originating from coral SML possess antibacterial properties (Ritchie 2006) that may assist the coral holobiont as a first line of defence against pathogens (Shnit-Orland & Kushmaro 2009). It has been suggested that these P a g e | 70

antimicrobial compounds are temperature sensitive (Ritchie 2006). Antibacterial activity was found to be optimal at 26 °C and slightly decreased at 30 °C, with partial inactivation occurring at 60 °C and complete loss of activity occurring at 80 °C (Shnit-Orland & Kushmaro 2009).

Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) are multiple enzymes that catalyse the oligomerization of small building blocks into complex structure such as active compounds (Donadio, Monciardini & Sosio 2007). NRPS usually works in conjunction with PKS to produce hybrid compounds which are significant in pharmaceutical products (Ansari et al. 2004). Both biosynthetic systems are responsible for synthesizing numerous biologically vital active metabolites from microorganisms (Ayuso-Sacido & Genilloud 2005).

Coral reefs are potentially significant sources of dissolved DMS and DMSP, particularly when corals are stressed or exposed during low tides (Broadbent & Jones 2004). The concentrations of DMS and DMSP measured in mucus are the highest reported in the marine environment, with values exceeding those reported from highly productive polar waters (Fogelqvist 1991; Trevena et al. 2000, 2003) and sea algal communities (Kirst et al. 1991; Levasseur, Gosselin & Michaud 1994; Trevena et al. 2003).

DMSP in the marine environment is degraded by bacteria via two possible pathways (González, Kiene & Moran 1999): the cleavage pathway which involves splitting of the DMSP molecule into acrylate and DMS (important in mediating organic sulfur emission into the atmosphere), and the demethylation pathway which involves the removal of a methyl group from DMSP to produce 3- methiolpropionate, which is then cleaved to methanethiol (MeSH). The high levels of DMSP and DMS produced by corals, coupled with involvement of bacteria in DMSP and DMS conversion, suggest that corals are likely to harbor bacterial species involved in the cycling of these compounds (Raina et al. 2009, 2010).

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4.2 MATERIALS AND METHODS 4.2.1 Study site and sample collection Field sampling took place at the Talang-talang reef in July 2011. Samples for this study were collected during the sampling. Figure 4.1 shows an overview of the sampling region. Recorded temperatures at Talang-talang reef indicates that it is exposed to temperatures between 28 to 31 °C throughout the year (Ng et al., pers. comm.), however coral assemblages are healthy and diverse (Kaie et al., pers. comm.) .

Figure 4.1: Overview of the Talang-talang Islands just off the shores of Kuching, Sarawak. Enlarged map indicates sampling area.

Sea water, sediment and coral mucus samples were streaked on marine agar at half strength and bacterial colonies were isolated based on their morphological differences. The coral mucus samples were isolated via two different methods. Mucus- associated bacteria were isolated using ultraviolet (UV) light exposure for 15 min as a form of sterilisation for the first layer of mucus to remove any possible surface microbes that may attach to it during the transfer (Chang et al. 1985). A second layer of coral mucus was streaked on top of the UV-exposed mucus, allowing only mucus-associated bacteria to grow on the mucus-regulated surface (Ritchie 2006). Mucus-attached bacteria were isolated without the UV exposure, allowing any bacteria that happened to be attached to the mucus at the time of collection to be P a g e | 72

grown. This approach is based on the hypothesis that true coral-associated bacteria will be impervious to the antibiotic properties of the mucus, while many attached bacteria may be sensitive to the bactericidal properties of the mucus.

Colonies were picked and purified by repeated streaking on plates. Pure cultures were preserved as a glycerol suspension (20%, w/v) at -70 °C.

4.2.2 DNA extraction and purification of cultured bacteria The isolates were grown in marine broth at half strength and pelleted by centrifugation at 13,000 rpm for 5 min. The pellets were then suspended in 50 µL of TE buffer (10 mM Tris-HC pH 8.0, 1 mM EDTA). Three cycles of freezing in a -80 °C freezer for 3 min and thawing in a 85 °C water bath for 3 min were conducted to release DNA from the microbial cells.

4.2.3 PCR amplification of bacterial 16S rRNA genes The bacterial DNA were amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of bacterial 16S rRNA genes was performed with broad-specificity primers 8F (Eden et al. 1991) and 519R (Lane et al. 1985). Amplification was performed by using RedTaqMix (Sigma Aldrich) using instructions provided by the Sigma Aldrich with the following cycling conditions: initial denaturation at 96 °C for 4 min, 40 cycles of 96 °C for 1 min, 55 °C for 1 min, extension at 72 °C for 2 min, and then a final elongation at 72 °C for 4 min. Samples of extracted DNA were analyzed on a 1% agarose gel containing 1 µg of ethidium bromide per mL.

4.2.4 Sequencing and phylogenetic analysis Sequences were analysed against the NCBI (USA) database (Zhang et al. 2000) using BLAST program packages and matched to known 16S rRNA gene sequences. Gene sequences were corrected manually. Results are shown in Appendix (see Table A.5). Sequences were aligned and phylogenetic trees were created with MEGA 6 (Tamura et al. 2013) using the maximum likelihood method, and are presented in Figures 4.3, 4.4 and 4.5. P a g e | 73

4.2.5 Nucleotide sequence accession numbers The nucleotide sequences obtained in the present study have been deposited in GenBank database (http://www.ncbi.nlm.nih.gov) under accession numbers KF373441 to KF373533.

4.2.6 PCR amplification of bacterial DMSP cleavage (dddP) and demethylation (dmdA) genes The bacterial DNA were amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of dddP genes was performed with degenerate dddP primers dddP_874F and dddP_971R (Levine et al. 2012) while amplification of dmdA genes was performed with universal dmdA primers dmdAUF160 and dmdAUR697 (Varaljay et al. 2010). Amplification was performed by using RedTaqMix (Sigma Aldrich) with the following cycling conditions: initial denaturation at 95 °C for 5 mins, 40 cycles of 95 °C for 30 s, 41 °C for 30 s, extension at 72 °C for 30 s, and then a final elongation at 72 °C for 4 min. Samples of extracted DNA were analyzed on a 1% agarose gel containing 1 µg of ethidium bromide per mL.

4.2.7 PCR amplification of bacterial polyketide synthase (PKS) and non- ribosomal peptide synthetase (NRPS) genes The bacterial DNA were amplified by polymerase chain reaction (PCR) and PCR products were purified using PureLink® PCR Purification Kit following the manufacturer’s protocol (Invitrogen Life Technologies). Amplification of PKS genes was performed with PKS degenerated primers KSDPQQF and KSHGTGR (Piel 2002) while amplification of NRPS genes was performed with NRPS degenerated primers A2gamF and A3gamR (Marahiel, Stachelhaus & Mootz 1997). Amplification was performed by using RedTaqMix (Sigma Aldrich) with the following cycling conditions for PKS: initial denaturation at 94 °C for 2 min, followed by 45 cycles of 94 °C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min, and then a final elongation at 72 °C for 4 min. The following are the cycling conditions for NRPS: initial denaturation at 94 °C for 2 min, followed by P a g e | 74

40 cycles of denaturation at 95 °C for 1 min, annealing at 70°C for 1 min and extension at 72 °C for 2 min and then a final elongation at 72 °C for 4 min.

4.2.8 Extraction of bioactive compounds The coral mucus bacterial isolates were grown in 20 ml of half strength marine broth at 28, 30 and 32 °C for three days. 20 ml of ethyl acetate was added into the bacterial broth and shaken on a rotary shaker overnight. The mixtures were poured into separating funnels and the broth layer was discarded while the layer containing the ethyl acetate phase was collected in pre-weighed beakers. Another 20 ml of ethyl acetate were added into the funnel and the extraction was repeated to rinse out any residue extract. The ethyl acetate extract was then dried in the fume hood to give a solid and oily residue. The dried extract was then kept in -20 °C until further use.

4.2.9 Well diffusion assay The dried extract was weighed and the extracted metabolite was diluted to 500 ppm using dimethyl sulfoxide (DMSO) (Matu et al. 2012). Coral mucus bacterial isolates (test organisms) were grown overnight in half strength marine broth at 28, 30 and 32 °C. Wells with a diameter of 5 mm were punched into half strength marine agar and the test organisms were swabbed onto the agar plates. 50 μl of extract from each bacterial culture were loaded each well. Chloramphenicol and DMSO adjusted to concentrations of 500 ppm were used as positive and negative controls. Chloramphenicol is a broad-spectrum antibiotic and is effective against a wide variety of Gram-positive and Gram-negative bacteria (Neu & Gootz 1996). The agar plates were incubated at 28, 30 and 32 °C for three days. The agar plates were observed for any zone of inhibitions and recorded.

4.3 RESULTS AND DISCUSSION 4.3.1 Diversity of culturable bacterial communities The present study provides what we believe is the first analysis of cultured bacterial communities from the reefs of Talang- Talang. The bacterial communities from coral mucus, reef sediment and water column were found to be diverse with representatives from several bacterial groups. The total bacterial assemblage had P a g e | 75

representatives within the Actinobacteria, Proteobacteria (Alpha- and Gammaproteobacteria), as well as Firmicutes (see Figure 4.2 for an overview of the major groups). The total number of bacterial isolates obtained and assemblages from the three reef environments are discussed in the following.

Figure 4.2: Pie charts illustrating the diversity of bacterial groups based on partial 16S rRNA gene sequences from bacteria isolated from (a) coral mucus, (b) water column and (c) sediment.

A total of 93 isolates were cultured from coral mucus, water column and reef sediment of the Talang-talang reef. Overall, 3% of the cultured bacteria were clustered within the Actinobacteria, 76% within the Gammaproteobacteria, 6% within the Alphaproteobacteria and 13% within the Firmicutes. From the coral mucus, 39 isolates were obtained with the majority clustered within the Gammaproteobacteria (64%), followed by Alphaproteobacteria (13%), Firmicutes (13%) and Actinobacteria (8%). There is an unknown isolate that was cultured from coral mucus. Its closest related sequence is unidentified (see Figure 4.3). Within the water column, 82% of the isolates were Gammaproteobacteria, 9% Actinobacteria and 9% Firmicutes. Isolates from reef sediment were less diverse with cultures from only two bacterial groups: the Gammaproteobacteria (86%) and Firmicutes (14%).

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Figure 4.3: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in coral mucus. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour- joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.1 substitution per site. Accession numbers for the reference sequences are indicated. P a g e | 77

Figure 4.4: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in water column. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour- joining method. Bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated.

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Figure 4.5: 16S rRNA gene-based phylogenetic tree representing bacterial sequences found in reef sediment. The phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour- joining method. Bootstrap values ≥50 are shown and the scale bar represents a P a g e | 79

difference of 0.05 substitution per site. Accession numbers for the reference sequences are indicated.

It has been established that mucus presents a specific environment which contains vast microbial communities (Sharon & Rosenberg 2008). Similar coral associated bacteria can be present in different species of corals that are also geographically distinct (Shnit-Orland & Kushmaro 2009). The coral mucus layer is in constant association with the surrounding water column, and bacteria may shift from the water column to the mucus and vice versa (Kooperman et al. 2007). Therefore, it is not surprising that there are overlaps between the mucus and its surrounding environment.

Figure 4.6: Percentage of Vibrio isolates in mucus attached and mucus associated communities.

The mucus associated isolates are related to representatives of bacteria documented in earlier studies, including a subset of Vibrio spp. consistently found in association with healthy corals (Ritchie & Smith 1995a, 1995b, 2004). Figure 4.6 shows that there is a higher percentage of Vibrios (91%) when comparing mucus attached isolates to mucus associated isolates (29%). This illustrates the defensive qualities of coral mucus, and how a potential composition shift from beneficial bacteria to Vibrio dominance (which are known to be opportunistic) under conditions of increased temperature can occur. P a g e | 80

The isolates linked to the phylum Gammaproteobacteria consist of members of the families Alteromonadaceae, Enterobacteriaceae, Moraxellaceae, Halomonadaceae and Vibrionaceae. Vibrionaceae members are present in high numbers in all three environments, where the predominant bacteria are related to Vibrio communis, Vibrio harveyi and Vibrio parahaemolyticus. Vibrio spp. are often associated with diseases in corals and other marine organisms (Rosenberg et al. 2007). Isolates related to known coral pathogens, Vibrio coralliilyticus strain LMG 21349 (GenBank accession number AJ440004) and Vibrio shiloi (GenBank accession number AF007115; 99% similarity) were cultured from mucus and sediment samples respectively. These findings indicate that the reef environment harbour potentially pathogenic bacteria which can cause disease under the right conditions. However, some Vibrios establish mutualistic partnerships with corals by providing nutrients and secondary metabolites to their hosts (Ritchie 2006). Vibrio spp. associated with the coral mucus are known to produce antibacterial compounds against several pathogens, thereby protecting the coral host against pathogens (Shnit-Orland & Kushmaro 2009).

Firmicutes are the second largest bacterial group in all three environments and are dominated by members of the Bacillus genera. Bacillus spp. present in the mucus of corals have been reported to exhibit antibacterial activity against pathogens (Shnit-Orland & Kushmaro 2009) and those of marine origin have been reported to produce unusual metabolites (Jensen & Fenical 1994) including peptide antibiotics such as bacitracin, gramicidin and polymyxin B (Wiese et al. 2009).

The Actinobacteria are known for their production of many bioactive compounds (Magarvey et al. 2004; Fiedler et al. 2005; Jensen et al. 2005) and may influence the susceptibility of corals to pathogens (Rohwer et al. 2002). Isolates related to this phylum are only found within the coral mucus. While the number of Actinobacteria in this study may not be high, other studies have found that they are generally found in corals (Nithyanand & Pandian 2009), and bacterial clone libraries of the coral species included a significant proportion of Actinobacteria (Lampert et al. 2008). The distribution of Actinomycetes in the sea remains largely undescribed and only a few of which were culturable (Webster et al. 2001). Only recently were P a g e | 81

novel marine Actinomycetes discovered in sponges (Webster et al. 2001) and ocean sediment (Mincer et al. 2002; Mincer, Fenical & Jensen 2005), and cultured from corals (Lampert et al. 2008; Nithyanand & Pandian 2009).

Isolates related to the Alphaproteobacteria are only found in coral mucus samples. A couple of the cultures are related to uncultured sequences (BCM 33 and 35-2; see Figure 4.3) suggesting a possible novel species. Isolates related to Roseobacter spp. and Sphingobium amiense strain D3AT58 (GenBank accession number JF459959; 97% similarity) were also obtained. Roseobacter spp. are widely associated with corals (Frias-Lopez et al. 2002; Rohwer et al. 2002; Bourne & Munn 2005; Kooperman et al. 2007; Bourne et al. 2008) and are potentially central to the health of corals. Antibacterial activities of Roseobacter have been observed against a wide range of marine pathogens (Hjelm et al. 2004). Coral associated bacteria from this genus have also been previously implicated in the degradation of DMSP (Raina et al. 2009, 2010). Thiotropocin, an antibiotic produced by Roseobacter, is a sulfur compound that might be derived from DMSP metabolism (Wagner-Döbler et al. 2004). These bacteria are suspected to be involved in a symbiotic relationship with coral-cultured zooxanthellae (Raina et al. 2009) which produces high concentrations of DMSP (Hill, Dacey & Krupp 1995; Broadbent, Jones & Jones 2002; Van Alstyne, Schupp & Slattery 2006). It is likely that the occurrence of Roseobacter spp. within the coral mucus may be due to the availability of DMSP produced by the zooxanthellae.

4.3.2 Bacterial strains with PKS and NRPS genes Not all isolates from the coral mucus were capable of amplifying PKS and NRPS gene fragments (see Figure 4.7). It appears that most of the isolates have the potential to produce NRPS compounds (51%) and only 8% have the ability to undergo PKS. Some isolates have the capability to form hybrids (18%) and may play a more important role in coral defence.

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Abundance of PKS and NRPS genes in bacteria from coral mucus

8% 23%

18% 51%

PKS NRPS Both None

Figure 4.7: Relative abundance of PKS and NRPS genes in cultured bacterial communities from coral mucus.

The Gammaproteobacteria, the largest faction within the coral mucus community is the only group with the potential ability to form PKS-NRPS hybrids (see Figure 4.8). The Alphaproteobacteria and Actinobacteria can only produce PKS compounds while Firmicutes appear to be more dominant in NRPS.

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% α-proteobacteria γ-proteobacteria Actinobacteria Firmicutes Unknown

PKS NRPS Both None

Figure 4.8: Presence of PKS and/or NRPS genes in bacterial isolates from coral mucus.

To estimate the ecological role of positive strains as well as their biotechnological potency, inhibitory tests were carried out against other coral associated bacteria.

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4.3.3 Role of mucus-associated bacteria in coral defence The mucus isolates were separated into two groups: mucus attached (11 isolates) and mucus associated (28 isolates).

14 12

10 8 6 4 Total Total inhibitions 2 0 BCM BCM BCM 23 BCM BCM BCM 25 BCM BCM BCM 27BCM 28BCM 29 22-1 22-2 24-1 24-2 26-1 26-2 Samples

28 °C 30 °C 32 °C

Figure 4.9: Total inhibitions of mucus attached bacteria at temperatures of 28, 30 and 32 °C.

12 10 8 6 4

Total Total inhibitions 2 0 BCM31 BCM32 BCM33 BCM34 BCM36 BCM37 BCM38 BCM39 BCM40 BCM41 BCM42 BCM43 BCM 44 BCM 45 BCM46 BCM48 BCM49 BCM50 BCM51 BCM52 BCM53 BCM54 BCM56 BCM57 BCM58 BCM59 BCM35-1 BCM35-2 Samples

28 °C 30 °C 32 °C

Figure 4.10: Total inhibitions of mucus associated bacteria at temperatures of 28, 30 and 32 °C.

The total inhibitions of mucus attached bacteria against all 39 coral mucus isolates increased with temperature (see Figure 4.9). A total of 44 inhibitions occurred at P a g e | 84

28 °C, 67 inhibitions at 30 °C and 69 inhibitions at 32 °C. For coral associated bacteria, inhibition activity is highest at 30 °C (163 inhibitions). This is followed by 141 inhibitions at 28 °C and 107 inhibitions at 30 °C. A few isolates did not show any inhibition at temperatures of 28 °C and/or 32 °C even though there was activity at 30 °C (see Figure 4.10).

1.2

1.0

0.8

0.6

0.4

0.2

0.0 BCM BCM BCM 23 BCM BCM BCM 25 BCM BCM BCM 27BCM 28BCM 29 Average Average zone of inhibition (cm) 22-1 22-2 24-1 24-2 26-1 26-2 Samples

28 °C 30 °C 32 °C

Figure 4.11: Average zone of inhibitions (cm) of mucus attached bacteria at temperatures of 28, 30 and 32 °C.

1.2 1.0 0.8 0.6 0.4 0.2 0.0 Average Average zone of inhibition (cm) BCM31 BCM32 BCM33 BCM34 BCM36 BCM37 BCM38 BCM39 BCM40 BCM41 BCM42 BCM43 BCM44 BCM45 BCM46 BCM48 BCM49 BCM50 BCM51 BCM52 BCM53 BCM54 BCM56 BCM57 BCM58 BCM59 BCM35-1 BCM35-2 Samples

28 °C 30 °C 32 °C

Figure 4.12: Average zone of inhibitions (cm) of mucus associated bacteria at temperatures of 28, 30 and 32 °C.

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Overall, inhibitory activities of the mucus attached bacteria appear to be most effective at 30 °C (see Figure 4.11). The largest inhibitory zone is 1.0 cm at 30 °C by isolate BCM 22-1. The second largest zone (0.9 cm) came from the extracts of BCM 25 (at 28 and 30 °C) and BCM 26-1 (at 32 °C). The inhibitory abilities of coral associated bacteria were observed to become less effective as temperatures increase (see Figure 4.12). Inhibition zones at 28 °C stay between 0.6 to 0.9 cm. Activity were erratic at 30 °C, with the largest zone at 1.0 cm (isolate BCM 35-2) and the smallest at 0.4 cm (isolates BCM 39, 44 and 58). At 32 °C, the zones were between 0.3 to 0.7 cm, with a couple of isolates (BCM 32 and 50) exhibiting larger zones at 0.9 cm.

Mucus attached isolates BCM 22-1 and 26-1 are closely matched with Vibrio parahaemolyticus strain DHC22 (GenBank accession number JQ904733; 99-100 % similarities) while isolate BCM 25 is closely matched with Vibrio harveyi isolate VHJR19 (GenBank accession number DQ995251; 99% similarity). Strains of Vibrio parahaemolyticus and Vibrio harveyi have previously been isolated through a similar method (Ritchie 2006) and were reported to have PKS and NRPS genes and exhibit inhibition activity against other coral-associated and pathogenic bacteria (Radjasa & Sabdono 2003). Other strains Vibrio parahaemolyticus have also been observed to be capable of producing a temperature regulated enzyme, superoxide dismutase (SOD) that detoxifies oxygen radicals which has been suggested to be a key virulence factor in the infection of corals (Banin et al. 2003). Vibrio harveyi strains have also been reported to be antibiotic-resistant (Sussman et al. 2009; Vizcaino et al. 2010) and implicated as part of bacterial consortiums that caused yellow band and black band diseases (Barneah et al. 2007; Cervino et al. 2008), and white plague (Sunagawa et al. 2009) in corals.

Mucus associated isolate BCM 35-2 is closely matched with an uncultured alpha proteobacterium clone FF-20 (GenBank accession number AY682051; 99% similarity). The isolate also showed a positive for NRPS genes. As not much is known about uncultured bacteria, this shows that the mucus regulated medium can be used to isolate previously unculturable bacteria. BCM 32 is closely matched with Bacillus arsenicus strain HLSB44 (GenBank accession number FJ999563; 95% P a g e | 86

similarity) and BCM 50 is closely matched with Brachybacterium paraconglomeratum (GenBank accession number AB362255; 100% similarity). B. arsenicus is an arsenic-resistant bacterium (Shivaji et al. 2005) which can possibly aid the coral against heavy metal pollution. Brachybacterium paraconglomeratum have previously been found in coral tissue (Nithyanand & Pandian 2009) and mucus (Wilson et al. 2012).

The surface of living corals is covered by a mucoid material. This mucopolysacchride layer provides a matrix for bacterial colonization, allowing establishment of a ‘normal bacterial community’ (Ducklow & Mitchell 1979; Ritchie et al. 1994). The normal bacterial flora may produce antimicrobial compounds that help the coral avoid infection by pathogens (Jensen & Fenical 1994). Worldwide coral decline have been attributed to the increase of sea surface temperatures, coastal degradation, pollution, diseases, ecosystem imbalance caused by anthropogenic influences, and the synergistic effect of multiple stressors (Harvell et al. 2002; Rosenberg & Ben-Haim 2002; Sutherland, Porter & Torres 2004). An explanation for the increased incidence of diseases in corals is its stress- induced susceptibility to opportunistic microbes trapped within the SML. It is acknowledged that stress conditions, particularly temperature, can cause certain bacteria to become virulent, by ‘turning on’ virulence genes (Colwell 1996; Patz et al. 1996). The mucus attached bacteria do not undergo mucus regulated selection and are therefore potentially invasive under the right conditions (Ritchie 2006).

In this study, we can conclude that the antibacterial properties of mucus attached and mucus associated bacteria have different optimum temperatures. Mucus associated bacteria work best at 28 °C while the mucus attached bacteria has the potential to take over at 30 °C. The attached bacteria also have more potential to produce bioactive compounds as 27% of them have NRPS genes while 64% of them have both PKS and NRPS genes (see Table 4.3). In contrast, the mucus associated isolates only has PKS (11%) or NRPS genes (61%), making them unable to form hybrids. This indicates the potential strength of the attached community to overcome resident mucus bacteria at elevated temperatures when they turn on their virulence genes. P a g e | 87

It is noteworthy that cultures related to the Vibrio coralliilyticus strain we isolated (98-99 % similarity; see Figure 4.3) is known to cause rapid tissue lysis in the stony coral Pocillopora damicornis by metalloproteinase at elevated temperatures (Ben-Haim et al. 2003; Ben-Haim, Zicherman-Keren & Rosenberg 2003; Rosenberg & Falkovitz 2004). Vibrio shiloi, is the causative agent of bacterial bleaching in the coral Occulina patagonica (Kushmaro et al. 2001). Similar to Vibrio coralliilyticus, it produces a proline-rich peptide that inhibits photosynthesis and a protease that lyses zooxanthellae (Ben-Haim et al. 1999; Banin, Israely, et al. 2000; Rosenberg & Falkovitz 2004).

Two of our Vibrio coralliilyticus -related isolates (BCM 38 and 39) has PKS genes while a third related isolate (BCM 45) has NRPS genes (see Table 4.3). These isolated were isolated from mucus regulated media, indicating that these bacteria are part of the mucus associated environment. Interestingly, these isolates also showed a slight decrease in inhibition activity at 30 and 32 °C (see Figure 4.10). It is possible that these isolates are not virulent strains and are part of the coral defence system at optimum temperatures.

Vibrio shiloi was isolated from reef sediment (BSD 16-11) and was used as a test organism along with the 39 coral mucus isolates. However, no inhibition zones were observed at the three temperatures we tested on, indicating that the isolate is resistant towards the antimicrobial properties of the SML bacteria and may pose a problem at elevated temperatures.

Laboratory studies had previously revealed that coral bleaching occurs when water temperature is increased roughly 1 °C above normal optimum temperatures of 26 to 27 °C during the warmest part of the year (Goreau & Hayes 2008). However, this was not the case for corals in the Talang-talang reef and our results indicate that these corals – and by extension, the bacterial community in the surrounding environment – may have a higher temperature threshold. It is possible that a more obvious demonstration of the antimicrobial properties of coral mucus isolates can be observed at lower and higher temperatures (i.e. ~26 and 34 °C). P a g e | 88

This study shows that the different groups of coral mucus isolates can dominate the SML environment at different periods depending on temperature, and that mucus attached isolates has a high chance of turning virulent against the mucus associated isolates and cause diseases which may lead to bleaching at elevated temperatures.

4.3.4 Bacterial strains with potential to metabolise DMS and/or demethylate DMSP To our knowledge, screening of dmdA and dddP genes in coral SML bacterial communities has not been done before. This preliminary study is part of our effort to understand the importance of bacteria in the region for the local sulphur cycle. Our isolates were not cultured in a method that involves specific selection for DMSP utilisation, therefore any presence of these genes in our isolates is most likely fundamental.

The dddP gene which is responsible for DMS production appears to be most abundant (26%) within the coral mucus bacterial community (see Figure 4.13). Many of our isolates also show potential in undergoing both DMSP degrading pathways as 20% of them have both dmdA and dddP genes.

Abundance of DMSP-degrading genes in bacteria from coral mucus

18% 36%

26%

20%

dmdA dddP Both None

Figure 4.13: Relative abundance of dmdA and dddP genes in cultured bacterial communities from coral mucus.

P a g e | 89

100% 90% 80%

70% 60% 50% 40% 30% Percentage Percentage (%) 20% 10% 0% α-proteobacteria γ-proteobacteria Actinobacteria Firmicutes Unknown

dmdA dddP Both None

Figure 4.14: Presence of dmdA and/or dddP genes in bacterial isolates from coral mucus.

The presence of DMSP degrading genes in the coral mucus bacterial groups is similar to their occurrence in bacterial communities in the Kuching area of the South China Sea (see Chapter 3) where their composition generally mirrored the bacterial community. The Gammaproteobacteria group is the largest identified fraction within the community with the potential for DMSP-assimilation, followed by the Alphaproteobacteria and Firmicutes.

Within the coral mucus, bacteria are extremely dependent on photosynthetic products produced by zooxanthellae which play a role in regulating microbial communities present in corals (Ritchie & Smith 2004). Studies into coral- associated bacteria capable of metabolizing DMSP and DMS have emerged only recently (Raina et al. 2009, 2010). Little information is available and the nature of their interactions with the coral host remains an important research question.

Roseobacter-related strains (BCM 37 and 56; 100% similarity) were isolated from the coral mucus. Both isolates may play a possible role in the the biogeochemical cycling of sulphur within the mucus as they appear to have both DMSP degrading genes. The Roseobacter genus is potentially central to the health of corals. The Roseobacter spp. are widely associated with corals (Frias-Lopez et al. 2002; Rohwer et al. 2002; Bourne & Munn 2005; Kooperman et al. 2007; Bourne et al. P a g e | 90

2008) and suspected to be involved in a symbiotic relationship with zooxanthellae (Raina et al. 2009). An antibiotic produced by Roseobacter, thiotropocin, is a sulphur compound derived from DMSP metabolism (Wagner-Döbler et al. 2004).

4.4 CONCLUSION The bacterial communities at the Talang-talang reef were different according to the environment (coral SML, water column and reef sediment). The coral mucus community is the most diverse with isolates playing potential roles in coral defence, while the community from reef sediment is dominated by potentially pathogenic Vibrio spp.. Two known coral pathogens, Vibrio coralliilyticus and Vibrio shiloi were successfully cultured from the coral reef environment. While the corals are healthy at the time of isolation, these opportunistic pathogens may pose a problem at elevated temperatures.

The coral mucus community also showed high potential in the production of PKS and NRPS compounds. The inhibitory results support the efficiency of PCR screening using specific PKS and NRPS primers, whereby PKS and/or NRPS strains exhibit substantial inhibition activity. Antimicrobial activities of mucus associated bacteria decrease as temperature increase while mucus attached bacteria are most effective at 30 °C. This study also confirms the coral mucus as a regulating media capable of choosing associated communities exhibiting antibacterial properties under optimum conditions.

The preliminary study on the potential role of coral SML bacterial communities in the local sulphur cycle revealed that the presence of DMSP degrading genes in the coral mucus bacterial groups mirrors the general bacterial community where the majority of gene abundance are within the Gammaproteobacteria, indicating a major role for the group. The majority of the SML isolates were observed to have both dmdA and/or dddP genes, showing potential in undergoing both DMSP degrading pathways depending on DMSP availability. Members of the Roseobacter genus which is widely associated with corals and DMSP degrading capabilities were successfully isolated from the coral SML, indicating possible roles (such as?) in the biogeochemical cycling of sulphur within the mucus. P a g e | 91

Further in-depth characterization of these communities through a combination of physical, chemical and molecular biological studies is however needed to improve our understanding of the role of bacteria in coral defence and especially in DMS(P) cycling.

4.5 ACKNOWLEDGEMENTS The authors would like to thank the Sarawak Forestry Department for their kind permission to conduct research at the Talang-Satang National Park (Permit No. NCCD.907.4.4 (Jld.VI)-104 and Park Permit No. 54/2011). Kuek FWI is funded by the Sarawak Foundation’s Tunku Abdul Rahman scholarship. The research leading to these results has received funding from the European Union's Seventh Framework Programme FP7/2007-2013 under grant agreement no. 226224 - SHIVA.

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CHAPTER 5

Summary and Future Work

This study has presented (i) an overview of culturable bacterial communities in waters of the South China Sea, Celebes Sea and a coral reef environment (Talang- talang reef), (ii) the potential roles of these communities in the marine DMS(P) cycle and (iii) the antimicrobial properties of cultured isolates from coral SML at elevated temperatures and their potential role in coral defence.

The bacterial communities in the waters of Kuching and Kota Kinabalu (within the South China Sea) and Semporna (within the Celebes Sea) are almost entirely unknown and have not been sampled by either culture or culture-independent techniques. Members of the Alphaproteobacteria, Gammaproteobacteria and Firmicutes were successfully cultured from all three sampling locations while isolates from Betaproteobacteria were only found in Semporna. Differences in bacterial communities between the three areas can partly be explained by differences in physico-chemical parameters. Kuching is dominated by potentially pathogenic Vibrio spp. possibly due to higher nutrients and riverine input at the sampling locations, while the community at Kota Kinabalu is more indicative of an open ocean environment. Bacterial communities from Kota Kinabalu and Semporna also show potential roles in bioremediation, nitrogen fixing and sulphate reduction.

The bacterial communities at the Talang-talang reef also show variations between environments (coral SML, water column and reef sediment). The isolated community from coral mucus is the most diverse of the three, with members from Actinobacteria, Alphaproteobacteria, Gammaproteobacteria and Firmicutes. Isolates from the SML also indicate potential roles in coral defence with strains related to antibiotic producers with tolerance towards elevated temperatures and heavy metal contamination, while the community from reef sediment is dominated by potentially pathogenic Vibrio spp.. P a g e | 93

Isolates from the SML isolates also displayed a high potential in the production of PKS and NRPS compounds. Strains that contained PKS and/or NRPS genes did exhibit substantial inhibition activity in the well diffusion assay. Antimicrobial properties of mucus associated bacteria were observed to decrease as temperature increase while mucus attached bacteria were most effective at 30 °C. This is an indication that different groups of coral mucus bacteria can dominate the SML environment at different periods depending on temperature, and that opportunistic pathogens can cause diseases which may lead to bleaching at elevated temperatures.

Two known coral pathogens, Vibrio coralliilyticus and Vibrio shiloi were successfully cultured from the coral reef environment, the latter showing resistance against the antimicrobial properties of the mucus associated bacterial community. While the corals are healthy at the time of isolation, these opportunistic pathogens may pose a problem at elevated temperatures.

In both open water and coral reef environments studied, the cultured bacterial communities displayed an abundance of DMSP degrading genes. Communities in this study have either dmdA or dddP or both genes when screened, showing high adaptability in DMS(P) utilisation which we believe is influenced by bacterial carbon and sulphur demands and by DMSP availability.

5.1 Future research Culturing and isolation of bacteria is necessary for detailed studies of physiology and ecological function. Culture-based methods used in this study enables us to further biochemically classify and analyse the bacterial portion of marine environment. Further in-depth characterization of these communities through a combination of physical, chemical and molecular biological studies is needed and will improve our understanding of the role of bacteria in DMS(P), coral defence and their impacts on climate change. Initial clone library from Kuching and Kota Kinabalu showed that culture-independent and cultured bacterial communities are very different, so further molecular-based studies are essential for a more complete assessment of their diversity. P a g e | 94

The use of an assortment of media types and growth condition variables can aid in increasing the diversity of microorganisms recovered by culturing and discovery of other specific properties fundamental to the species. Studies by Vila-Costa et al. 2010 have successfully utilised DMSP enriched media to select for bacteria capable of degrading DMSP into DMS from the natural environment. The approach used in this study did uncover the existence of dmdA and dddP genes in species that were previously involved in DMSP degradation (i.e. Alcaligenes faecalis), confirming their potential role in our waters. However, our understanding of the role of the genes in the various isolates (i.e. gene activity, conditions for ‘bacterial switch’) is limited and further studies are needed to reveal their role in the sulphur cycle. Partial sequencing of the 16S gene is insufficient for a thorough identification of the bacterial isolates; therefore these isolates will require further genetic delineation using gene specific primers.

After final identification it would also be of interest to see if the isolates that are related to Vibrio coralliilyticus and Vibrio shiloi actually do cause diseases on corals; if the disease symptoms differ or even why the corals in our reef are healthy despite enhanced temperatures and existence of potentially pathogenic strains. Furthermore, some of the isolates that have displayed enhanced antibiotic activity at higher temperatures could be tested on corals and see if they develop diseases.

P a g e | 95

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APPENDIX

Table A.1: 16S rRNA gene sequence analysis of bacterial cultures from Kuching 1611, based on BLAST analysis.

GenBank Sequence accession Closest match Identities Phylogenetic division number Gammaproteobacteria; Pseudomonas 1611-S1- 460/460 Pseudomonadales; KF373266 aeruginosa strain 11.2 01-1 (100%) Pseudomonadaceae; [JX286673] Pseudomonas 1611-S1- Vibrio harveyi strain 462/463 Gammaproteobacteria; KF373267 01-1.3 E385 [JX290081] (99%) Vibrionales; Vibrionaceae; Vibrio Gammaproteobacteria; 1611-S1- Pseudomonas sp. 462/462 Pseudomonadales; KF373268 01-2 Mexd38 [JX436405] (100%) Pseudomonadaceae; Pseudomonas Vibrio 1611-S1- parahaemolyticus 475/475 Gammaproteobacteria; KF373269 05-1 strain 448 (100%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Vibrio 1611-S1- parahaemolyticus 476/476 Gammaproteobacteria; KF373270 05-2.1 strain 448 (100%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Vibrio 1611-S1- parahaemolyticus 474/475 Gammaproteobacteria; KF373271 05-2.2 strain 448 (99%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Vibrio 1611-S1- parahaemolyticus 474/475 Gammaproteobacteria; KF373272 05-3 strain 448 (99%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Vibrio alginolyticus 1611-S1- 397/464 Gammaproteobacteria; KF373273 strain XSBZ14 10-1.1.1 (86%) Vibrionales; Vibrionaceae; Vibrio [JX221045] Shewanella chilikensis Gammaproteobacteria; 1611-S1- 461/463 KF373274 strain JC5 Alteromonadales; 10-1.1.2.1 (99%) [HM016088] Shewanellaceae; Shewanella Gammaproteobacteria; 1611-S1- Photobacterium sp. 473/473 KF373275 Vibrionales; Vibrionaceae; 10-1.1.2.2 TKY4 [AB583193] (100%) Photobacterium Bacillus sphaericus 1611-S1- 475/475 Firmicutes; Bacilli; Bacillales; KF373276 isolate BS11 10-1.2.1 (100%) Bacillaceae; Lysinibacillus [AM269451] Gammaproteobacteria; 1611-S1- Oceanimonas sp. 425/462 KF373277 Aeromonadales; 10-2.1 D6083 [FJ161317] (92%) Aeromonadaceae; Oceanimonas Gammaproteobacteria; 1611-S2- Shewanella haliotis 425/426 KF373278 Alteromonadales; 01-1.1.2 strain Z4 [JX286502] (99%) Shewanellaceae; Shewanella 1611-S2- Shewanella haliotis 467/467 Gammaproteobacteria; KF373279 01-2.1 strain Z4 [JX286502] (100%) Alteromonadales; P a g e | 122

Shewanellaceae; Shewanella Shewanella haliotis Gammaproteobacteria; 1611-S2- 463/466 KF373280 strain MS41 Alteromonadales; 01-2.2 (99%) [FN997635] Shewanellaceae; Shewanella Shewanella haliotis Gammaproteobacteria; 1611-S2- 469/469 KF373281 strain MS41 Alteromonadales; 01-2.3 (100%) [FN997635] Shewanellaceae; Shewanella Gammaproteobacteria; 1611-S2- Pseudomonas sp. 461/461 Pseudomonadales; KF373282 01-3.2 Mexd38 [JX436405] (100%) Pseudomonadaceae; Pseudomonas Shewanella haliotis Gammaproteobacteria; 1611-S2- 468/468 KF373283 strain MS41 Alteromonadales; 05-1.1 (100%) [FN997635] Shewanellaceae; Shewanella Vibrio parahaemolyticus 1611-S2- 474/474 Gammaproteobacteria; KF373284 strain 05-1.2 (100%) Vibrionales; Vibrionaceae; Vibrio Aj2010072802A90 [JF432066] Gammaproteobacteria; 1611-S2- Shewanella haliotis 467/467 KF373285 Alteromonadales; 05-2 strain Z4 [JX286502] (100%) Shewanellaceae; Shewanella Gammaproteobacteria; 1611-S2- Oceanimonas sp. 461/461 KF373286 Aeromonadales; 10-1 D6083 [FJ161317] (100%) Aeromonadaceae; Oceanimonas 1611-S2- Bacillus sp. 1-1(2012) 461/461 Firmicutes; Bacilli; Bacillales; KF373287 10-2 [JN942108] (100%) Bacillaceae; Bacillus Vibrio 1611-S4- parahaemolyticus 460/464 Gammaproteobacteria; KF373288 01-1 isolate Mm004 (99%) Vibrionales; Vibrionaceae; Vibrio [FR686998] Gammaproteobacteria; 1611-S4- Oceanimonas sp. 463/463 KF373289 Aeromonadales; 01-1.1 D6083 [FJ161317] (100%) Aeromonadaceae; Oceanimonas Vibrio 1611-S4- parahaemolyticus 471/474 Gammaproteobacteria; KF373290 01-2.1.1 strain 448 (99%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Vibrio natriegens 1611-S4- 458/465 Gammaproteobacteria; KF373291 strain AUCASVE5 01-2.2 (98%) Vibrionales; Vibrionaceae; Vibrio [JQ277719] Vibrio 1611-S4- parahaemolyticus 471/472 Gammaproteobacteria; KF373292 01-3.1 strain 448 (99%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Vibrio 1611-S4- parahaemolyticus 471/473 Gammaproteobacteria; KF373293 01-3.1.2 strain 448 (99%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Vibrio 1611-S4- parahaemolyticus 472/473 Gammaproteobacteria; KF373294 01-3.2 strain 448 (99%) Vibrionales; Vibrionaceae; Vibrio [JN188417] Roseovarius pacificus Alphaproteobacteria; 1611-S4- 405/405 KF373295 strain 81-2 Rhodobacterales; 05-1 (100%) [NR_043564] Rhodobacteraceae; Roseovarius 1611-S4- Roseovarius pacificus 408/408 Alphaproteobacteria; KF373296 05-2 strain 81-2 (100%) Rhodobacterales; P a g e | 123

[NR_043564] Rhodobacteraceae; Roseovarius 1611-S4- Vibrio harveyi isolate 475/477 Gammaproteobacteria; KF373297 05-3 VHJR6 [DQ995240] (99%) Vibrionales; Vibrionaceae; Vibrio 1611-S4- Vibrio azureus strain 473/473 Gammaproteobacteria; KF373298 10-1.2 M2-164 [JQ810832] (100%) Vibrionales; Vibrionaceae; Vibrio Vibrio 1611-S4- parahaemolyticus 477/479 Gammaproteobacteria; KF373299 10-2 strain RW1 (99%) Vibrionales; Vibrionaceae; Vibrio [FJ172044] 1611-S4- Vibrio rotiferianus 469/473 Gammaproteobacteria; KF373300 10-3.1 strain 5S [JF792070] (99%) Vibrionales; Vibrionaceae; Vibrio Vibrio natriegens 1611-S4- 470/473 Gammaproteobacteria; KF373301 strain AUCASVE5 10-3.2 (99%) Vibrionales; Vibrionaceae; Vibrio [JQ277719] 1611-S5- Bacillus subtilis strain 453/475 Firmicutes; Bacilli; Bacillales; KF373302 01-1 y86-7 [FJ460478] (95%) Bacillaceae; Bacillus Vibrio natriegens 1611-S5- 473/473 Gammaproteobacteria; KF373303 strain CM3 05-1 (100%) Vibrionales; Vibrionaceae; Vibrio [EU660320] Vibrio 1611-S5- parahaemolyticus 473/473 Gammaproteobacteria; KF373304 05-3.1 isolate Mm004 (100%) Vibrionales; Vibrionaceae; Vibrio [FR686998] Burzellia piscidermidis 1611-S5- 464/464 KF373305 strain P6-6 Gammaproteobacteria; Burzellia 05-3.2 (100%) [EU127296] Burzellia piscidermidis 1611-S5- 463/464 KF373306 strain P6-6 Gammaproteobacteria; Burzellia 10-1 (99%) [EU127296] 1611-S5- Vibrio azureus strain 472/473 Gammaproteobacteria; KF373307 10-2 F77118 [HQ908716] (99%) Vibrionales; Vibrionaceae; Vibrio Vibrio sinaloensis 1611-S6- 469/475 Gammaproteobacteria; KF373308 strain CAIM 1068 01-1.1 (99%) Vibrionales; Vibrionaceae; Vibrio [HM584056] Pseudoalteromonas Gammaproteobacteria; 1611-S6- maricaloris strain 458/458 Alteromonadales; KF373309 01-1.2 KMM636 (100%) Pseudoalteromonadaceae; [NR_025009] Pseudoalteromonas Rhodobacteraceae Alphaproteobacteria; 1611-S6- 360/375 KF373310 bacterium SCSWE04 Rhodobacterales; 01-2 (96%) [FJ461471] Rhodobacteraceae Gammaproteobacteria; Stenotrophomonas Xanthomonadales; 1611-S6- maltophilia strain 471/471 Xanthomonadaceae; KF373311 05-1.1 BQAPs-03d (100%) Stenotrophomonas; [FJ217200] Stenotrophomonas maltophilia group 1611-S6- Vibrio harveyi isolate 472/475 Gammaproteobacteria; KF373312 05-1.2 VHJR6 [DQ995240] (99%) Vibrionales; Vibrionaceae; Vibrio 1611-S6- Vibrio harveyi isolate 474/475 Gammaproteobacteria; KF373313 05-2.1 VHJR14 [EF011651] (99%) Vibrionales; Vibrionaceae; Vibrio 1611-S6- Vibrio harveyi strain 475/475 Gammaproteobacteria; KF373314 05-2.2 HL19 [JQ948038] (100%) Vibrionales; Vibrionaceae; Vibrio 1611-S6- Vibrio harveyi isolate 468/472 Gammaproteobacteria; KF373315 05-3.2 VHJR6 [DQ995240] (99%) Vibrionales; Vibrionaceae; Vibrio 1611-S6- Vibrio harveyi isolate 471/472 Gammaproteobacteria; KF373316 10-1.1 VHJR6 [DQ995240] (99%) Vibrionales; Vibrionaceae; Vibrio 1611-S6- KF373317 Vibrio harveyi isolate 477/477 Gammaproteobacteria; P a g e | 124

10-1.2 VHJR12 [DQ995245] (100%) Vibrionales; Vibrionaceae; Vibrio Vibrio campbellii 1611-S6- 475/476 Gammaproteobacteria; KF373318 strain CAIM 886 10-2 (99%) Vibrionales; Vibrionaceae; Vibrio [HM584033]

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Table A.2: 16S rRNA gene sequence analysis of bacterial cultures from Kuching 1911, based on BLAST analysis.

GenBank Sequence accession Closest match Identities Phylogenetic division number Vibrio orientalis strain Gammaproteobacteria; 1911-S1- 465/468 KF373319 JC97, isolate Pkl-17 Vibrionales; Vibrionaceae; 01-1.2.1 (99%) [FR837599] Vibrio Rhodobacter Alphaproteobacteria; 1911-S1- 434/440 KF373320 capsulatus strain PSB- Rhodobacterales; 01-1.2.2 (99%) 06 [FJ866784] Rhodobacteraceae; Rhodobacter Rhodobacter Alphaproteobacteria; 1911-S1- 440/455 KF373321 capsulatus strain PSB- Rhodobacterales; 01-2 (97%) 06 [FJ866784] Rhodobacteraceae; Rhodobacter Rhodobacter Alphaproteobacteria; 1911-S1- 433/440 KF373322 capsulatus strain PSB- Rhodobacterales; 01-3 (98%) 06 [FJ866784] Rhodobacteraceae; Rhodobacter Gammaproteobacteria; Pseudomonas 1911-S1- 456/457 Pseudomonadales; KF373323 oleovorans strain 05-2 (99%) Pseudomonadaceae; HNS030 [JN128264] Pseudomonas; Pseudomonas Gammaproteobacteria; Pseudomonas 1911-S1- 459/460 Pseudomonadales; KF373324 oleovorans strain 07-1 (99%) Pseudomonadaceae; HNS030 [JN128264] Pseudomonas; Pseudomonas Vibrio alginolyticus Gammaproteobacteria; 1911-S2- 476/478 KF373325 isolate Va150 Vibrionales; Vibrionaceae; 01-1 (99%) [EU155497] Vibrio Vibrio alginolyticus Gammaproteobacteria; 1911-S2- 471/474 KF373326 strain HZBC71 Vibrionales; Vibrionaceae; 05-1 (99%) [JN188402] Vibrio Vibrio alginolyticus Gammaproteobacteria; 1911-S2- 473/475 KF373327 strain HZBC71 Vibrionales; Vibrionaceae; 07-1 (99%) [JN188402] Vibrio Vibrio Gammaproteobacteria; 1911-S2- parahaemolyticus 471/473 KF373328 Vibrionales; Vibrionaceae; 07-2 isolate Vp481 (99%) Vibrio [EU155540] Gammaproteobacteria; Pseudoalteromonas 1911-S3- 464/465 Alteromonadales; KF373329 ganghwensis 01-1.1.1 (99%) Pseudoalteromonadaceae; [DQ011614] Pseudoalteromonas Vibrio Gammaproteobacteria; 1911-S3- parahaemolyticus 319/402 KF373330 Vibrionales; Vibrionaceae; 01-1.1.2 strain VPMP55 (79%) Vibrio [JQ663925] Vibrio alginolyticus Gammaproteobacteria; 1911-S3- 474/475 KF373331 strain P61224 Vibrionales; Vibrionaceae; 01-1.2 (99%) [AJ704375] Vibrio Vibrio diabolicus strain Gammaproteobacteria; 1911-S3- 475/478 KF373332 KM30-12-3 Vibrionales; Vibrionaceae; 01-2 (99%) [JQ670740] Vibrio Vibrio Gammaproteobacteria; 1911-S3- parahaemolyticus 474/476 KF373333 Vibrionales; Vibrionaceae; 05-1 strain 93A-5807 (99%) Vibrio [DQ497398] 1911-S3- KF373334 Vibrio 470/473 Gammaproteobacteria; P a g e | 126

05-2 parahaemolyticus (99%) Vibrionales; Vibrionaceae; strain 93A-5807 Vibrio [DQ497398] Gammaproteobacteria; 1911-S3- Vibrio harveyi strain 473/474 KF373335 Vibrionales; Vibrionaceae; 10-1.1 IS01 [GU974342] (99%) Vibrio Vibrio campbellii Gammaproteobacteria; 1911-S3- 473/475 KF373336 strain CAIM 886 Vibrionales; Vibrionaceae; 10-1.2 (99%) [HM584033] Vibrio Gammaproteobacteria; 1911-S3- Vibrio rotiferianus 475/478 KF373337 Vibrionales; Vibrionaceae; 10-2.1 strain BV1 [JN391272] (99%) Vibrio Gammaproteobacteria; Pseudoalteromonas 1911-S4- 462/463 Alteromonadales; KF373338 ganghwensis 01-1 (99%) Pseudoalteromonadaceae; [DQ011614] Pseudoalteromonas Vibrio alginolyticus Gammaproteobacteria; 1911-S4- 474/475 KF373339 strain H050815-1 Vibrionales; Vibrionaceae; 01-1.1 (99%) [EF219054] Vibrio Alphaproteobacteria; Thalassospira 1911-S4- 411/416 Rhodospirillales; KF373340 xiamenensis strain 01-2.2 (99%) Rhodospirillaceae; PTG4-18 [EU603449] Thalassospira Citrobacter freundii Gammaproteobacteria; 1911-S4- 461/462 KF373341 strain AIMST Ehe5 Enterobacteriales; 05-1.1 (99%) [JQ312038] Enterobacteriaceae; Citrobacter Leclercia Gammaproteobacteria; 1911-S4- adecarboxylata strain 461/462 KF373342 Enterobacteriales; 05-1.2 AIMST Ehe6 (99%) Enterobacteriaceae; Leclercia [JQ312039] Gammaproteobacteria; 1911-S4- Vibrio azureus strain 452/468 KF373343 Vibrionales; Vibrionaceae; 05-2 41113 [HM032787] (97%) Vibrio Vibrio alginolyticus Gammaproteobacteria; 1911-S4- 472/473 KF373344 strain H050815-1 Vibrionales; Vibrionaceae; 10-2.1 (99%) [EF219054] Vibrio Vibrio natriegens Gammaproteobacteria; 1911-S5- 471/472 KF373345 strain AUCASVE1 Vibrionales; Vibrionaceae; 01-1 (99%) [JQ043186] Vibrio Vibrio natriegens Gammaproteobacteria; 1911-S5- 474/475 KF373346 strain AUCASVE1 Vibrionales; Vibrionaceae; 01-2.1 (99%) [JQ043186] Vibrio Vibrio natriegens Gammaproteobacteria; 1911-S5- 472/473 KF373347 strain AUCASVE1 Vibrionales; Vibrionaceae; 01-2.2 (99%) [JQ043186] Vibrio Citrobacter freundii Gammaproteobacteria; 1911-S5- 462/463 KF373348 strain AIMST Ehe5 Enterobacteriales; 05-1.1.2 (99%) [JQ312038] Enterobacteriaceae; Citrobacter Vibrio natriegens Gammaproteobacteria; 1911-S5- 472/473 KF373349 strain AUCASVE1 Vibrionales; Vibrionaceae; 05-1.2 (99%) [JQ043186] Vibrio Gammaproteobacteria; 1911-S5- Vibrio azureus strain 473/473 KF373350 Vibrionales; Vibrionaceae; 05-1.2.1 F77118 [HQ908716] (100%) Vibrio Vibrio Gammaproteobacteria; 1911-S5- 474/475 KF373351 parahaemolyticus Vibrionales; Vibrionaceae; 05-2 (99%) strain 448 [JN188417] Vibrio 1911-S5- KF373352 Vibrio natriegens 471/472 Gammaproteobacteria; P a g e | 127

05-3 strain AUCASVE1 (99%) Vibrionales; Vibrionaceae; [JQ043186] Vibrio Gammaproteobacteria; 1911-S5- Vibrio azureus strain 471/474 KF373353 Vibrionales; Vibrionaceae; 10-1 41113 [HM032787] (99%) Vibrio Vibrio splendidus Gammaproteobacteria; 1911-S5- 469/471 KF373354 strain AP625 Vibrionales; Vibrionaceae; 10-2 (99%) [GQ254509] Vibrio

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Table A.3: 16S rRNA gene sequence analysis of bacterial cultures from Kota Kinabalu, based on BLAST analysis.

GenBank Sequence accession Closest match Identities Phylogenetic division number Gammaproteobacteria; Pseudomonas 2311-S1- 452/453 Pseudomonadales; KF373355 oleovorans strain 01-1.1 (99%) Pseudomonadaceae; HNS030 [JN128264] Pseudomonas; Pseudomonas Shewanella haliotis Gammaproteobacteria; 2311-S1- 461/461 KF373356 strain MS41 Alteromonadales; 01-1.2 (100%) [FN997635] Shewanellaceae; Shewanella Shewanella haliotis Gammaproteobacteria; 2311-S1- 469/469 KF373357 strain MS41 Alteromonadales; 01-2.1 (100%) [FN997635] Shewanellaceae; Shewanella Shewanella haliotis Gammaproteobacteria; 2311-S1- 467/467 KF373358 strain MS41 Alteromonadales; 01-2.2 (100%) [FN997635] Shewanellaceae; Shewanella Shewanella haliotis Gammaproteobacteria; 2311-S1- 466/466 KF373359 strain MS41 Alteromonadales; 01-3.1 (100%) [FN997635] Shewanellaceae; Shewanella Exiguobacterium Firmicutes; Bacilli; Bacillales; 2311-S1- 485/485 KF373360 aurantiacum var. Colo. Bacillales Family XII. Incertae 05-1 (100%) Road [AY047481] Sedis; Exiguobacterium Oceanimonas smirnovii Gammaproteobacteria; 2311-S1- 442/461 KF373361 strain 31-13 Aeromonadales; 05-2 (96%) [NR_042963] Aeromonadaceae; Oceanimonas Gammaproteobacteria; 2311-S1- Vibrio rotiferianus 466/470 KF373362 Vibrionales; Vibrionaceae; 10-1 strain 5S [JF792070] (99%) Vibrio Brevibacillus 2311-S2- 472/472 Firmicutes; Bacilli; Bacillales; KF373363 laterosporus strain 01-1 (100%) Paenibacillaceae; Brevibacillus GZUB11 [FJ434663] Vibrio splendidus Gammaproteobacteria; 2311-S2- 414/453 KF373364 strain AP625 Vibrionales; Vibrionaceae; 10-1 (91%) [GQ254509] Vibrio Bacillus sphaericus 2311-S3- 431/456 Firmicutes; Bacilli; Bacillales; KF373365 clone 7-16 01-1.1 (95%) Bacillaceae; Lysinibacillus [DQ364585] Shewanella Gammaproteobacteria; 2311-S3- 455/461 KF373366 putrefaciens strain Alteromonadales; 01-1.2 (99%) R1418 [AB208055] Shewanellaceae; Shewanella Shewanella Gammaproteobacteria; 2311-S3- 459/462 KF373367 putrefaciens strain Alteromonadales; 01-2 (99%) R1418 [AB208055] Shewanellaceae; Shewanella Gammaproteobacteria; 2311-S3- Vibrio vulnificus strain 473/473 KF373368 Vibrionales; Vibrionaceae; 01-3 W045 [EF114147] (100%) Vibrio Gammaproteobacteria; Enterobacter ludwigii 2311-S3- 463/463 Enterobacteriales; KF373369 strain KW 93 05-1 (100%) Enterobacteriaceae; [JX262395] Enterobacter Pseudomonas Gammaproteobacteria; 2311-S3- plecoglossicida strain 459/459 Pseudomonadales; KF373370 05-2.1 AIMST Aie20 (100%) Pseudomonadaceae; [JQ312025] Pseudomonas 2311-S3- KF373371 Thalassospira sp. SKUK 417/417 Alphaproteobacteria; P a g e | 129

10-1 MB1005 [EU907920] (100%) Rhodospirillales; Rhodospirillaceae; Thalassospira Bacillus malacitensis 2311-S3- 404/408 Firmicutes; Bacilli; Bacillales; KF373372 strain TP12 10-2.1 (99%) Bacillaceae; Bacillus [FJ887890] Vibrio natriegens Gammaproteobacteria; 2311-S3- 471/472 KF373373 strain AUCASVE5 Vibrionales; Vibrionaceae; 10-2.2 (99%) [JQ277719] Vibrio Providencia sp. Gammaproteobacteria; 2311-S4- 456/460 KF373374 Sam130-9A Enterobacteriales; 01-1 (99%) [FJ418577] Enterobacteriaceae; Providencia Nitratireductor Alphaproteobacteria; 2311-S4- 409/409 KF373375 basaltis strain J3 Rhizobiales; Phyllobacteriaceae; 05-1 (100%) [NR_044414] Nitratireductor Oceanimonas smirnovii Gammaproteobacteria; 2311-S4- 463/468 KF373376 strain 31-13 Aeromonadales; 10-1 (99%) [NR_042963] Aeromonadaceae; Oceanimonas Oceanimonas smirnovii Gammaproteobacteria; 2311-S4- 463/468 KF373377 strain 31-13 Aeromonadales; 10-2.1.1 (99%) [NR_042963] Aeromonadaceae; Oceanimonas Lysinibacillus 2311-S4- 476/476 Firmicutes; Bacilli; Bacillales; KF373378 fusiformis strain R3 10-2.1.3 (100%) Bacillaceae; Lysinibacillus [JQ991002] Exiguobacterium Firmicutes; Bacilli; Bacillales; 2311-S4- 489/490 KF373379 aurantiacum var. Colo. Bacillales Family XII. Incertae 10-2.2 (99%) Road [AY047481] Sedis; Exiguobacterium Oceanimonas smirnovii Gammaproteobacteria; 2311-S4- 460/465 KF373380 strain 31-13 Aeromonadales; 10-2.3 (99%) [NR_042963] Aeromonadaceae; Oceanimonas Gammaproteobacteria; 2311-S4- Vibrio vulnificus strain 475/475 KF373381 Vibrionales; Vibrionaceae; 18-1.1 W045 [EF114147] (100%) Vibrio Oceanimonas smirnovii Gammaproteobacteria; 2311-S4- 436/447 KF373382 strain 31-13 Aeromonadales; 18-1.2 (98%) [NR_042963] Aeromonadaceae; Oceanimonas Gammaproteobacteria; Pseudoalteromonas 2311-S5- 464/465 Alteromonadales; KF373383 lipolytica strain ZR064 01-1.2 (99%) Pseudoalteromonadaceae; [JX173567] Pseudoalteromonas Gammaproteobacteria; Pseudoalteromonas 2311-S5- 463/463 Alteromonadales; KF373384 lipolytica strain ZR064 01-2.1 (100%) Pseudoalteromonadaceae; [JX173567] Pseudoalteromonas Gammaproteobacteria; Pseudomonas stutzeri 2311-S5- 453/454 Pseudomonadales; KF373385 strain UP-1 01-2.2 (99%) Pseudomonadaceae; [AY364327] Pseudomonas Gammaproteobacteria; Pseudomonas stutzeri 2311-S5- 458/459 Pseudomonadales; KF373386 strain UP-1 01-2.3 (99%) Pseudomonadaceae; [AY364327] Pseudomonas Alphaproteobacteria; Brevundimonas 2311-S5- 405/406 Caulobacterales; KF373387 diminuta strain c138 01-3.1.1 (99%) Caulobacteraceae; [FJ950570] Brevundimonas 2311-S5- Exiguobacterium 438/479 Firmicutes; Bacilli; Bacillales; KF373388 01-3.1.2 arabatum [JF758868] (91%) Bacillales Family XII. Incertae P a g e | 130

Sedis; Exiguobacterium Alphaproteobacteria; Brevundimonas 2311-S5- 407/407 Caulobacterales; KF373389 diminuta strain 01-3.2 (100%) Caulobacteraceae; KSC_AK3a [EF191247] Brevundimonas Vibrio natriegens Gammaproteobacteria; 2311-S5- 472/472 KF373390 strain AUCASVE5 Vibrionales; Vibrionaceae; 01B-1 (100%) [JQ277719] Vibrio Vibrio splendidus Gammaproteobacteria; 2311-S5- 472/473 KF373391 strain AP625 Vibrionales; Vibrionaceae; 05-1 (99%) [GQ254509] Vibrio Vibrio splendidus Gammaproteobacteria; 2311-S5- 470/472 KF373392 strain AP625 Vibrionales; Vibrionaceae; 05-2 (99%) [GQ254509] Vibrio

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Table A.4: 16S rRNA gene sequence analysis of bacterial cultures from Semporna, based on BLAST analysis.

GenBank Sequence accession Closest match Identities Phylogenetic division number Alcaligenes faecalis Betaproteobacteria; 2611-S1- 410/465 KF373393 strain OCEN2DBT Burkholderiales; 01-1.1 (88%) [JF264463] Alcaligenaceae; Alcaligenes Gammaproteobacteria; 2611-S1- Vibrio communis strain 472/472 KF373394 Vibrionales; Vibrionaceae; 01-1.2 F75216 [HQ161743] (100%) Vibrio Exiguobacterium Firmicutes; Bacilli; Bacillales; 2611-S1- lactigenes strain: 483/483 KF373395 Bacillales Family XII. Incertae 05-1.1 HYS0503-MK66 (100%) Sedis; Exiguobacterium [AB259161] Oceanimonas smirnovii Gammaproteobacteria; 2611-S1- 463/468 KF373396 strain 31-13 Aeromonadales; 05-1.2 (99%) [NR_042963] Aeromonadaceae; Oceanimonas Gammaproteobacteria; 2611-S2- Vibrio natriegens strain 473/475 KF373397 Vibrionales; Vibrionaceae; 01-1 CM3 [EU660320] (99%) Vibrio Gammaproteobacteria; 2611-S2- Vibrio furnissii strain 451/473 KF373398 Vibrionales; Vibrionaceae; 01-3 MM5 [FJ906812] (95%) Vibrio Allomonas enterica Gammaproteobacteria; 2611-S2- 473/475 KF373399 strain JC74, isolate R2A Vibrionales; Vibrionaceae; 05-1.1 (99%) [FR837595] Allomonas Allomonas enterica Gammaproteobacteria; 2611-S2- 476/478 KF373400 strain JC74, isolate R2A Vibrionales; Vibrionaceae; 05-1.2 (99%) [FR837595] Allomonas Gammaproteobacteria; Bowmanella 2611-S2- 448/459 Alteromonadales; KF373401 denitrificans strain BD1 05-2.2 (98%) Alteromonadaceae; [NR_043738] Bowmanella Allomonas enterica Gammaproteobacteria; 2611-S2- 470/474 KF373402 strain JC74, isolate R2A Vibrionales; Vibrionaceae; 05-3 (99%) [FR837595] Allomonas Gammaproteobacteria; Pseudomonas 2611-S2- 459/459 Pseudomonadales; KF373403 plecoglossicida strain 10-2 (100%) Pseudomonadaceae; R8-591-1 [JQ659971] Pseudomonas Gammaproteobacteria; 2611-S3- Vibrio splendidus strain 472/473 KF373404 Vibrionales; Vibrionaceae; 01-1 AP625 [GQ254509] (99%) Vibrio Vibrio alginolyticus Gammaproteobacteria; 2611-S3- 472/472 KF373405 strain XHS1-3 Vibrionales; Vibrionaceae; 01-2.2 (100%) [JN188407] Vibrio Oceanimonas smirnovii Gammaproteobacteria; 2611-S3- 449/464 KF373406 strain 31-13 Aeromonadales; 05-1 (97%) [NR_042963] Aeromonadaceae; Oceanimonas Gammaproteobacteria; 2611-S4- Vibrio harveyi isolate 473/473 KF373407 Vibrionales; Vibrionaceae; 01-1 VHJR19 [DQ995251] (100%) Vibrio Gammaproteobacteria; 2611-S4- Vibrio rotiferianus 471/474 KF373408 Vibrionales; Vibrionaceae; 01-2 strain BV1 [JN391272] (99%) Vibrio 2611-S4- KF373409 Pseudomonas fulva 458/458 Gammaproteobacteria; P a g e | 132

01-2.1 strain SMA24 (100%) Pseudomonadales; [JQ618288] Pseudomonadaceae; Pseudomonas Gammaproteobacteria; 2611-S4- Vibrio harveyi isolate 470/472 KF373410 Vibrionales; Vibrionaceae; 01-2.2 VHJR19 [DQ995251] (99%) Vibrio Pseudidiomarina Gammaproteobacteria; 2611-S4- 440/461 KF373411 sediminum strain c121 Alteromonadales; 01-4 (95%) [NR_044176] Idiomarinaceae; Idiomarina Pseudomonas Gammaproteobacteria; 2611-S4- pseudoalcaligenes 460/461 Pseudomonadales; KF373412 01A-2 strain K29411 (99%) Pseudomonadaceae; [DQ298030] Pseudomonas; Pseudomonas Allomonas enterica Gammaproteobacteria; 2611-S4- 472/473 KF373413 strain JC74, isolate R2A Vibrionales; Vibrionaceae; 01B-1 (99%) [FR837595] Allomonas Gammaproteobacteria; 2611-S4- Shewanella sp. 428/428 KF373414 Alteromonadales; 01B-2.1 UMS11/10 [JQ231163] (100%) Shewanellaceae; Shewanella Oceanimonas smirnovii Gammaproteobacteria; 2611-S4- 454/459 KF373415 strain 31-13 Aeromonadales; 01B-2.2 (99%) [NR_042963] Aeromonadaceae; Oceanimonas Exiguobacterium Firmicutes; Bacilli; Bacillales; 2611-S4- 474/476 KF373416 profundum strain Bacillales Family XII. Incertae 01B-3 (99%) SigaKolEp3 [JX987048] Sedis; Exiguobacterium Allomonas enterica Gammaproteobacteria; 2611-S4- 474/475 KF373417 strain JC74, isolate R2A Vibrionales; Vibrionaceae; 01C-1 (99%) [FR837595] Allomonas Allomonas enterica Gammaproteobacteria; 2611-S4- 472/473 KF373418 strain JC74, isolate R2A Vibrionales; Vibrionaceae; 01C-2 (99%) [FR837595] Allomonas 2611-S4- Bacillus cereus strain 329/433 Firmicutes; Bacilli; Bacillales; KF373419 05-2 14B [JX901104] (76%) Bacillaceae; Bacillus Gammaproteobacteria; 2611-S4- Shewanella sp. 460/468 KF373420 Alteromonadales; 06A-1 UMS11/10 [JQ231163] (98%) Shewanellaceae; Shewanella Pseudidiomarina Gammaproteobacteria; 2611-S5- 423/463 KF373421 sediminum strain c121 Alteromonadales; 01-1 (91%) [NR_044176] Idiomarinaceae; Idiomarina Pseudomonas Gammaproteobacteria; 2611-S5- pseudoalcaligenes 437/438 Pseudomonadales; KF373422 05A-1 strain K29411 (99%) Pseudomonadaceae; [DQ298030] Pseudomonas; Pseudomonas Gammaproteobacteria; 2611-S5- Pseudoalteromonas sp. 465/466 Alteromonadales; KF373423 05B-1.1 S187 [FJ457123] (99%) Pseudoalteromonadaceae; Pseudoalteromonas Gammaproteobacteria; 2611-S5- Photobacterium sp. 473/473 KF373424 Vibrionales; Vibrionaceae; 05B-1.2 MM14 [JN791371] (100%) Photobacterium Gammaproteobacteria; 2611-S5- Shewanella sp. 460/465 KF373425 Alteromonadales; 05B-3.2.1 UMS11/10 [JQ231163] (99%) Shewanellaceae; Shewanella Gammaproteobacteria; 2611-S5- Shewanella sp. 464/465 KF373426 Alteromonadales; 05B-3.2.2 UMS11/10 [JQ231163] (99%) Shewanellaceae; Shewanella 2611-S5- Photobacterium sp. 477/477 Gammaproteobacteria; KF373427 05C-2 MM14 [JN791371] (100%) Vibrionales; Vibrionaceae; P a g e | 133

Photobacterium Nitratireductor Alphaproteobacteria; 2611-S5- 404/406 KF373428 aquimarinus CL-SC21 Rhizobiales; Phyllobacteriaceae; 10-2 (99%) [HQ176467] Nitratireductor Pseudomonas Gammaproteobacteria; 2611-S6- pseudoalcaligenes 347/410 Pseudomonadales; KF373429 01-1 strain K29411 (85%) Pseudomonadaceae; [DQ298030] Pseudomonas; Pseudomonas Pseudomonas Gammaproteobacteria; 2611-S6- pseudoalcaligenes 450/450 Pseudomonadales; KF373430 01-1.1 strain K29411 (100%) Pseudomonadaceae; [DQ298030] Pseudomonas; Pseudomonas Gammaproteobacteria; 2611-S6- Vibrio campbellii strain 463/467 KF373431 Vibrionales; Vibrionaceae; 01-1.2 CAIM 886 [HM584033] (99%) Vibrio Gammaproteobacteria; 2611-S6- Vibrio alginolyticus 475/475 KF373432 Vibrionales; Vibrionaceae; 01-3 strain 486 [JN188409] (100%) Vibrio Oceanimonas smirnovii Gammaproteobacteria; 2611-S6- 445/449 KF373433 strain 31-13 Aeromonadales; 05-1.1 (99%) [NR_042963] Aeromonadaceae; Oceanimonas Alphaproteobacteria; Rhodobacter capsulatus 2611-S6- 468/468 Rhodobacterales; KF373434 strain PSB-06 05-2 (100%) Rhodobacteraceae; [FJ866784] Rhodobacter Vibrio Gammaproteobacteria; 2611-S6- 474/475 KF373435 parahaemolyticus Vibrionales; Vibrionaceae; 09-1 (99%) strain 448 [JN188417] Vibrio Vibrio Gammaproteobacteria; 2611-S6- 471/472 KF373436 parahaemolyticus Vibrionales; Vibrionaceae; 09-2 (99%) strain 448 [JN188417] Vibrio Vibrio Gammaproteobacteria; 2611-S7- 475/476 KF373437 parahaemolyticus Vibrionales; Vibrionaceae; 01-1 (99%) strain 448 [JN188417] Vibrio Vibrio parahaemolyticus Gammaproteobacteria; 2611-S7- 475/475 KF373438 strain S9-891- Vibrionales; Vibrionaceae; 01-2 (100%) B0919354-5-8F Vibrio [KC520577] Gammaproteobacteria; 2611-S8- Vibrio alginolyticus 471/472 KF373439 Vibrionales; Vibrionaceae; 01-1.1 strain 486 [JN188409] (99%) Vibrio Gammaproteobacteria; 2611-S8- Vibrio communis strain 474/474 KF373440 Vibrionales; Vibrionaceae; 01-3 F75216 [HQ161743] (100%) Vibrio

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Table A.5: 16S rRNA gene sequence analysis of bacterial cultures from Talang- talang reef and its surrounding waters, based on BLAST analysis.

GenBank Sequence accession Closest match Identities Phylogenetic division number Gammaproteobacteria; Vibrio parahaemolyticus 451/451 BCM 22-1 KF373441 Vibrionales; Vibrionaceae; strain DHC22 [JQ904733] (100%) Vibrio Gammaproteobacteria; Vibrio harveyi strain 459/467 BCM 22-2 KF373442 Vibrionales; Vibrionaceae; F75032 [HQ161747] (98%) Vibrio Gammaproteobacteria; Vibrio parahaemolyticus 418/418 BCM 23 KF373443 Vibrionales; Vibrionaceae; strain DHC22 [JQ904733] (100%) Vibrio Gammaproteobacteria; Vibrio harveyi isolate 475/475 BCM 24-1 KF373444 Vibrionales; Vibrionaceae; VHJR19 [DQ995251] (100%) Vibrio Gammaproteobacteria; Vibrio harveyi isolate 472/472 BCM 24-2 KF373445 Vibrionales; Vibrionaceae; VHJR19 [DQ995251] (100%) Vibrio Gammaproteobacteria; Vibrio harveyi isolate 474/475 BCM 25 KF373446 Vibrionales; Vibrionaceae; VHJR19 [DQ995251] (99%) Vibrio Gammaproteobacteria; Vibrio parahaemolyticus 472/474 BCM 26-1 KF373447 Vibrionales; Vibrionaceae; strain DHC22 [JQ904733] (99%) Vibrio Gammaproteobacteria; Vibrio harveyi isolate 472/475 BCM 26-2 KF373448 Vibrionales; Vibrionaceae; VHJR19 [DQ995251] (99%) Vibrio Gammaproteobacteria; Vibrio harveyi isolate 475/476 BCM 27 KF373449 Vibrionales; Vibrionaceae; VHJR12 [DQ995245] (99%) Vibrio Gammaproteobacteria; Vibrio azureus strain M2- 472/473 BCM 28 KF373450 Vibrionales; Vibrionaceae; 164 [JQ810832] (99%) Vibrio Gammaproteobacteria; Halomonas aquamarina 460/460 Oceanospirillales; BCM 29 KF373451 strain Ve1-10-83 (100%) Halomonadaceae; [EU684464] Halomonas Bacillus arsenicus strain 455/466 Firmicutes; Bacillales; BCM 31 KF373452 HLSB44 [FJ999563] (98%) Bacillaceae; Bacillus Bacillus arsenicus strain 444/468 Firmicutes; Bacillales; BCM 32 KF373453 HLSB44 [FJ999563] (95%) Bacillaceae; Bacillus Uncultured alpha 382/384 Alphaproteobacteria; BCM 33 KF373454 proteobacterium clone (99%) environmental samples FF-20 [AY682051] Gammaproteobacteria; Psychrobacter celer 453/454 Pseudomonadales; BCM 34 KF373455 strain K-W15 [JQ799068] (99%) Moraxellaceae; Psychrobacter Mucus bacterium 108 377/406 BCM 35-1 KF373456 Bacteria [AY654761] (93%) Uncultured alpha 382/385 Alphaproteobacteria; BCM 35-2 KF373457 proteobacterium clone (99%) environmental samples FF-20 [AY682051] BCM 36 KF373458 Staphylococcus 474/474 Firmicutes; Bacilli; P a g e | 135

lugdunensis strain NBL01 (100%) Bacillales; Staphylococcus [JX629460] Alphaproteobacteria; Roseobacter sp. H454 406/406 Rhodobacterales; BCM 37 KF373459 [AY368572] (100%) Rhodobacteraceae; Roseobacter Vibrio coralliilyticus Gammaproteobacteria; 467/472 BCM 38 KF373460 strain LMG 21349 Vibrionales; Vibrionaceae; (99%) [AJ440004] Vibrio Vibrio coralliilyticus Gammaproteobacteria; 472/477 BCM 39 KF373461 strain LMG 21349 Vibrionales; Vibrionaceae; (99%) [AJ440004] Vibrio Gammaproteobacteria; Vibrio brasiliensis strain 472/477 BCM 40 KF373462 Vibrionales; Vibrionaceae; HQSB7 [JF721971] (99%) Vibrio Gammaproteobacteria; Vibrio parahaemolyticus 470/472 BCM 41 KF373463 Vibrionales; Vibrionaceae; strain DHC22 [JQ904733] (99%) Vibrio Gammaproteobacteria; Vibrio parahaemolyticus 478/480 BCM 42 KF373464 Vibrionales; Vibrionaceae; strain DHC22 [JQ904733] (99%) Vibrio Gammaproteobacteria; Klebsiella oxytoca strain 458/460 Enterobacteriales; BCM 43 KF373465 AIMST 10.Pl.3 (99%) Enterobacteriaceae; [HQ683968] Klebsiella Gammaproteobacteria; Microbulbifer sp. 2ta13 455/455 Alteromonadales; BCM 44 KF373466 [FJ952779] (100%) Alteromonadaceae; Microbulbifer Vibrio coralliilyticus Gammaproteobacteria; 470/479 BCM 45 KF373467 strain LMG 21349 Vibrionales; Vibrionaceae; (98%) [AJ440004] Vibrio Gammaproteobacteria; Vibrio harveyi strain 469/472 BCM 46 KF373468 Vibrionales; Vibrionaceae; F75087 [HQ161750] (99%) Vibrio Photobacterium jeanii Gammaproteobacteria; 471/477 BCM 48 KF373469 strain R-21419 Vibrionales; Vibrionaceae; (99%) [GU065212] Photobacterium Actinobacteria; Actinobacteridae; Kocuria rosea strain 442/446 BCM 49 KF373470 Actinomycetales; CT22 [EU660350] (99%) Micrococcineae; Micrococcaceae; Kocuria Actinobacteria; Actinobacteridae; Brachybacterium 441/441 Actinomycetales; BCM 50 KF373471 paraconglomeratum (100%) Micrococcineae; [AB362255] Dermabacteraceae; Brachybacterium Actinobacteria; Actinobacteridae; Kocuria rosea strain 441/445 BCM 51 KF373472 Actinomycetales; CT22 [EU660350] (99%) Micrococcineae; Micrococcaceae; Kocuria Alteromonadales 465/465 Gammaproteobacteria; BCM 52 KF373473 bacterium fav-2-10-05 (100%) Alteromonadales [FJ041083] BCM 53 KF373474 Sphingobium amiense 397/408 Alphaproteobacteria; P a g e | 136

strain D3AT58 (97%) Sphingomonadales; [JF459959] Sphingomonadaceae; Sphingobium Gammaproteobacteria; Vibrio harveyi isolate 473/475 BCM 54 KF373475 Vibrionales; Vibrionaceae; VHJR12 [DQ995245] (99%) Vibrio Alphaproteobacteria; Roseobacter sp. NY93C 407/407 Rhodobacterales; BCM 56 KF373476 [EU660505] (100%) Rhodobacteraceae; Roseobacter Gammaproteobacteria; Klebsiella oxytoca strain 463/463 Enterobacteriales; BCM 57 KF373477 AIMST 10.Pl.3 (100%) Enterobacteriaceae; [HQ683968] Klebsiella Bacillus firmus strain 471/471 Firmicutes; Bacillales; BCM 58 KF373478 AIR-NUS-07 [JQ413263] (100%) Bacillaceae; Bacillus Bacillus sp. WRB-4 459/473 Firmicutes; Bacillales; BCM 59 KF373479 [EF636891] (97%) Bacillaceae; Bacillus Gammaproteobacteria; BSD 128-4- Vibrio harveyi 474/475 KF373480 Vibrionales; Vibrionaceae; 1 L [EU373091] (99%) Vibrio Gammaproteobacteria; BSD 128-4- Vibrio harveyi strain 477/477 KF373481 Vibrionales; Vibrionaceae; 2 090212 [GU262992] (100%) Vibrio Gammaproteobacteria; Vibrio harveyi strain A3 424/428 BSD 128-5 KF373482 Vibrionales; Vibrionaceae; [JN391271] (99%) Vibrio Vibrio parahaemolyticus Gammaproteobacteria; 475/475 BSD 128-6 KF373483 isolate Mm007 Vibrionales; Vibrionaceae; (100%) [FR686999] Vibrio Gammaproteobacteria; BSD 128-7 Vibrio communis strain 474/474 KF373484 Vibrionales; Vibrionaceae; L F75216 [HQ161743] (100%) Vibrio Gammaproteobacteria; BSD 128-8- Vibrio ponticus strain 478/478 KF373485 Vibrionales; Vibrionaceae; 1-1 AN62 [JQ409384] (100%) Vibrio Gammaproteobacteria; BSD 128-8- Vibrio campbellii strain 475/476( KF373486 Vibrionales; Vibrionaceae; 1-2 L VSD807 [KC534398] 99%) Vibrio Gammaproteobacteria; Vibrio communis strain 477/478 BSD 13 KF373487 Vibrionales; Vibrionaceae; P274 [JF836181] (99%) Vibrio Gammaproteobacteria; Vibrio azureus strain 475/476 BSD 14 KF373488 Vibrionales; Vibrionaceae; F77118 [HQ908716] (99%) Vibrio Gammaproteobacteria; Vibrio parahaemolyticus 473/475 BSD 15 KF373489 Vibrionales; Vibrionaceae; strain 448 [JN188417] (99%) Vibrio Firmicutes; Bacillales; Bacillus cereus strain B3 475/475 BSD 16-10 KF373490 Bacillaceae; Bacillus; [JN252053] (100%) Bacillus cereus group Gammaproteobacteria; 472/475 BSD 16-11 KF373491 Vibrio shiloi [AF007115] Vibrionales; Vibrionaceae; (99%) Vibrio Gammaproteobacteria; Vibrio communis strain 476/477 BSD 16-2-1 KF373492 Vibrionales; Vibrionaceae; F75214 [HQ161741] (99%) Vibrio BSD 16-2-2 KF373493 Lysinibacillus fusiformis 476/476 Firmicutes; Bacillales; P a g e | 137

[JQ897408] (100%) Bacillaceae; Lysinibacillus Gammaproteobacteria; Vibrio ponticus strain 476/476 BSD 16-3 KF373494 Vibrionales; Vibrionaceae; AN62 [JQ409384] (100%) Vibrio Alteromonadales 460/460 Gammaproteobacteria; BSD 16-5 KF373495 bacterium fav-2-10-05 (100%) Alteromonadales [FJ041083] Alteromonadales 459/459 Gammaproteobacteria; BSD 16-7 KF373496 bacterium fav-2-10-05 (100%) Alteromonadales [FJ041083] Gammaproteobacteria; Vibrio communis strain 476/477 BSD 16-8 L KF373497 Vibrionales; Vibrionaceae; F75214 [HQ161741] (99%) Vibrio Gammaproteobacteria; Vibrio rotiferianus strain 476/476 BSD 2-10 L KF373498 Vibrionales; Vibrionaceae; HT110622 [JQ792238] (100%) Vibrio Gammaproteobacteria; Vibrio communis strain 473/474 BSD 2-6 L KF373499 Vibrionales; Vibrionaceae; F75214 [HQ161741] (99%) Vibrio Lysinibacillus fusiformis 474/474 Firmicutes; Bacillales; BSD 2-7-1 KF373500 [JQ897408] (100%) Bacillaceae; Lysinibacillus Vibrionaceae bacterium 431/433 Gammaproteobacteria; BSD 2-7-2 KF373501 PaD2.06 [GQ406614] (99%) Vibrionales; Vibrionaceae Vibrio parahaemolyticus Gammaproteobacteria; 475/475 BSD 2-8 L KF373502 isolate Mm007 Vibrionales; Vibrionaceae; (100%) [FR686999] Vibrio Gammaproteobacteria; Vibrio azureus strain 472/476 BSD 2-9-1 KF373503 Vibrionales; Vibrionaceae; HNS029 [JN128263] (99%) Vibrio Gammaproteobacteria; Vibrio natriegens strain 474/475 BSD 2-9-2 KF373504 Vibrionales; Vibrionaceae; AUCASVE1 [JQ043186] (99%) Vibrio Firmicutes; Bacillales; Bacillus anthracis strain: 474/474 BSD 256-5 KF373505 Bacillaceae; Bacillus; PD7-4 [AB506122] (100%) Bacillus cereus group Gammaproteobacteria; Vibrio natriegens strain 474/475( BSD 32-5 L KF373506 Vibrionales; Vibrionaceae; AUCASVE1 [JQ043186] 99%) Vibrio Gammaproteobacteria; Vibrio harveyi 476/476 BSD 32-6-1 KF373507 Vibrionales; Vibrionaceae; [EU373091] (100%) Vibrio Gammaproteobacteria; Ferrimonas sp. A3B-58 466/469 Alteromonadales; BSD 32-6-2 KF373508 [AB193755] (99%) Ferrimonadaceae; Ferrimonas Gammaproteobacteria; Vibrio harveyi isolate 477/477 BSD 4-4 KF373509 Vibrionales; Vibrionaceae; VHJR19 [DQ995251] (100%) Vibrio Gammaproteobacteria; Vibrio harveyi isolate 475/476 BSD 4-5 KF373510 Vibrionales; Vibrionaceae; VHJR19 [DQ995251] (99%) Vibrio Vibrionaceae bacterium 433/433 Gammaproteobacteria; BSD 4-7 KF373511 PaD2.06 [GQ406614] (100%) Vibrionales; Vibrionaceae Vibrio parahaemolyticus Gammaproteobacteria; 475/475 BSD 4-8 L KF373512 isolate Mm007 Vibrionales; Vibrionaceae; (100%) [FR686999] Vibrio Vibrio harveyi strain 474/474 Gammaproteobacteria; BSD 4-9 KF373513 S090801 [HM236045] (100%) Vibrionales; Vibrionaceae; P a g e | 138

Vibrio Firmicutes; Bacillales; Bacillus cereus strain 2 467/467 BSD 64-1-1 KF373514 Bacillaceae; Bacillus; [JX439638] (100%) Bacillus cereus group Gammaproteobacteria; Vibrio harveyi strain 474/475 BSD 64-1-2 KF373515 Vibrionales; Vibrionaceae; S090801 [HM236045] (99%) Vibrio Firmicutes; Bacillales; Bacillus anthracis strain: 477/477 BSD 64-2-1 KF373516 Bacillaceae; Bacillus; PD7-4 [AB506122] (100%) Bacillus cereus group Gammaproteobacteria; Vibrio fortis strain 477/478 BSD 64-2-2 KF373517 Vibrionales; Vibrionaceae; VPMP50 [JQ663920] (99%) Vibrio Gammaproteobacteria; Vibrio harveyi strain BK2 473/475 BSD 8-2 L KF373518 Vibrionales; Vibrionaceae; [HM355956] (99%) Vibrio Gammaproteobacteria; Vibrio azureus strain 475/476 BSD 8-3 KF373519 Vibrionales; Vibrionaceae; HNS022 [JN128256] (99%) Vibrio Gammaproteobacteria; Vibrio harveyi strain 475/476 BSD 8-4 KF373520 Vibrionales; Vibrionaceae; S090801 [HM236045] (99%) Vibrio Vibrionaceae bacterium 432/433 Gammaproteobacteria; BSD 8-5 KF373521 PaD2.06 [GQ406614] (99%) Vibrionales; Vibrionaceae Gammaproteobacteria; Vibrio communis strain 471/472 BSD 8-6 L KF373522 Vibrionales; Vibrionaceae; F75214 [HQ161741] (99%) Vibrio Lysinibacillus 473/473 Firmicutes; Bacillales; BSF 11 KF373523 boronitolerans (100%) Bacillaceae; Lysinibacillus [FJ237498] Gammaproteobacteria; Halomonas sp. 612M-23 451/452 Oceanospirillales; BSF 12 KF373524 [GU371676] (99%) Halomonadaceae; Halomonas Gammaproteobacteria; Halomonas sp. 612M-23 449/450 Oceanospirillales; BSF 14 KF373525 [GU371676] (99%) Halomonadaceae; Halomonas Alphaproteobacteria; Rhodobacter capsulatus 463/463 Rhodobacterales; BWC 04-1 KF373526 strain PSB-06 [FJ866784] (100%) Rhodobacteraceae; Rhodobacter Gammaproteobacteria; Halomonas sp. 612M-23 449/450 Oceanospirillales; BWC 13 KF373527 [GU371676] (99%) Halomonadaceae; Halomonas Gammaproteobacteria; Vibrio harveyi strain 473/473 BWC 14 KF373528 Vibrionales; Vibrionaceae; S090801 [HM236045] (100%) Vibrio Gammaproteobacteria; Alteromonas macleodii 457/457 Alteromonadales; BWC 15 KF373529 [AB238950] (100%) Alteromonadaceae; Alteromonas Gammaproteobacteria; Vibrio harveyi strain 474/475 BWC 16 L KF373530 Vibrionales; Vibrionaceae; HL19 [JQ948038] (99%) Vibrio Vibrionaceae bacterium 423/424 Gammaproteobacteria; BWC 17 KF373531 PaD2.06 [GQ406614] (99%) Vibrionales; Vibrionaceae P a g e | 139

Gammaproteobacteria; Vibrio harveyi strain 473/476 BWC 18 KF373532 Vibrionales; Vibrionaceae; HL19 [JQ948038] (99%) Vibrio Gammaproteobacteria; Vibrio harveyi strain 475/476 BWC 19 L KF373533 Vibrionales; Vibrionaceae; HL19 [JQ948038] (99%) Vibrio

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Table A.6: Screening of antimicrobial (PKS/NRPS) and DMSP-degrading (dmdA/dddP) genes in coral mucus isolates. Presence of genes Samples PKS NRPS dmdA dddP BCM 22-1 + + - - BCM 22-2 - + + - BCM 23 + + - + BCM 24-1 + + - + BCM 24-2 + + + + BCM 25 + + + + BCM 26-1 - + - + BCM 26-2 - + - + BCM 27 + + - - BCM 28 - - + + BCM 29 + + + - BCM 31 + - - + BCM 32 - - + - BCM 33 - - - + BCM 34 - + - - BCM 35-1 - + + - BCM 35-2 - + - - BCM 36 - - - - BCM 37 - + - + BCM 38 + - - + BCM 39 + - - - BCM 40 - + - - BCM 41 - + + - BCM 42 - + - + BCM 43 - + - + BCM 44 - + - - BCM 45 - + - - BCM 46 - + + - BCM 48 - - + + BCM 49 - + - - BCM 50 - - - - BCM 51 - - - - BCM 52 - + - - BCM 53 - + + + BCM 54 - - - - BCM 56 - + + + BCM 57 - + + + BCM 58 - + + - BCM 59 - - + + “+” indicates positive presence of the genes, “-“ indicates negative presence of genes.

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Table A.7: Total inhibition and inhibition zones of mucus attached isolates at 28, 30 and 32 °C.

Temperature 28 °C 30 °C 32 °C Samples Average Average Average Total Total Total zone size zone size zone size inhibition inhibition inhibition (cm) (cm) (cm) Positive - 2.8 - 2.7 - 2.7 Negative - 0.0 - 0.0 - 0.0 BCM 22-1 2 0.7 5 1.0 6 0.7 BCM 22-2 2 0.7 6 0.8 5 0.8 BCM 23 3 0.7 7 0.7 5 0.8 BCM 24-1 3 0.7 8 0.6 6 0.8 BCM 24-2 4 0.7 5 0.8 5 0.7 BCM 25 2 0.9 7 0.9 6 0.6 BCM 26-1 8 0.8 11 0.8 11 0.9 BCM 26-2 4 0.5 5 0.7 5 0.6 BCM 27 5 0.5 4 0.6 6 0.6 BCM 28 6 0.5 5 0.6 6 0.6 BCM 29 5 0.5 4 0.7 8 0.5

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Table A.8: Total inhibition and inhibition zones of mucus associated isolates at 28, 30 and 32 °C.

Temperature 28 °C 30 °C 32 °C Samples Average Average Average Total Total Total zone size zone size zone size inhibition inhibition inhibition (cm) (cm) (cm) Positive - 2.7 - 2.6 - 2.7 Negative - 0.0 - 0.0 - 0.0 BCM 31 5 0.5 3 0.9 6 0.6 BCM 32 9 0.8 9 0.8 11 0.9 BCM 33 7 0.7 5 0.9 4 0.4 BCM 34 8 0.7 5 0.5 4 0.4 BCM 35-1 5 0.7 8 0.7 3 0.3 BCM 35-2 5 0.7 5 1.0 4 0.6 BCM 36 6 0.7 6 0.9 3 0.5 BCM 37 6 0.8 8 0.8 4 0.4 BCM 38 10 0.9 11 0.9 9 0.8 BCM 39 4 1.0 5 0.4 3 0.7 BCM 40 5 0.8 5 0.5 3 0.4 BCM 41 5 0.9 6 0.6 4 0.6 BCM 42 4 0.6 6 0.5 2 0.3 BCM 43 5 0.9 7 0.5 4 0.5 BCM 44 6 0.8 5 0.4 2 0.4 BCM 45 6 0.9 9 0.8 9 0.8 BCM 46 4 0.7 6 0.7 4 0.6 BCM 48 5 0.6 7 0.6 4 0.5 BCM 49 4 0.8 6 0.7 3 0.7 BCM 50 7 0.6 7 0.6 2 0.9 BCM 51 7 0.6 8 0.6 2 0.7 BCM 52 6 0.7 7 0.7 4 0.5 BCM 53 10 0.9 10 0.8 13 0.9 BCM 54 0 0.0 2 0.9 0 0.0 BCM 56 1 0.7 1 0.6 0 0.0 BCM 57 1 0.8 1 0.9 0 0.0 BCM 58 0 0.0 3 0.4 0 0.0 BCM 59 0 0.0 2 0.9 0 0.0