Coastal Bacterial Communities_Their Potential Roles in DIMETHYLSULPHIDE (DMS)

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

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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

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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.

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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)

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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

List of Tables

1 Introduction

1.1 Microbes in the ocean

1.2 South China Sea and the Celebes Sea

1.3 Surface microbes and their roles in the DMS(P) cycle

1.4 Coral reefs

1.4.1 Coral reefs of Malaysia

1.5 Coral bleaching

1.6 Coral Surface Mucus Layer (SML) and associated microbes

1.7 Coral diseases and the role of microbes in the SML

1.8 Polyketide synthase (PKS) and non-ribosomal peptide

synthetase (NRPS)

1.9 Significance and aims of the present study and dissertation

outline

2 Methodology

2.1 Field sampling

2.1.1 Reef samples

2.2 Laboratory procedures

2.2.1 Isolation of bacteria

2.2.2 Molecular characterisation

2.2.3 Clone libraries from water samples

2.2.3.1 Extraction of genomic DNA from sea water

2.2.3.2 DNA cloning and plasmid extraction

2.2.3.3 Bacterial communities based in the

construction of clone libraries

2.2.4 PCR amplification of bacterial DMSP cleavage (dddP)

and demethylation (dmdA) genes

2.2.5 PCR amplification of bacterial antimicrobial PKS and

NRPS genes

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2.2.6 Antimicrobial tests

2.2.6.1 Extraction of bioactive compounds

2.2.6.2 Inhibitory interaction tests (well diffusion

assay)

3 Bacterial Communities from the eastern South China Sea and

the Celebes Sea, and Their Potential Role in the DMS(P) Cycle

3.1 Introduction

3.2 Materials and Methods

3.2.1 Study site and sample collection

3.2.2 DNA extraction and purification of cultured bacteria

3.2.3 PCR amplification of bacterial 16S rRNA genes

3.2.4 Sequencing and phylogenetic analysis

3.2.5 Nucleotide sequence accession numbers



3.3 Results and Discussion

3.3.1 Physico-chemical parameters

3.3.2 Diversity of culturable bacterial communities

3.3.3 Variations in the bacterial communities in Kuching,

Kota Kinabalu and Semporna waters

3.3.4 Bacterial strains with potential to metabolise DMS

and/or demethylate DMSP

3.4 Conclusion

3.5 Acknowledgement

4 Bacterial Communities from Talang-Talang Reef and Their

Potential Role in Coral Defence and the DMS(P) Cycle

4.1 Introduction

4.2 Materials and Methods






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4.2.7 PCR amplification of bacterial polyketide synthase

(PKS) and non-ribosomal peptide synthetase (NRPS)

genes

4.2.8 Extraction of bioactive compounds

4.2.9 Well diffusion assay

4.3 Results and Discussion


4.3.2 Bacterial strains with PKS and NRPS genes

4.3.3 Role of mucus-associated bacteria in coral defence

4.3.4 Bacterial strains with potential to metabolise DMS

and/or demethylate DMSP

4.4 Conclusion

4.5 Acknowledgement

5 Summary and Future Work

5.1 Future Research

References

Appendix

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List of Figures

Figure Page

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

1.2 Overview of DMSP catabolic pathways in marine bacteria and the

fates of carbon and sulphur (taken from Reisch, Moran & Whitman

2011).

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).

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).

1.5 Distribution of coral reefs in the East Asian Seas (source:

http://www.ncdc.noaa.gov/paleo/outreach/coral/sor/sor_asia.html).

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

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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).

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).

2.1 Overview of the SHIVA cruise (source: http://shiva.iup.uni-

heidelberg.de/a_activities.html).

2.2 Overview of the Talang-talang Islands just off the shores of Kuching,

Sarawak (source: Yahya, Hassan & Husaini 2012)

2.3 Collection of fresh coral mucus.

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.

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

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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.

2.6 PCR-based screening of dmdA genes. Bands highlighted in this figure

indicate presence of the genes.

2.7 PCR-based screening of dddP genes. Bands highlighted in this figure


2.8 PCR-based screening of PKS genes. Bands highlighted in this figure


2.9 PCR-based screening of NRPS genes. Bands highlighted in this figure


2.10 Inhibition zones from bioactive compoinds with antimicrobial

properties.

3.1 The RV Sonne ship track leading from Singapore to Manila between

November 15-29, 2011 during the SHIVA SO 218 cruise.

3.2 Locations of sampling stations in Kuching, Sarawak.

3.3 Locations of sampling stations in Kota Kinabalu, Sabah.

3.4 Locations of sampling stations in Semporna, Sabah.

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.

3.6 Phylogenetic groups of isolates from the waters of Kuching, Kota

Kinabalu and Semporna at depths of 1, 5 and 10 m.


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.


sequences found in Kuching 1911. The phylogenetic tree was

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are indicated.


sequences found in Kota Kinabalu. The phylogenetic tree was





are indicated.


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.

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.

3.12 Presence of dmdA and/or dddP genes in bacterial isolates from the

waters of Kuching, Kota Kinabalu and Semporna.

3.13 Relative abundance of dmdA and dddP genes in isolated

Gammaproteobacteria from Kuching.


Gammaproteobacteria from Kota Kinabalu.


Gammaproteobacteria from Semporna.

4.1 Overview of the Talang-talang Islands just off the shores of Kuching,

Sarawak. Enlarged map indicates sampling area.

4.2 Pie charts illustrating the diversity of bacterial groups based on

partial 16S rRNA gene sequences from bacteria isolated from (a)

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coral mucus, (b) water column and (c) sediment.


sequences found in coral mucus The phylogenetic tree was





are indicated.


sequences found in water column. The phylogenetic tree was





are indicated.


sequences found in reef sediment. The phylogenetic tree was





are indicated.

4.6 Percentage of Vibrio isolates in mucus attached and mucus

associated communities.

4.7 Relative abundance of PKS and NRPS genes in cultured bacterial

communities from coral mucus.

4.8 Presence of PKS and/or NRPS genes in bacterial isolates from coral

mucus.

4.9 Total inhibitions of mucus attached bacteria at temperatures of 28,

30 and 32 °C.

4.10 Total inhibitions of mucus associated bacteria at temperatures of 28,

30 and 32 °C.

4.11 Average zone of inhibitions (cm) of mucus attached bacteria at

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temperatures of 28, 30 and 32 °C.

4.12 Average zone of inhibitions (cm) of mucus associated bacteria at


4.13 Relative abundance of dmdA and dddP genes in cultured bacterial


4.14 Presence of dmdA and/or dddP genes in bacterial isolates from coral

mucus.

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List of Tables

Table Page

1.1 Regional distribution of coral reefs (source: Veron & Stafford-Smith

2000).

3.1 Locations of sampling stations at Kuching, Kota Kinabalu and

Semporna.

3.2 Physico-chemical parameters measured from Kuching and Kota

Kinabalu at depths of 1 and 5 m.

3.3 Indices used to quantify the diversity of bacterial communities at

Kuching, Kota Kinabalu and Semporna.

A.1 16S rRNA gene sequence analysis of bacterial cultures from Kuching

1611, based on BLAST analysis.

A.2 16S rRNA gene sequence analysis of bacterial cultures from Kuching


A.3 16S rRNA gene sequence analysis of bacterial cultures from Kota

Kinabalu, based on BLAST analysis.

A.4 16S rRNA gene sequence analysis of bacterial cultures from

Semporna, based on BLAST analysis.

A.5 16S rRNA gene sequence analysis of bacterial cultures from Talang-

talang reef and its surrounding waters, based on BLAST analysis.

A.6 Screening of antimicrobial (PKS/NRPS) and DMSP-degrading

(dmdA/dddP) genes in coral mucus isolates.

A.7 Total inhibition and inhibition zones of mucus attached isolates at

28, 30 and 32 °C.

A.8 Total inhibition and inhibition zones of mucus associated isolates at

28, 30 and 32 °C.

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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

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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

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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).

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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;

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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).

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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

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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

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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).

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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,

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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.

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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).

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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).





40 cycles of:

- 96 °C for 1 min.

- 55 °C for 2 min.

- 72 °C for 3 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.

P a g e | 27

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


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.



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


P a g e | 30

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


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


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).





45 cycles of:

- 94 °C for 1 min.

- 55 °C for 1 min.

- 72 °C for 2 min.


P a g e | 31


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


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


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).

P a g e | 33

Figure 2.10: Inhibition zones from bioactive compounds with antimicrobial

properties.

P a g e | 34

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


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 stations

GPS coordinates

Kuching (1611) Kuching (1911) Kota Kinabalu Semporna

Station 1 1°38'36.24"N, 110°30'5.28"E

1°39'28.81"N, 110°31'24.42"E

6° 3'4.56"N, 116° 5'54.60"E

4°35'15.96"N, 118°32'58.14"E

Station 2 1°39'44.82"N, 110°32'7.26"E

1°42'44.24"N, 110°33'23.46"E

6° 3'5.82"N, 116° 4'1.45"E

4°38'37.86"N, 118°20'25.44"E

Station 3 1°42'2.80"N, 110°37'12.36"E

1°45'32.93"N, 110°35'16.86"E

6° 3'4.02"N, 116° 0'2.77"E

4°42'31.68"N, 118°23'19.38"E

Station 4 1°42'46.62"N, 110°39'17.40"E

1°48'2.16"N, 110°37'51.53"E

6° 2'49.85"N, 115°57'38.26"E

4°40'42.48"N, 118°32'11.34"E

Station 5 1°45'49.07"N, 110°41'27.77"E

1°50'54.15"N, 110°40'11.26"E

6° 4'23.64"N, 115°54'36.42"E

4°37'31.26"N, 118°41'5.99"E

Station 6 – – – 4°35'56.76"N, 118°43'19.14"E

Station 7 – – – 4°35'30.66"N, 118°42'17.10"E

Station 8 – – – 4°33'17.83"N, 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.

P a g e | 40

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

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


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.






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.


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.


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.






P a g e | 42




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.

Station Depth

(m) Temp

(°C) pH

Salinity (ppt)

Nitrate (ppm)

Phosphate (ppm)

Nitrite (ppm)

Silicate (ppm)

KCH-1 1 29.06 7.90 28.48 9.13 0.60 1.46 23.47 5 29.34 8.10 30.59 BD BD BD BD

KCH-2 1 28.98 8.25 30.65 2.02 0.33 0.60 7.48 5 29.11 8.25 30.89 BD BD BD BD

KCH-3 1 29.05 8.33 31.18 0.85 0.15 0.03 2.97 5 29.16 8.30 30.53 BD BD BD BD

KCH-4 1 29.00 8.33 31.07 0.49 0.10 0.00 4.52 5 29.10 8.29 30.52 BD BD BD BD

KCH-5 1 29.27 8.31 31.61 0.15 0.06 0.00 5.86 5 29.40 8.29 31.85 0.15 0.05 0.00 2.21

KCH mean

29.15 8.24 30.74 2.13 0.22 0.35 7.75

KK-1 1 29.80 8.44 31.85 1.04 0.15 BD 3.47 5 29.90 8.37 32.04 BD BD BD BD

KK-2 1 29.73 8.36 31.44 0.25 0.17 BD 3.21 5 29.78 8.33 31.95 BD BD BD BD

KK-3 1 29.55 8.34 31.88 0.23 0.11 BD 2.67 5 29.54 8.33 31.87 BD BD BD BD

KK-4 1 29.52 8.36 31.93 BD BD BD BD 5 29.45 8.34 31.91 BD BD BD BD

KK-5 1 29.68 8.38 32.03 0.13 0.03 BD 2.74 5 29.50 8.37 31.92 0.15 0.02 BD 2.79

P a g e | 43

KK mean

29.65 8.36 31.88 0.36 0.10 BD 2.98

*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).


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).

P a g e | 45

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.

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.

0

5

10

15

20

25

30

35

40

1m 5m ≥10m 1m 5m ≥10m 1m 5m ≥10m

Kuching Kota Kinabalu Semporna

Nu

mb

er

of

iso

late

s

α-proteobacteria β-proteobacteria γ-proteobacteria Firmicutes

P a g e | 46

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.

P a g e | 47

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.


sequences found in Kuching 1911. The phylogenetic tree was generated with



P a g e | 49




sequences found in Kota Kinabalu. The phylogenetic tree was generated with


P a g e | 50




P a g e | 51


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.

P a g e | 60

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).

P a g e | 62

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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ob

acte

ria

Fir

mic

ute

s

Kuching Kota Kinabalu Semporna

Pe

rce

nta

ge

(%

)

Only dmdA Only dddP Both None

P a g e | 63

Figure 3.13: Relative abundance of dmdA and dddP genes in isolated

Gammaproteobacteria from Kuching.


Gammaproteobacteria from Kota Kinabalu.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

dmdA dddP Both

Vibrio sp.

Stenotrophomonas sp.

Shewanella sp.

Pseudomonas sp.

Pseudoalteromonas sp.

Oceanimonas sp.

Citrobacter sp

Burzellia sp.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

dmdA dddP Both

Vibrio sp.

Shewanella sp.

Pseudomonas sp.

Oceanimonas sp.

Enterobacter sp.

P a g e | 64


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.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

dmdA dddP Both

Vibrio sp.

Shewanella sp.

Pseudomonas sp.

Photobacterium sp.

Oceanimonas sp.

Bowmanella sp.

Allomonas sp

P a g e | 65

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.

P a g e | 68

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).

P a g e | 71

4.2 MATERIALS AND METHODS


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.


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.






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.


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


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.










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



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).


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


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%).

P a g e | 76


sequences found in coral mucus. The phylogenetic tree was generated with





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sequences found in water column. The phylogenetic tree was generated with





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sequences found in reef sediment. The phylogenetic tree was generated with



P a g e | 79



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.

P a g e | 82

Figure 4.7: Relative abundance of PKS and NRPS genes in cultured bacterial


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.

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.

8%

51% 18%

23%

Abundance of PKS and NRPS genes in bacteria from coral mucus

PKS NRPS Both None

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

α-proteobacteria γ-proteobacteria Actinobacteria Firmicutes Unknown

PKS NRPS Both None

P a g e | 83

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).

Figure 4.9: Total inhibitions of mucus attached bacteria at temperatures of 28, 30

and 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

0

2

4

6

8

10

12

14

BCM22-1

BCM22-2

BCM 23 BCM24-1

BCM24-2

BCM 25 BCM26-1

BCM26-2

BCM 27BCM 28BCM 29

To

tal

inh

ibit

ion

s

Samples

28 °C 30 °C 32 °C

0

2

4

6

8

10

12

14

BC

M 3

1

BC

M 3

2

BC

M 3

3

BC

M 3

4

BC

M 3

5-1

BC

M 3

5-2

BC

M 3

6

BC

M 3

7

BC

M 3

8

BC

M 3

9

BC

M 4

0

BC

M 4

1

BC

M 4

2

BC

M 4

3

BC

M 4

4

BC

M 4

5

BC

M 4

6

BC

M 4

8

BC

M 4

9

BC

M 5

0

BC

M 5

1

BC

M 5

2

BC

M 5

3

BC

M 5

4

BC

M 5

6

BC

M 5

7

BC

M 5

8

BC

M 5

9

To

tal

inh

ibit

ion

s

Samples

28 °C 30 °C 32 °C

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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).

Figure 4.11: Average zone of inhibitions (cm) of mucus attached bacteria at


Figure 4.12: Average zone of inhibitions (cm) of mucus associated bacteria at


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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).

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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.

Figure 4.13: Relative abundance of dmdA and dddP genes in cultured bacterial


18%

26%

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36%

Abundance of DMSP-degrading genes in bacteria from coral mucus

dmdA dddP Both None

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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.

0%

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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..

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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


Sequence GenBank accession number

Closest match Identities Phylogenetic division

1611-S1-01-1

KF373266 Pseudomonas aeruginosa strain 11.2 [JX286673]

460/460 (100%)

Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas

1611-S1-01-1.3

KF373267 Vibrio harveyi strain E385 [JX290081]

462/463 (99%)

Gammaproteobacteria; Vibrionales; Vibrionaceae; Vibrio

1611-S1-01-2

KF373268 Pseudomonas sp. Mexd38 [JX436405]

462/462 (100%)


1611-S1-05-1

KF373269

Vibrio parahaemolyticus strain 448 [JN188417]

475/475 (100%)


1611-S1-05-2.1

KF373270


476/476 (100%)


1611-S1-05-2.2

KF373271


474/475 (99%)


1611-S1-05-3

KF373272


474/475 (99%)


1611-S1-10-1.1.1

KF373273 Vibrio alginolyticus strain XSBZ14 [JX221045]

397/464 (86%)


1611-S1-10-1.1.2.1

KF373274 Shewanella chilikensis strain JC5 [HM016088]

461/463 (99%)

Gammaproteobacteria; Alteromonadales; Shewanellaceae; Shewanella

1611-S1-10-1.1.2.2

KF373275 Photobacterium sp. TKY4 [AB583193]

473/473 (100%)

Gammaproteobacteria; Vibrionales; Vibrionaceae; Photobacterium

1611-S1-10-1.2.1

KF373276 Bacillus sphaericus isolate BS11 [AM269451]

475/475 (100%)

Firmicutes; Bacilli; Bacillales; Bacillaceae; Lysinibacillus

1611-S1-10-2.1

KF373277 Oceanimonas sp. D6083 [FJ161317]

425/462 (92%)

Gammaproteobacteria; Aeromonadales; Aeromonadaceae; Oceanimonas

1611-S2-01-1.1.2

KF373278 Shewanella haliotis strain Z4 [JX286502]

425/426 (99%)


1611-S2-01-2.1


467/467 (100%)

Gammaproteobacteria; Alteromonadales;

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Shewanellaceae; Shewanella

1611-S2-01-2.2

KF373280 Shewanella haliotis strain MS41 [FN997635]

463/466 (99%)


1611-S2-01-2.3


469/469 (100%)


1611-S2-01-3.2

KF373282 Pseudomonas sp. Mexd38 [JX436405]

461/461 (100%)


1611-S2-05-1.1


468/468 (100%)


1611-S2-05-1.2

KF373284

Vibrio parahaemolyticus strain Aj2010072802A90 [JF432066]

474/474 (100%)


1611-S2-05-2


467/467 (100%)


1611-S2-10-1


461/461 (100%)


1611-S2-10-2

KF373287 Bacillus sp. 1-1(2012) [JN942108]

461/461 (100%)

Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus

1611-S4-01-1

KF373288

Vibrio parahaemolyticus isolate Mm004 [FR686998]

460/464 (99%)


1611-S4-01-1.1


463/463 (100%)


1611-S4-01-2.1.1

KF373290


471/474 (99%)


1611-S4-01-2.2

KF373291 Vibrio natriegens strain AUCASVE5 [JQ277719]

458/465 (98%)


1611-S4-01-3.1

KF373292


471/472 (99%)


1611-S4-01-3.1.2

KF373293


471/473 (99%)


1611-S4-01-3.2

KF373294


472/473 (99%)


1611-S4-05-1

KF373295 Roseovarius pacificus strain 81-2 [NR_043564]

405/405 (100%)

Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae; Roseovarius

1611-S4-05-2

KF373296 Roseovarius pacificus strain 81-2

408/408 (100%)

Alphaproteobacteria; Rhodobacterales;

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[NR_043564] Rhodobacteraceae; Roseovarius 1611-S4-05-3

KF373297 Vibrio harveyi isolate VHJR6 [DQ995240]

475/477 (99%)


1611-S4-10-1.2

KF373298 Vibrio azureus strain M2-164 [JQ810832]

473/473 (100%)


1611-S4-10-2

KF373299

Vibrio parahaemolyticus strain RW1 [FJ172044]

477/479 (99%)


1611-S4-10-3.1

KF373300 Vibrio rotiferianus strain 5S [JF792070]

469/473 (99%)


1611-S4-10-3.2


470/473 (99%)


1611-S5-01-1

KF373302 Bacillus subtilis strain y86-7 [FJ460478]

453/475 (95%)


1611-S5-05-1

KF373303 Vibrio natriegens strain CM3 [EU660320]

473/473 (100%)


1611-S5-05-3.1

KF373304

Vibrio parahaemolyticus isolate Mm004 [FR686998]

473/473 (100%)


1611-S5-05-3.2

KF373305 Burzellia piscidermidis strain P6-6 [EU127296]

464/464 (100%)

Gammaproteobacteria; Burzellia

1611-S5-10-1

KF373306 Burzellia piscidermidis strain P6-6 [EU127296]

463/464 (99%)

Gammaproteobacteria; Burzellia

1611-S5-10-2

KF373307 Vibrio azureus strain F77118 [HQ908716]

472/473 (99%)


1611-S6-01-1.1

KF373308 Vibrio sinaloensis strain CAIM 1068 [HM584056]

469/475 (99%)


1611-S6-01-1.2

KF373309

Pseudoalteromonas maricaloris strain KMM636 [NR_025009]

458/458 (100%)

Gammaproteobacteria; Alteromonadales; Pseudoalteromonadaceae; Pseudoalteromonas

1611-S6-01-2

KF373310 Rhodobacteraceae bacterium SCSWE04 [FJ461471]

360/375 (96%)

Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae

1611-S6-05-1.1

KF373311

Stenotrophomonas maltophilia strain BQAPs-03d [FJ217200]

471/471 (100%)

Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Stenotrophomonas; Stenotrophomonas maltophilia group

1611-S6-05-1.2


472/475 (99%)


1611-S6-05-2.1

KF373313 Vibrio harveyi isolate VHJR14 [EF011651]

474/475 (99%)


1611-S6-05-2.2

KF373314 Vibrio harveyi strain HL19 [JQ948038]

475/475 (100%)


1611-S6-05-3.2


468/472 (99%)


1611-S6-10-1.1


471/472 (99%)


1611-S6- KF373317 Vibrio harveyi isolate 477/477 Gammaproteobacteria;

P a g e | 124

10-1.2 VHJR12 [DQ995245] (100%) Vibrionales; Vibrionaceae; Vibrio

1611-S6-10-2

KF373318 Vibrio campbellii strain CAIM 886 [HM584033]

475/476 (99%)


P a g e | 125

Table A.2: 16S rRNA gene sequence analysis of bacterial cultures from Kuching




1911-S1-01-1.2.1

KF373319 Vibrio orientalis strain JC97, isolate Pkl-17 [FR837599]

465/468 (99%)


1911-S1-01-1.2.2

KF373320 Rhodobacter capsulatus strain PSB-06 [FJ866784]

434/440 (99%)

Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae; Rhodobacter

1911-S1-01-2


440/455 (97%)


1911-S1-01-3


433/440 (98%)


1911-S1-05-2

KF373323 Pseudomonas oleovorans strain HNS030 [JN128264]

456/457 (99%)

Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas; Pseudomonas

1911-S1-07-1


459/460 (99%)


1911-S2-01-1

KF373325 Vibrio alginolyticus isolate Va150 [EU155497]

476/478 (99%)


1911-S2-05-1

KF373326 Vibrio alginolyticus strain HZBC71 [JN188402]

471/474 (99%)


1911-S2-07-1

KF373327 Vibrio alginolyticus strain HZBC71 [JN188402]

473/475 (99%)


1911-S2-07-2

KF373328

Vibrio parahaemolyticus isolate Vp481 [EU155540]

471/473 (99%)


1911-S3-01-1.1.1

KF373329 Pseudoalteromonas ganghwensis [DQ011614]

464/465 (99%)


1911-S3-01-1.1.2

KF373330

Vibrio parahaemolyticus strain VPMP55 [JQ663925]

319/402 (79%)


1911-S3-01-1.2

KF373331 Vibrio alginolyticus strain P61224 [AJ704375]

474/475 (99%)


1911-S3-01-2

KF373332 Vibrio diabolicus strain KM30-12-3 [JQ670740]

475/478 (99%)


1911-S3-05-1

KF373333

Vibrio parahaemolyticus strain 93A-5807 [DQ497398]

474/476 (99%)


1911-S3- KF373334 Vibrio 470/473 Gammaproteobacteria;

P a g e | 126

05-2 parahaemolyticus strain 93A-5807 [DQ497398]

(99%) Vibrionales; Vibrionaceae; Vibrio

1911-S3-10-1.1

KF373335 Vibrio harveyi strain IS01 [GU974342]

473/474 (99%)


1911-S3-10-1.2


473/475 (99%)


1911-S3-10-2.1

KF373337 Vibrio rotiferianus strain BV1 [JN391272]

475/478 (99%)


1911-S4-01-1

KF373338 Pseudoalteromonas ganghwensis [DQ011614]

462/463 (99%)


1911-S4-01-1.1

KF373339 Vibrio alginolyticus strain H050815-1 [EF219054]

474/475 (99%)


1911-S4-01-2.2

KF373340 Thalassospira xiamenensis strain PTG4-18 [EU603449]

411/416 (99%)

Alphaproteobacteria; Rhodospirillales; Rhodospirillaceae; Thalassospira

1911-S4-05-1.1

KF373341 Citrobacter freundii strain AIMST Ehe5 [JQ312038]

461/462 (99%)

Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Citrobacter

1911-S4-05-1.2

KF373342

Leclercia adecarboxylata strain AIMST Ehe6 [JQ312039]

461/462 (99%)

Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Leclercia

1911-S4-05-2

KF373343 Vibrio azureus strain 41113 [HM032787]

452/468 (97%)


1911-S4-10-2.1

KF373344 Vibrio alginolyticus strain H050815-1 [EF219054]

472/473 (99%)


1911-S5-01-1


471/472 (99%)


1911-S5-01-2.1


474/475 (99%)


1911-S5-01-2.2


472/473 (99%)


1911-S5-05-1.1.2

KF373348 Citrobacter freundii strain AIMST Ehe5 [JQ312038]

462/463 (99%)

Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Citrobacter

1911-S5-05-1.2


472/473 (99%)


1911-S5-05-1.2.1

KF373350 Vibrio azureus strain F77118 [HQ908716]

473/473 (100%)


1911-S5-05-2

KF373351 Vibrio parahaemolyticus strain 448 [JN188417]

474/475 (99%)


1911-S5- KF373352 Vibrio natriegens 471/472 Gammaproteobacteria;

P a g e | 127

05-3 strain AUCASVE1 [JQ043186]

(99%) Vibrionales; Vibrionaceae; Vibrio

1911-S5-10-1

KF373353 Vibrio azureus strain 41113 [HM032787]

471/474 (99%)


1911-S5-10-2

KF373354 Vibrio splendidus strain AP625 [GQ254509]

469/471 (99%)


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Table A.3: 16S rRNA gene sequence analysis of bacterial cultures from Kota

Kinabalu, based on BLAST analysis.



2311-S1-01-1.1


452/453 (99%)


2311-S1-01-1.2


461/461 (100%)


2311-S1-01-2.1


469/469 (100%)


2311-S1-01-2.2


467/467 (100%)


2311-S1-01-3.1


466/466 (100%)


2311-S1-05-1

KF373360 Exiguobacterium aurantiacum var. Colo. Road [AY047481]

485/485 (100%)

Firmicutes; Bacilli; Bacillales; Bacillales Family XII. Incertae Sedis; Exiguobacterium

2311-S1-05-2

KF373361 Oceanimonas smirnovii strain 31-13 [NR_042963]

442/461 (96%)


2311-S1-10-1

KF373362 Vibrio rotiferianus strain 5S [JF792070]

466/470 (99%)


2311-S2-01-1

KF373363 Brevibacillus laterosporus strain GZUB11 [FJ434663]

472/472 (100%)

Firmicutes; Bacilli; Bacillales; Paenibacillaceae; Brevibacillus

2311-S2-10-1


414/453 (91%)


2311-S3-01-1.1

KF373365 Bacillus sphaericus clone 7-16 [DQ364585]

431/456 (95%)


2311-S3-01-1.2

KF373366 Shewanella putrefaciens strain R1418 [AB208055]

455/461 (99%)


2311-S3-01-2

KF373367 Shewanella putrefaciens strain R1418 [AB208055]

459/462 (99%)


2311-S3-01-3

KF373368 Vibrio vulnificus strain W045 [EF114147]

473/473 (100%)


2311-S3-05-1

KF373369 Enterobacter ludwigii strain KW 93 [JX262395]

463/463 (100%)

Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Enterobacter

2311-S3-05-2.1

KF373370

Pseudomonas plecoglossicida strain AIMST Aie20 [JQ312025]

459/459 (100%)


2311-S3- KF373371 Thalassospira sp. SKUK 417/417 Alphaproteobacteria;

P a g e | 129

10-1 MB1005 [EU907920] (100%) Rhodospirillales; Rhodospirillaceae; Thalassospira

2311-S3-10-2.1

KF373372 Bacillus malacitensis strain TP12 [FJ887890]

404/408 (99%)


2311-S3-10-2.2


471/472 (99%)


2311-S4-01-1

KF373374 Providencia sp. Sam130-9A [FJ418577]

456/460 (99%)

Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Providencia

2311-S4-05-1

KF373375 Nitratireductor basaltis strain J3 [NR_044414]

409/409 (100%)

Alphaproteobacteria; Rhizobiales; Phyllobacteriaceae; Nitratireductor

2311-S4-10-1


463/468 (99%)


2311-S4-10-2.1.1


463/468 (99%)


2311-S4-10-2.1.3

KF373378 Lysinibacillus fusiformis strain R3 [JQ991002]

476/476 (100%)


2311-S4-10-2.2

KF373379 Exiguobacterium aurantiacum var. Colo. Road [AY047481]

489/490 (99%)


2311-S4-10-2.3


460/465 (99%)


2311-S4-18-1.1

KF373381 Vibrio vulnificus strain W045 [EF114147]

475/475 (100%)


2311-S4-18-1.2


436/447 (98%)


2311-S5-01-1.2

KF373383 Pseudoalteromonas lipolytica strain ZR064 [JX173567]

464/465 (99%)


2311-S5-01-2.1

KF373384 Pseudoalteromonas lipolytica strain ZR064 [JX173567]

463/463 (100%)


2311-S5-01-2.2

KF373385 Pseudomonas stutzeri strain UP-1 [AY364327]

453/454 (99%)


2311-S5-01-2.3

KF373386 Pseudomonas stutzeri strain UP-1 [AY364327]

458/459 (99%)


2311-S5-01-3.1.1

KF373387 Brevundimonas diminuta strain c138 [FJ950570]

405/406 (99%)

Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Brevundimonas

2311-S5-01-3.1.2

KF373388 Exiguobacterium arabatum [JF758868]

438/479 (91%)

Firmicutes; Bacilli; Bacillales; Bacillales Family XII. Incertae

P a g e | 130

Sedis; Exiguobacterium

2311-S5-01-3.2

KF373389 Brevundimonas diminuta strain KSC_AK3a [EF191247]

407/407 (100%)

Alphaproteobacteria; Caulobacterales; Caulobacteraceae; Brevundimonas

2311-S5-01B-1


472/472 (100%)


2311-S5-05-1


472/473 (99%)


2311-S5-05-2


470/472 (99%)


P a g e | 131

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

based on BLAST analysis.



2611-S1-01-1.1

KF373393 Alcaligenes faecalis strain OCEN2DBT [JF264463]

410/465 (88%)

Betaproteobacteria; Burkholderiales; Alcaligenaceae; Alcaligenes

2611-S1-01-1.2

KF373394 Vibrio communis strain F75216 [HQ161743]

472/472 (100%)


2611-S1-05-1.1

KF373395

Exiguobacterium lactigenes strain: HYS0503-MK66 [AB259161]

483/483 (100%)


2611-S1-05-1.2


463/468 (99%)


2611-S2-01-1

KF373397 Vibrio natriegens strain CM3 [EU660320]

473/475 (99%)


2611-S2-01-3

KF373398 Vibrio furnissii strain MM5 [FJ906812]

451/473 (95%)


2611-S2-05-1.1

KF373399 Allomonas enterica strain JC74, isolate R2A [FR837595]

473/475 (99%)

Gammaproteobacteria; Vibrionales; Vibrionaceae; Allomonas

2611-S2-05-1.2


476/478 (99%)


2611-S2-05-2.2

KF373401 Bowmanella denitrificans strain BD1 [NR_043738]

448/459 (98%)

Gammaproteobacteria; Alteromonadales; Alteromonadaceae; Bowmanella

2611-S2-05-3


470/474 (99%)


2611-S2-10-2

KF373403 Pseudomonas plecoglossicida strain R8-591-1 [JQ659971]

459/459 (100%)


2611-S3-01-1


472/473 (99%)


2611-S3-01-2.2

KF373405 Vibrio alginolyticus strain XHS1-3 [JN188407]

472/472 (100%)


2611-S3-05-1


449/464 (97%)


2611-S4-01-1


473/473 (100%)


2611-S4-01-2

KF373408 Vibrio rotiferianus strain BV1 [JN391272]

471/474 (99%)


2611-S4- KF373409 Pseudomonas fulva 458/458 Gammaproteobacteria;

P a g e | 132

01-2.1 strain SMA24 [JQ618288]

(100%) Pseudomonadales; Pseudomonadaceae; Pseudomonas

2611-S4-01-2.2


470/472 (99%)


2611-S4-01-4

KF373411 Pseudidiomarina sediminum strain c121 [NR_044176]

440/461 (95%)

Gammaproteobacteria; Alteromonadales; Idiomarinaceae; Idiomarina

2611-S4-01A-2

KF373412

Pseudomonas pseudoalcaligenes strain K29411 [DQ298030]

460/461 (99%)


2611-S4-01B-1


472/473 (99%)


2611-S4-01B-2.1

KF373414 Shewanella sp. UMS11/10 [JQ231163]

428/428 (100%)


2611-S4-01B-2.2


454/459 (99%)


2611-S4-01B-3

KF373416 Exiguobacterium profundum strain SigaKolEp3 [JX987048]

474/476 (99%)


2611-S4-01C-1


474/475 (99%)


2611-S4-01C-2


472/473 (99%)


2611-S4-05-2

KF373419 Bacillus cereus strain 14B [JX901104]

329/433 (76%)


2611-S4-06A-1


460/468 (98%)


2611-S5-01-1

KF373421 Pseudidiomarina sediminum strain c121 [NR_044176]

423/463 (91%)

Gammaproteobacteria; Alteromonadales; Idiomarinaceae; Idiomarina

2611-S5-05A-1

KF373422


437/438 (99%)


2611-S5-05B-1.1

KF373423 Pseudoalteromonas sp. S187 [FJ457123]

465/466 (99%)


2611-S5-05B-1.2

KF373424 Photobacterium sp. MM14 [JN791371]

473/473 (100%)


2611-S5-05B-3.2.1


460/465 (99%)


2611-S5-05B-3.2.2


464/465 (99%)


2611-S5-05C-2

KF373427 Photobacterium sp. MM14 [JN791371]

477/477 (100%)

Gammaproteobacteria; Vibrionales; Vibrionaceae;

P a g e | 133

Photobacterium

2611-S5-10-2

KF373428 Nitratireductor aquimarinus CL-SC21 [HQ176467]

404/406 (99%)

Alphaproteobacteria; Rhizobiales; Phyllobacteriaceae; Nitratireductor

2611-S6-01-1

KF373429


347/410 (85%)


2611-S6-01-1.1

KF373430


450/450 (100%)


2611-S6-01-1.2


463/467 (99%)


2611-S6-01-3

KF373432 Vibrio alginolyticus strain 486 [JN188409]

475/475 (100%)


2611-S6-05-1.1


445/449 (99%)


2611-S6-05-2


468/468 (100%)


2611-S6-09-1


474/475 (99%)


2611-S6-09-2


471/472 (99%)


2611-S7-01-1


475/476 (99%)


2611-S7-01-2

KF373438

Vibrio parahaemolyticus strain S9-891-B0919354-5-8F [KC520577]

475/475 (100%)


2611-S8-01-1.1

KF373439 Vibrio alginolyticus strain 486 [JN188409]

471/472 (99%)


2611-S8-01-3


474/474 (100%)


P a g e | 134

Table A.5: 16S rRNA gene sequence analysis of bacterial cultures from Talang-

talang reef and its surrounding waters, based on BLAST analysis.



BCM 22-1 KF373441 Vibrio parahaemolyticus strain DHC22 [JQ904733]

451/451 (100%)


BCM 22-2 KF373442 Vibrio harveyi strain F75032 [HQ161747]

459/467 (98%)


BCM 23 KF373443 Vibrio parahaemolyticus strain DHC22 [JQ904733]

418/418 (100%)


BCM 24-1 KF373444 Vibrio harveyi isolate VHJR19 [DQ995251]

475/475 (100%)



472/472 (100%)


BCM 25 KF373446 Vibrio harveyi isolate VHJR19 [DQ995251]

474/475 (99%)


BCM 26-1 KF373447 Vibrio parahaemolyticus strain DHC22 [JQ904733]

472/474 (99%)



472/475 (99%)



475/476 (99%)


BCM 28 KF373450 Vibrio azureus strain M2-164 [JQ810832]

472/473 (99%)


BCM 29 KF373451 Halomonas aquamarina strain Ve1-10-83 [EU684464]

460/460 (100%)

Gammaproteobacteria; Oceanospirillales; Halomonadaceae; Halomonas

BCM 31 KF373452 Bacillus arsenicus strain HLSB44 [FJ999563]

455/466 (98%)

Firmicutes; Bacillales; Bacillaceae; Bacillus

BCM 32 KF373453 Bacillus arsenicus strain HLSB44 [FJ999563]

444/468 (95%)


BCM 33 KF373454 Uncultured alpha proteobacterium clone FF-20 [AY682051]

382/384 (99%)

Alphaproteobacteria; environmental samples

BCM 34 KF373455 Psychrobacter celer strain K-W15 [JQ799068]

453/454 (99%)

Gammaproteobacteria; Pseudomonadales; Moraxellaceae; Psychrobacter

BCM 35-1 KF373456 Mucus bacterium 108 [AY654761]

377/406 (93%)

Bacteria

BCM 35-2 KF373457 Uncultured alpha proteobacterium clone FF-20 [AY682051]

382/385 (99%)

Alphaproteobacteria; environmental samples

BCM 36 KF373458 Staphylococcus 474/474 Firmicutes; Bacilli;

P a g e | 135

lugdunensis strain NBL01 [JX629460]

(100%) Bacillales; Staphylococcus

BCM 37 KF373459 Roseobacter sp. H454 [AY368572]

406/406 (100%)

Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae; Roseobacter

BCM 38 KF373460 Vibrio coralliilyticus strain LMG 21349 [AJ440004]

467/472 (99%)



472/477 (99%)


BCM 40 KF373462 Vibrio brasiliensis strain HQSB7 [JF721971]

472/477 (99%)



470/472 (99%)



478/480 (99%)


BCM 43 KF373465 Klebsiella oxytoca strain AIMST 10.Pl.3 [HQ683968]

458/460 (99%)

Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Klebsiella

BCM 44 KF373466 Microbulbifer sp. 2ta13 [FJ952779]

455/455 (100%)

Gammaproteobacteria; Alteromonadales; Alteromonadaceae; Microbulbifer


470/479 (98%)


BCM 46 KF373468 Vibrio harveyi strain F75087 [HQ161750]

469/472 (99%)


BCM 48 KF373469 Photobacterium jeanii strain R-21419 [GU065212]

471/477 (99%)


BCM 49 KF373470 Kocuria rosea strain CT22 [EU660350]

442/446 (99%)

Actinobacteria; Actinobacteridae; Actinomycetales; Micrococcineae; Micrococcaceae; Kocuria

BCM 50 KF373471 Brachybacterium paraconglomeratum [AB362255]

441/441 (100%)

Actinobacteria; Actinobacteridae; Actinomycetales; Micrococcineae; Dermabacteraceae; Brachybacterium

BCM 51 KF373472 Kocuria rosea strain CT22 [EU660350]

441/445 (99%)

Actinobacteria; Actinobacteridae; Actinomycetales; Micrococcineae; Micrococcaceae; Kocuria

BCM 52 KF373473 Alteromonadales bacterium fav-2-10-05 [FJ041083]

465/465 (100%)

Gammaproteobacteria; Alteromonadales

BCM 53 KF373474 Sphingobium amiense 397/408 Alphaproteobacteria;

P a g e | 136

strain D3AT58 [JF459959]

(97%) Sphingomonadales; Sphingomonadaceae; Sphingobium


473/475 (99%)


BCM 56 KF373476 Roseobacter sp. NY93C [EU660505]

407/407 (100%)

Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae; Roseobacter

BCM 57 KF373477 Klebsiella oxytoca strain AIMST 10.Pl.3 [HQ683968]

463/463 (100%)

Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Klebsiella

BCM 58 KF373478 Bacillus firmus strain AIR-NUS-07 [JQ413263]

471/471 (100%)


BCM 59 KF373479 Bacillus sp. WRB-4 [EF636891]

459/473 (97%)


BSD 128-4-1 L

KF373480 Vibrio harveyi [EU373091]

474/475 (99%)


BSD 128-4-2

KF373481 Vibrio harveyi strain 090212 [GU262992]

477/477 (100%)


BSD 128-5 KF373482 Vibrio harveyi strain A3 [JN391271]

424/428 (99%)


BSD 128-6 KF373483 Vibrio parahaemolyticus isolate Mm007 [FR686999]

475/475 (100%)


BSD 128-7 L


474/474 (100%)


BSD 128-8-1-1

KF373485 Vibrio ponticus strain AN62 [JQ409384]

478/478 (100%)


BSD 128-8-1-2 L

KF373486 Vibrio campbellii strain VSD807 [KC534398]

475/476(99%)


BSD 13 KF373487 Vibrio communis strain P274 [JF836181]

477/478 (99%)


BSD 14 KF373488 Vibrio azureus strain F77118 [HQ908716]

475/476 (99%)


BSD 15 KF373489 Vibrio parahaemolyticus strain 448 [JN188417]

473/475 (99%)


BSD 16-10 KF373490 Bacillus cereus strain B3 [JN252053]

475/475 (100%)

Firmicutes; Bacillales; Bacillaceae; Bacillus; Bacillus cereus group

BSD 16-11 KF373491 Vibrio shiloi [AF007115] 472/475 (99%)


BSD 16-2-1 KF373492 Vibrio communis strain F75214 [HQ161741]

476/477 (99%)


BSD 16-2-2 KF373493 Lysinibacillus fusiformis 476/476 Firmicutes; Bacillales;

P a g e | 137

[JQ897408] (100%) Bacillaceae; Lysinibacillus

BSD 16-3 KF373494 Vibrio ponticus strain AN62 [JQ409384]

476/476 (100%)


BSD 16-5 KF373495 Alteromonadales bacterium fav-2-10-05 [FJ041083]

460/460 (100%)


BSD 16-7 KF373496 Alteromonadales bacterium fav-2-10-05 [FJ041083]

459/459 (100%)


BSD 16-8 L KF373497 Vibrio communis strain F75214 [HQ161741]

476/477 (99%)


BSD 2-10 L KF373498 Vibrio rotiferianus strain HT110622 [JQ792238]

476/476 (100%)



473/474 (99%)


BSD 2-7-1 KF373500 Lysinibacillus fusiformis [JQ897408]

474/474 (100%)

Firmicutes; Bacillales; Bacillaceae; Lysinibacillus

BSD 2-7-2 KF373501 Vibrionaceae bacterium PaD2.06 [GQ406614]

431/433 (99%)

Gammaproteobacteria; Vibrionales; Vibrionaceae

BSD 2-8 L KF373502 Vibrio parahaemolyticus isolate Mm007 [FR686999]

475/475 (100%)


BSD 2-9-1 KF373503 Vibrio azureus strain HNS029 [JN128263]

472/476 (99%)


BSD 2-9-2 KF373504 Vibrio natriegens strain AUCASVE1 [JQ043186]

474/475 (99%)


BSD 256-5 KF373505 Bacillus anthracis strain: PD7-4 [AB506122]

474/474 (100%)


BSD 32-5 L KF373506 Vibrio natriegens strain AUCASVE1 [JQ043186]

474/475(99%)


BSD 32-6-1 KF373507 Vibrio harveyi [EU373091]

476/476 (100%)


BSD 32-6-2 KF373508 Ferrimonas sp. A3B-58 [AB193755]

466/469 (99%)

Gammaproteobacteria; Alteromonadales; Ferrimonadaceae; Ferrimonas

BSD 4-4 KF373509 Vibrio harveyi isolate VHJR19 [DQ995251]

477/477 (100%)


BSD 4-5 KF373510 Vibrio harveyi isolate VHJR19 [DQ995251]

475/476 (99%)


BSD 4-7 KF373511 Vibrionaceae bacterium PaD2.06 [GQ406614]

433/433 (100%)


BSD 4-8 L KF373512 Vibrio parahaemolyticus isolate Mm007 [FR686999]

475/475 (100%)


BSD 4-9 KF373513 Vibrio harveyi strain S090801 [HM236045]

474/474 (100%)

Gammaproteobacteria; Vibrionales; Vibrionaceae;

P a g e | 138

Vibrio

BSD 64-1-1 KF373514 Bacillus cereus strain 2 [JX439638]

467/467 (100%)


BSD 64-1-2 KF373515 Vibrio harveyi strain S090801 [HM236045]

474/475 (99%)


BSD 64-2-1 KF373516 Bacillus anthracis strain: PD7-4 [AB506122]

477/477 (100%)


BSD 64-2-2 KF373517 Vibrio fortis strain VPMP50 [JQ663920]

477/478 (99%)


BSD 8-2 L KF373518 Vibrio harveyi strain BK2 [HM355956]

473/475 (99%)


BSD 8-3 KF373519 Vibrio azureus strain HNS022 [JN128256]

475/476 (99%)


BSD 8-4 KF373520 Vibrio harveyi strain S090801 [HM236045]

475/476 (99%)


BSD 8-5 KF373521 Vibrionaceae bacterium PaD2.06 [GQ406614]

432/433 (99%)



471/472 (99%)


BSF 11 KF373523 Lysinibacillus boronitolerans [FJ237498]

473/473 (100%)

Firmicutes; Bacillales; Bacillaceae; Lysinibacillus

BSF 12 KF373524 Halomonas sp. 612M-23 [GU371676]

451/452 (99%)


BSF 14 KF373525 Halomonas sp. 612M-23 [GU371676]

449/450 (99%)


BWC 04-1 KF373526 Rhodobacter capsulatus strain PSB-06 [FJ866784]

463/463 (100%)


BWC 13 KF373527 Halomonas sp. 612M-23 [GU371676]

449/450 (99%)


BWC 14 KF373528 Vibrio harveyi strain S090801 [HM236045]

473/473 (100%)


BWC 15 KF373529 Alteromonas macleodii [AB238950]

457/457 (100%)

Gammaproteobacteria; Alteromonadales; Alteromonadaceae; Alteromonas

BWC 16 L KF373530 Vibrio harveyi strain HL19 [JQ948038]

474/475 (99%)


BWC 17 KF373531 Vibrionaceae bacterium PaD2.06 [GQ406614]

423/424 (99%)


P a g e | 139

BWC 18 KF373532 Vibrio harveyi strain HL19 [JQ948038]

473/476 (99%)


BWC 19 L KF373533 Vibrio harveyi strain HL19 [JQ948038]

475/476 (99%)


P a g e | 140

Table A.6: Screening of antimicrobial (PKS/NRPS) and DMSP-degrading (dmdA/dddP) genes in coral mucus isolates.

Samples Presence of genes

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.

P a g e | 141

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

and 32 °C.

Samples

Temperature

28 °C 30 °C 32 °C

Total inhibition

Average zone size

(cm)

Total inhibition

Average zone size

(cm)

Total inhibition

Average zone size

(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

P a g e | 142

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

30 and 32 °C.

Samples

Temperature

28 °C 30 °C 32 °C

Total inhibition

Average zone size

(cm)

Total inhibition

Average zone size

(cm)

Total inhibition

Average zone size

(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

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Coastal Bacterial Communities_Their Potential Roles in DIMETHYLSULPHIDE (DMS)

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