Dilution-to-extinction culturing of SAR11 members and other marine bacteria from the Red Sea Thesis written by Roslinda Mohamed In Partial Fulfillment of the Requirements For the Degree of Master of Science (MSc.) in Marine Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia December 2013 2 The thesis of Roslinda Mohamed is approved by the examination committee. Committee Chairperson: Ulrich Stingl Committee Co-Chair: NIL Committee Members: Pascal Saikaly David Ngugi King Abdullah University of Science and Technology 2013 3 Copyright © December 2013 Roslinda Mohamed All Rights Reserved 4 ABSTRACT Dilution-to-extinction culturing of SAR11 members and other marine bacteria from the Red Sea Roslinda Mohamed Life in oceans originated about 3.5 billion years ago where microbes were the only life form for two thirds of the planet’s existence. Apart from being abundant and diverse, marine microbes are involved in nearly all biogeochemical processes and are vital to sustain all life forms. With the overgrowing number of data arising from culture-independent studies, it became necessary to improve culturing techniques in order to obtain pure cultures of the environmentally significant bacteria to back up the findings and test hypotheses. Particularly in the ultra-oligotrophic Red Sea, the ubiquitous SAR11 bacteria has been reported to account for more than half of the surface bacterioplankton community. It is therefore highly likely that SAR11, and other microbial life that exists have developed special adaptations that enabled them to thrive successfully. Advances in conventional culturing have made it possible for abundant, unculturable marine bacteria to be grown in the lab. In this study, we analyzed the effectiveness of the media LNHM and AMS1 in isolating marine bacteria from the Red Sea, particularly members of the SAR11 clade. SAR11 strains obtained from this study AMS1, and belonged to subgroup 1a and phylotype 1a.3. We also obtained other interesting strains which should be followed up with in the future. In the long run, results from this study will enhance our knowledge of the pelagic ecosystem and allow the impacts of rising temperatures on marine life to be understood. 5 ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my supervisor Dr Ulrich Stingl who has supported me throughout this thesis. I appreciate his support and guidance, and value the freedom he gives to work independently. I attribute the completion of this thesis to his encouragement and effort, and I could not have wished for a better supervisor. I am also truly grateful to Dr Saikaly and Dr Ngugi for accepting the invitation to being part of my thesis defense committee and appreciate his valuable time and feedbacks. I would also like to express my appreciation to the Coastal Marine Resources Core Lab (CMOR) crew at KAUST for their assistance in getting the seawater samples used in this study. Principally, Ioannis Georgakakis, for his help in making physical measurements of the water each time of sampling. My special thanks goes out to the RSRC family, especially to members of the Stingl team, for creating a warm working environment throughout my stay in KAUST. I give special thanks to Francy Jimenez for her time and help in various laboratory experiments, and for her patience in guiding me throughout this project. Also, I am truly grateful to Zahraa Alhashem for her contributions in looking at the nitrogen-fixers. I would definitely not have been able to complete this study single-handedly. Finally, I would like to thank my family and friends for their endless encouragement throughout the years, especially to my parents for supporting me emotionally and understanding of my visions in life. 6 TABLE OF CONTENTS EXAMINATION COMMITTEE APPROVALS ___________________________ 2 COPYRIGHT ____________________________________________________ 3 ABSTRACT _____________________________________________________ 4 ACKNOWLEDGEMENTS __________________________________________ 5 TABLE OF CONTENTS ___________________________________________ 6 LIST OF ABBREVIATIONS _________________________________________ 8 LIST OF FIGURES _______________________________________________ 9 LIST OF TABLES _______________________________________________ 10 1. Introduction ________________________________________________ 11 1.1 The Red Sea ___________________________________________ 11 1.2 Composition of Marine Microbial Communities _________________ 13 1.3 Microbes and Their Roles in the Marine Food Web ______________ 14 1.4 The Ubiquitous SAR11 Clade ______________________________ 17 1.5 Microdiversity Within the SAR11 Clade _______________________ 19 1.6 Microbial Communities and SAR11 in the Red Sea ______________ 20 1.7 Proteorhodopsin _________________________________________ 22 1.8 Significance of Culturing Marine Bacteria ______________________ 23 1.9 Dilution-to-extinction Culturing ______________________________ 24 1.10 Research Objectives ____________________________________ 26 2. Material and Methods ________________________________________ 27 2.1 Collection of Seawater ___________________________________ 27 2.2 Preparation of Low Nutrient Heterotrophic Media (LNHM) ________ 27 2.3 Preparation of Artificial Seawater Medium (AMS1) ______________ 28 2.4 Preparation of Vessels for Dilution-to-extinction Culturing ________ 29 2.5 Inoculation of Media _____________________________________ 30 2.6 Screening of Cell Cultures for Growth ________________________ 31 2.7 DNA Extraction and Gene-specific Sanger Sequencing __________ 32 2.8 Long-term Storage ______________________________________ 33 2.9 Sequence Assembly and BLAST Searches ___________________ 33 7 2.10 Phylogenetic Analysis ___________________________________ 34 3. Results ____________________________________________________ 35 3.1 Physical Properties of the Water Column ______________________ 35 3.2 Identification of Isolates Grown in LNHM and AMS1 _____________ 35 3.2.1 Growth Behaviors _____________________________________ 35 3.2.2 DNA Extraction and Gene Amplifications ___________________ 36 3.2.3 Total Isolates Identified _________________________________ 38 3.2.4 SAR11 Isolates _______________________________________ 40 3.2.5 Phylogeny of SAR11 Isolates Based on 16S rRNA Gene ______ 42 3.2.6 Phylogeny of SAR11 Isolates Based on ITS Sequences _______ 44 3.2.7 Phylogeny of SAR11 Strains Based on PR Gene ____________ 46 3.2.8 Growth Profile of the SAR11 Isolate- SAR08 ________________ 47 3.3 Identification of Isolates Grown In Nitrogen-deficient Media _______ 48 3.3.1 Growth Behaviors _____________________________________ 48 3.3.2 Total Isolates Identified _________________________________ 51 4. Discussion _________________________________________________ 52 4.1 Effects of Media, Sampling Depth and Cell Densities on Cell Culture Growth _____________________________________ 52 4.2 Isolation of DNA from Cell Cultures __________________________ 55 4.3 Red Sea Bacteria Isolates and Their Possible Functions __________ 56 5. Conclusion and Future Directions ______________________________ 59 6. References _________________________________________________ 60 Appendices ____________________________________________________ 67 8 LIST OF ABBREVIATIONS 3-PGA 3-phosphoglyceric acid ABI Application binary interface Ac-CoA acetyl CoA AMS1 artificial seawater media ATP adenosine triphosphate BLAST Basic Local Alignment Search Tool DMSO dimethyl sulfoxide DMSP dimethylsulfoniopropionate DNA deoxyribonucleic acid DOC dissolved organic carbon DOM dissolved organic matter GBT glycine betaine Gly glycine GOS Global Ocean Sampling HNF heterotrophic nanoflagellates ITS internal transcribed spacer IUPAC International Union of Pure and Applied Chemistry KAUST King Abdullah University of Science and Technology LAS linear alkylbenzenesulfonate LNHM low nutrient heterotrophic media LTP living tree project Mbp mega basepairs MPN most probable count technique NCBI National Centre for Biotechnology Information NJ neighbour-joining PR proteorhodopsin rRNA ribosomal RNA RSRC Red Sea Research Centre SINA SILVA Incremental Aligner TCA tricarboxylic acid THF tetrahydrofolate TMAO trimethylamine oxide UV ultra-violet 9 LIST OF FIGURES Figure 1 Map of the Red Sea 11 Figure 2 Schematic diagram of the microbial loop 15 Central carbon, sulfur and glycine-serine Figure 3 18 metabolism in SAR11 Schematic plan for our 10 m, 100 m and 500 m Figure 4 30 cell cultures Optimization of PCR amplification cycles of Figure 5 38 deep-sea DNA. Figure 6 Taxonomic classification of cultured isolates 39 Phylogenetic tree based on 16S rRNA gene Figure 7 42 sequences Phylogenetic tree based on ITS gene Figure 8 43 sequences Figure 9 Growth curve of SAR08 47 10 LIST OF TABLES List of compounds and their final concentrations in Table 1 28 AMS1 List of primers and conditions used in amplifying Table 2 32 genesof interest Number of strains exhibiting growth relative to total Table 3 35 number of wells per cell density, per depth Number of strains successfully amplified by 16S Table 4 rRNA amplification relative to the total number of 37 strains where DNA were extracted Table 5 List of all 8 strains of SAR11 isolated from this study 40 Table 6 Results from PR-based BLAST search 46 Number of strains exhibiting growth after 2 weeks of Table 7 48 growing in nitrogen-deficient media Number of strains exhibiting growth after second Table 8 49 transfer to light and dark Number of strains exhibiting growth in media with