Isolation and Purification of Planctomycetes Associated with Harbor and Lagoon Seagrasses of the Red Sea
Total Page：16
File Type：pdf, Size：1020Kb
Isolation and Purification of Planctomycetes associated with Harbor and Lagoon Seagrasses of the Red Sea Thesis by Holly Bream In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia 2 EXAMINATION COMMITTEE APPROVALS FORM The thesis of Holly Bream is approved by the examination committee. Committee Chairperson Dr. Vladimir Bajic Committee Member Dr. Uli Stingl Committee Member Dr. Pascal Saikaly Committee Member Dr. Feras Lafi 3 ABSTRACT Isolation and Purification of Planctomycetes associated with Harbor and Lagoon Seagrasses of the Red Sea Holly Bream Planctomycetes are members of a unique superphylum along with Verrucomicrobia and Chlamydia, situated in the domain Bacteria. They have distinct structural and morphological features, and discoveries made through phylogenetic analysis indicate their important role in nutrient cycling. Their known relationships with marine photosynthetic organisms led to the formation of this study's hypothesis, namely, that Planctomycetes can be isolated from the biofilm of seagrass species of the Red Sea using cultivation techniques adapted for these organisms. Preparation of solid and liquid media using M13 with both agar and gellan, and 2216 Difco Marine Broth full- strength, 1/10-strength, and with antibiotics, resulted in the successful isolation of Planctomycetes as confirmed by morphological examination and transmission electron microscopy. The work performed in this study provides a solid foundation for further studies to elucidate the metabolic pathways and ecological significance of Planctomycetes. 4 ACKNOWLEDGEMENTS Many thanks to Dr. Vladimir Bajic, my committee chair, for his support of the microbiological work we were able to perform in the CBRC lab, and for patiently awaiting the arrival of data that the CBRC team can play with. Thank you to Dr. Feras Lafi, without whom this work would not have been possible, from the conception of the project through the outline of the thesis. I would also like to thank the invaluable members of my thesis committee, Dr. Uli Stingl and Dr. Pascal Saikaly, for their insight and constructive criticism of the draft stages of this work, and for being willing to help me improve it even over the course of a very hot Saudi summer. I much appreciate the time and effort put in by Dr. Abdulaziz Al-Suwailem and Dr. Zenon Batang of the CMOR Core Lab. Their professionalism and knowledge greatly enhanced the collection and analysis phases of the experiment. A special thanks goes to Jay Larson, who reminded me when it was appropriate to eat meals, shower, and occasionally take breaks during the preparation of this thesis. I may not have survived through its completion otherwise. To the newly married Mrs. Soha Alamoudi, I would like to extend my appreciation for keeping me company during the long hours we shared under the fume hood, and until all hours of the night after the collection trips. She inspired me to work harder every day and is a model of strength and discipline. Last but not least, I would like to thank my family. They may be 7,500 miles away physically, but were with me in spirit throughout the writing of this thesis as I missed birthdays, graduations, and weddings. I dedicate this to you. 5 TABLE OF CONTENTS Page EXAMINATION COMMITTEE APPROVALS FORM ...................................................... 2 ABSTRACT ................................................................................................................. 3 ACKNOWLEDGEMENTS ............................................................................................. 4 TABLE OF CONTENTS ................................................................................................ 5 LIST OF ABBREVIATIONS ........................................................................................... 6 LIST OF FIGURES ....................................................................................................... 9 LIST OF TABLES ......................................................................................................... 11 CHAPTER 1: INTRODUCTION……………………………………………………………………….. ......... 13 CHAPTER 2: METHODOLOGY……………………………………………………………………….. ........ 49 CHAPTER 3: RESULTS AND DISCUSSION……………………………………………………….. ........ 64 CHAPTER 4: CONCLUSION……………………………………………………………………….. ............. 79 REFERENCES………………………………………………………………………………………………………….. 83 APPENDICES…………………………………………………………………………………………………………... 92 6 LIST OF ABBREVIATIONS Acetyl-CoA acetyl co-enzyme A ATP adenosine triphosphate β-RFAP β-ribofuranosylaminobenzene 5ʹ-phosphate B (Appendix B) broth C1 one-carbon molecule CCD charge-coupled device CMOR Coastal and Marine Resources Core Lab dd H2O double-distilled water EDTA ethylenediaminetetraacetic acid F (Appendix B) flask Fae formaldehyde-activating enzyme FISH fluorescence in situ hybridization Fmd formyl methanofuran dehydrogenase Ftr formylmethanofuran-tetrahydromethanopterin N- formyltransferase G (Appendix B) glycerol H4F tetrahydrofolate H4MPT methylene tetrahydromethanopterin HAO hydroxylamine:oxygen oxidoreductase HD hydrazine dehydrogenase HH hydrazine hydrolase 7 ICM intracytoplasmic membrane KAUST King Abdullah University of Science and Technology LECA last eukaryotic common ancestor LGT lateral gene transfer LUCA last universal common ancestor MC membrane coat Mch methenyl-H4MPT cyclohydrolyase MtdA methylene-H4MPT dehydrogenase A NIR nitrite oxidoreductase NPC nuclear pore complex NTA nitrilotriacetate Omp2 outer membrane protein 2 PCR polymerase chain reaction PMF proton motive force PVC Planctomycetes-Verrucomicrobia-Chlamydiae PVC polyvinyl chloride (in Methodology only) redox reduction oxidation RO research objective rpm revolutions per minute S (Appendix B) sludge sed (Appendix B) sediment SRSW sterilized Red Sea water 8 TEM transmission electron microscopy TRFLP terminal restriction fragment length polymorphism 9 LIST OF FIGURES Figure 1. Location of Al Kharrar Lagoon........................................................................... 16 Figure 2. Location of Rabigh Harbor................................................................................ 19 Figure 3. C. serrulata as depicted by Seagrass-Watch…………………………………………………..23 Figure 4. H. uninervis as documented by the Systematic Marine Biodiversity Inventory System………………………………………………………………………………………………………………….………24 Figure 5. H. stipulacea as identified by DAISIE Species Factsheet…………………………….……25 Figure 6. Diagram of the cellular organization of Pirellula and Isosphaera species, and “Candidatus” Brocadia anammoxidans and Gemmata species from Fuerst....................32 Figure 7. Schematic of possible anammoxosome membrane reactions involved in catabolism and reverse electron transport chain.............................................................44 Figure 8. Map of sampling sites at Al Kharrar Lagoon and Rabigh Harbor.......................57 Figure 9. Seagrass samples collected in Rabigh Harbor and Al Kharrar Lagoon with their taxonomic diagnosis displayed.........................................................................................65 Figure 10. Photo of 2216 plate inoculated with undiluted H. uninervis biofilm sample...67 Figure 11. Bacteria present in the white rosette-like flakes accompanying the ball-like structures identified in enrichment broth........................................................................70 Figure 12. Specimens from a colony picked on an anti-2216 plate that was inoculated with sludge swabbed from the side of a flask enriching H. stipulacea abnormal biofilm sample………………………………….......................................................................................... 71 Figure 13. Specimens processed from sample grown on an M13 plate which was streaked from broth enrichment of sediment collected near H. stipulacea and observed using TEM after negative staining with uranyl acetate.....................................................72 Figure 14. Comparison of suspected Pirellula species with confirmed Pirellula strains…………………………………………….................................................................................76 Figure 15. Flagella and crateriform structures clearly visible in cell from FHS-sed2 sample............................................................................................................................ 125 10 Figure 16. Close-up of complex sheathed flagella......................................................... 126 Figure 17. Specimen displaying polar fibrillar appendages............................................ 127 Figure 18. Two cells attached to each other by some unknown mechanism, both displaying polar fibrillar appendages............................................................................. 128 11 LIST OF TABLES Table 1.Rabigh Harbor and Al Kharrar Lagoon Environmental Parameters…………………..58 Table 2. Description of colonies growing from all three concentrations of H. uninervis inoculum on all five media types as of 29/4/12, with updates on isolates that were picked previously.............................................................................................................94 Table 3. Description of colonies growing from all three concentrations of C. serrulata inoculum on all five media types as of 29/4/12, with updates on isolates that were picked previously………………............................................................................................95 Table 4. Description