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Isolation and Physiological Characterization of Microorganisms THÈSE / UNIVERSITÉ DE BRETAGNE OCCIDENTALE présentée par sous le sceau de l’Université européenne de Bretagne Junwei CAO pour obtenir le titre de Préparée au Laboratoire de Microbiologie des DOCTEUR DE L’UNIVERSITÉ DE BRETAGNE OCCIDENTALE Environnements Extrêmes (LM2E, UMR6197 UBO- Mention : Microbiologie) CNRS-Ifremer) de l'Institut Universitaire Européen de École Doctorale des Sciences de la Mer la MER (IUEM) Thèse soutenue le 23 mars 2016 devant le jury composé de: Isolation and physiological Anna-Louise REYSENBACH Professeur, Université de Portland (USA)/ Rapporteur characterization of Robert DURAN Professeur, Université de Pau et des Pays de l'Adour/ Rapporteur microorganisms involved in Vianney PICHEREAU Professeur, Université de Bretagne Occidentale/ Examinateur the sulfur cycle, from under- Zongze SHAO Professeur, 3e Institut d'Océanographie de Xiamen (Chine)/ explored deep-sea Examinateur Karine ALAIN hydrothermal vents Chargée de Recherche, CNRS Brest/ Directrice de thèse Mohamed JEBBAR Professeur, Université de Bretagne Occidentale/ Directeur de thèse Acknowledgements Thank you to the members of my dissertation committee: Christian Jeanthon and Christine baysse, for helping and supporting me for the completion of this dissertation work. I would like to thank my advisors, Prof. Mohamed Jebbar, Dr. Karine Alain, and Prof. Zongze Shao, for giving me the opportunity to study this topic, for giving me helpful advices, and for always supporting and encouraging me. I am extremely thankful to Dr. Karine Alain, for being my direct advisor, for always being there whenever I need help. I want to thank Yann Moalic and Tiphaine Birien, for the help on the genetic manipulation experiments. Many thanks to Myriam Georges, Stéphanie Renard, Stéphane L’Haridon, Samuel Dupont, Cécile Dalmasso, Coraline Mercier, Lois Maignien, Claire Geslin, Alexandre Garlaschelli, Frederique Duthoit, Nadège Bienvenu, Marc Le Romancer, Florian Trigodet, Gwenaelle Le Blay, Odile Trambouze, Matthieu Landreau, Gwendoline Selva, Damien Courtine, Clarisse Lemonnier, and other members of the lab, for being so reliably helpful in the laboratory, anytime. I also would like to express my gratitude to my colleagues from Key Laboratory of Marine Genetic Resources in Xiamen and Harbin Institute of Technology, for helping me grow in many ways during the past years. I am very grateful to the R/V “Xiang Yang Hong Jiu Hao” and “Da Yang Yi Hao” operation teams for helping us to collect the hydrothermal samples. Lastly I would like to express my deepest gratitude to my friends and family, for staying around and supporting me throughout my studying in France. This work was supported by the EU program MaCuMBA, the COMRA project (DY125-15-R- 01), the PICS-InEE Phypress, the PHC Cai Yuanpei Pandore (N° 30412WG), the PHC Cai Yuanpei Provirvent (N° 34634WE) and the National Natural Science Foundation of China (41411130113). 1 Contains Acknowledgements .................................................................................................................... 1 Contains ..................................................................................................................................... 2 Figures ........................................................................................................................................ 4 Tables ......................................................................................................................................... 5 1 Introduction ............................................................................................................................. 6 1.1 Deep-sea hydrothermal vent systems ........................................................................... 6 1.2 Physical and chemical characteristics of deep-sea hydrothermal vents ....................... 7 1.3 Diversity of prokaryotes isolated from deep-sea hydrothermal vents .......................... 8 1.3.1 Archaea ............................................................................................................. 8 1.3.2 Bacteria ........................................................................................................... 15 1.4 Sulfate and sulfur-reducing prokaryotes .................................................................... 33 1.4.1 Sulfate-reducing prokaryotes .......................................................................... 34 1.4.2 Sulfur-reducing prokaryotes ............................................................................ 35 1.5 Cultivation efforts remain crucial .............................................................................. 36 1.6 Genomic studies of deep-sea hydrothermal vents ...................................................... 38 2 Study scope and objectives ................................................................................................... 41 3 Materials and Methods .......................................................................................................... 42 3.1 Collection of bulk samples ......................................................................................... 42 3.2 Solutions and Media ................................................................................................... 42 3.3 Enrichment cultures and isolation of microorganisms ............................................... 44 3.4 DNA extraction .......................................................................................................... 44 3.5 Analysis of the 16S rDNA sequence .......................................................................... 45 3.6 Phenotypic, physiological and chemotaxonomical characterization .......................... 45 3.7 Determination of the pressure range for growth ........................................................ 47 3.8 Genome sequencing and analysis ............................................................................... 47 3.9 Genetic manipulations of Palaeococcus pacificus ..................................................... 47 3.9.1 Construction of a suicide vector ...................................................................... 47 2 3.9.2 Transformation of Palaeococcus pacificus ..................................................... 48 4 Results ................................................................................................................................... 49 4.1 Isolates........................................................................................................................ 49 4.1.1 Isolate from the serpentinized peridotite deep-sea site 30I-TVG05 ................ 49 4.1.2 Isolates from the deep-sea sulfide site JL-Dive94-S01 ................................... 49 4.1.3 Isolates from the deep-sea sulfide site JL-Dive90-S01 ................................... 50 4.2 Desulfovibrio indicus sp. nov. .................................................................................... 52 4.3 Desulfurobacterium indicum sp. nov. ........................................................................ 63 4.4 Thermodesulfatator lithotrophica sp. nov. ................................................................. 71 4.5 Determination of the pressure range for growth of isolates from the deep-sea .......... 79 4.5.1 Piezophilic bacterium Anoxybacter fermentans DY22613T ............................ 79 4.5.2 Piezosensitive bacterium Caloranaerobacter ferrireducens DY22619T ......... 79 4.6 Genetic manipulations of Palaeococcus pacificus DY20341T ................................... 81 4.6.1 Construction of the suicide vector ................................................................... 81 4.6.2 Transformation of Palaeococcus pacificus ..................................................... 83 5 Discussion and Conclusion ................................................................................................... 84 5.1 Isolates from the deep-sea hydrothermal samples ...................................................... 84 5.2 The cultivation of novel microorganisms ................................................................... 86 5.3 Novel microorganisms from underexplored deep-sea vents and their potential ........ 87 5.4 Perspectives ................................................................................................................ 87 6 Annexes ................................................................................................................................. 89 6.1 Annex 1: Article Anoxybacter fermentans gen. nov., sp. nov., a piezophilic, thermophilic, anaerobic, fermentative bacterium isolated from a deep-sea hydrothermal vent. ................................................................................................................................ 90 6.2 Annex 2: Article Physiological features of Halomonas lionensis sp. nov., a novel bacterium isolated from a Mediterranean Sea sediment................................................. 96 References .............................................................................................................................. 107 Abstrcact ................................................................................................................................ 129 3 Figures Figure 1 | Global distribution of known hydrothermal vent fields (Source: InterRidge). ........ 6 Figure 2 | Hydrothermal circulation. ........................................................................................ 7 Figure 3 | The sulfur cycle.
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