Sevasti Filippidou

Sevasti Filippidou University of Neuchatel, 2016 Sporulation Capability and Metabolic Mechanisms of Endospore-Forming Firmicutes under Conditions Limiting for Growth and Survival A dissertation submitted to the University of Neuchatel for the degree of Docteure ès Sciences by Sevasti Filippidou, MSc Molecular Genetics Accepted by the Jury: Prof. Pilar Junier, thesis director, University of Neuchatel Prof. Maarten Voordow, University of Neuchatel Prof. Melanie Blokesch, EPFL, Lausanne Dr. David Russel Johnson, Eawag, Dübendorf Dr. Paul Herron, University of Strathclyde, Glasgow, UK Defended the 11th April 2016 University of Neuchatel Faculté des sciences Secrétariat-décanat de Faculté Rue Emile-Argand 11 2000 Neuchâtel - Suisse Tél: + 41 (0)32 718 2100 E-mail: [email protected] IMPRIMATUR POUR THESE DE DOCTORAT La Faculté des sciences de l'Université de Neuchâtel autorise l'impression de la présente thèse soutenue par Madame Sevasti Filippidou Titre: “Sporulation capability and metabolic mechanisms of endospore-forming Firmicutes under conditions limiting for growth and survival” sur le rapport des membres du jury composé comme suit: - Prof. Pilar Junier, directrice de thèse, Université de Neuchâtel, Suisse - Prof. ass. Maarten Voordouw, Université de Neuchâtel, Suisse - Prof. Melanie Blokesch, EPF Lausanne, Suisse - Dr David R. Johnson, EAWAG, Dübendorf, Suisse - Dr Paul Herron, University of Strathclyde, Glasgow, UK Neuchâtel, le 28 avril 2016 Le Doyen, Prof. B. Colbois Imprimatur pour thèse de doctorat www.unine.ch/sciences This work is dedicated to The memory of Marilena Vourkou, for her inspiration to life, Entropia, that has shaped me to what I am, Dimitris Serafis, without whom I wouldn’t have reached that far. Acknowledgements Firstly, I would like to thank my professor Pilar Junier for being a very supportive tutor and a real mentor throughout all these years. Pilar has taught me, guided me, encouraged me, and gave me opportunities I never imagined I could have. She is constantly showing how academic science should be and inspires her team, being a reassurance for brighter days. I would also like to thank Mrs. Nicole Jeanneret for having introduced me to the lab and who has been a good-hearted advisor on techniques, laboratory practices and French since day one.Many thanks also go to all my lab collaborators, and more specifically the ‘spores’ team, Dr. Matthieu Bueche, Dr. Ludovic Roussel-Delif, Dr. Sathiya Ganesan, Mr. Loic Sauvain, Mrs. Marion Jaussi and Mr. Christophe Paul for their help, contribution and pleasant atmosphere in the group. In particular, Dr. Tina Wunderlin has generously coached, advised, supported, and shared with me in any possible way. I thank her for being there to encourage me since I could never imagine for a better colleague, office mate and friend. I am most grateful to Dr. Thomas Junier for almost all bioinformatic analysis and for patiently spending his time teaching me phylogenetic tools and rules of scientific writing. Moreover, I would like to thank Dr. Radu Slobodeanu for his statistics advice for the article presented in chapter three. Finally, I am greatful to all our collaborators, and especially the team of Dr. Patrick S. Chain, without whose valuable contributions, this work would never be completed. Thanks also go to all the students who have worked with me, during their learning stage: Mrs. Amandine Pillonel, Mr. Andrej Al-Dourobi, Mrs. Ilona Palmieri, technicians; Mr. Patricio Munoz-Ruffat, undergraduate student and Mr. Fabio Palmieri, master student. I hope their learning procedure was as constructive as mine during our collaboration. Special thanks to Mrs. Anna Voulgari for being the perfect ecological eye during sampling campaigns, and who, along with Mrs. Vassiliki Mpelogianni, Dr. Tina Wunderlin and Dr. Nikos Mitakidis have critically read my general introduction. Mrs. Monica Albini has patiently helped me during the last hours before submission and I thank her for that. Dr. Vincent Hervé has translated my summary into “good” French, for which I am most grateful. I thank my thesis jury for accepting to evaluate this work and provide feedback. Special thanks go to what I call “an army of people” that helped and supported me throughout these years and which include my family and dearest friends. Although some of them are already acknowledged, all of them have always been available to support and encourage me in any possible manner. Cover photo: Endospore-forming Firmicutes forming a sphere with copper carbonate crystals. Scanning Electron Microscopy. Photo taken by S. Filippidou. LAMUN 2012 Summary Life, as we know it, has physical and chemical limits. More precisely, physical and chemical environmental parameters set the thresholds for reproduction, metabolism and survival for the organisms known to date. Environmental conditions unsuitable for survival and development are the rule rather than the exception in most habitats. Microorganisms have developed various strategies to withstand environmental conditions that limit active growth. A microbial group that displays a large array of strategies to resist adversity is endospore‐forming Firmicutes. These strategies range from the formation of resting states (endospores), to biofilms and metabolic adaptation. These strategies are a costly biological investment and therefore might affect their success. Endospore‐forming Firmicutes have been isolated from various environments, including extreme habitats. Paradoxically, in diversity studies they are either absent from these environments or they represent a small fraction of the microbial community. Using a profile analysis of the spo0A gene, unique to endospore‐forming Firmicutes, we have shown that they cannot be found in publically available metagenomic datasets, despite the fact that many of these datasets correspond to well‐known habitats for endospore‐ forming Firmicutes. We have shown that this bias is likely due to the fact that commonly used methodological approaches are inefficient. Moreover, we have shown experimentally that an improved DNA extraction can improve detection in amplicon sequencing; however, this was not the case for shotgun classification. Although this group is known to colonize every habitat on Earth, endospore‐firming Firmicutes are not prevalent in all habitats. Their energy‐demanding survival strategies become an actual benefit only when multiple physical and chemical limits of life are present. Extremity favors the presence of the survival strategies deployed by endospore‐forming Firmicutes. More specifically, we have shown that extreme environmental conditions are an important factor for the survival strategy of sporulation to evolve. Two novel discoveries support this suggestion. Firstly, we have shown that extremity is a driving force for sporulation in a species that was not known to sporulate, Serratia ureilytica. Secondly, we demonstrated that the ability to sporulate is not lost when there is environmental pressure. That was the case of Kurthia spp. a previously known asporogenic genus. The common ancestor of Kurthia, and the endospore‐forming Firmicutes was able to produce spores, however sporulation is considered to have been lost within the lineage of Kurthia. The genomic analysis and the microscopic observation of a Kurthia sp. strain isolated from a geothermal reservoir reveal that the sporulation pathway has not been lost, and that Kurthia is not an asporogenic but rather a cryptosporulating genus. The survival strategy of sporulation makes endospore‐forming Firmicutes capable of tolerating adverse conditions and thriving in extreme environments. A novel species that has a particular ecological niche in geothermal reservoirs was discovered; Anoxybacillus geothermalis was revived under laboratory conditions and is hypothesized to have remained inactive in the reservoir since the Permian age. More isolates belonging to the same species were also discovered in different geothermal reservoirs. 1 Extreme environments do not only allow endospore‐forming Firmicutes to deploy their sporulation strategy but also their high metabolic diversity. Manganese oxidation and copper reduction of endospore‐forming Firmicutes in natural, uncontaminated environments, were studied leading to the conclusion that metal tolerance is a widespread phenomenon in unrelated aerobic endospore‐forming Firmicutes from natural uncontaminated environments. Finally, in saline habitats, both metabolic strategies are deployed, resulting in an impressive diversity of endospore‐forming Firmicutes. Biochemical, genomic, ecological and environmental data are pieces to fill in the puzzle of adaptations of endospore‐forming Firmicutes in extreme habitats. Keywords: Microbial Ecology, Firmicutes, endospore‐forming Firmicutes, sporulation, Kurthia, Serratia, Anoxybacillus geothermalis, Manganese Oxidation, Copper Tolerance, Halophiles. 2 Résumé La vie, telle que nous la connaissons, possède des limites physiques et chimiques. Plus précisément, des paramètres environnementaux physiques et chimiques limitent la reproduction, le métabolisme et la survie des organismes vivants décrits à ce jour. Des conditions environnementales défavorables à la survie et au développement biologique sont la règle plutôt que l'exception dans la plupart des habitats. Les microorganismes ont cependant développé différentes stratégies pour résister aux conditions environnementales qui limitent leur croissance. Parmi ces microorganismes, le groupe des Firmicutes endosporulantes présente

Load more