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Approches Moléculaires De La Diversité Microbienne De Deux Environnements Extrêmes : Les Sources Hydrothermales Profondes Et Les Réservoirs Pétroliers Erwan Corre

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Approches Moléculaires De La Diversité Microbienne De Deux Environnements Extrêmes : Les Sources Hydrothermales Profondes Et Les Réservoirs Pétroliers Erwan Corre Approches moléculaires de la diversité microbienne de deux environnements extrêmes : les sources hydrothermales profondes et les réservoirs pétroliers Erwan Corre To cite this version: Erwan Corre. Approches moléculaires de la diversité microbienne de deux environnements extrêmes : les sources hydrothermales profondes et les réservoirs pétroliers. Biologie moléculaire. Université de Bretagne Occidentale, 2000. Français. tel-01115381 HAL Id: tel-01115381 https://hal.sorbonne-universite.fr/tel-01115381 Submitted on 12 Feb 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Avertissement Au vu de la législation sur les droits d'auteur, ce travail de thèse demeure la propriété de son auteur, et toute reproduction de cette oeuvre doit faire l'objet d'une autorisation de l'auteur. (cf Loi n°92-597; 1/07/1992. Journal Officiel, 2/07/1992) THESE présentée pour l'obtention du grade de Docteur de l'Université de Bretagne Occidentale (Mention Microbiologie) Erwan CORRE Approches moléculaires de la diversité microbienne de deux environnements extrêmes : les sources hydrothermales profondes et les réservoirs pétroliers Soutenue le Mercredi 2 Février 2000 devant le jury composé de : MM. P. CAUMETTE, Professeur, Université de Pau Examinateur M. MAGOT, Responsable de Laboratoire, Sanofi, Labège Rapporteur P. NORMAND, Directeur de Recherche CNRS, Université Lyon I Rapporteur B. PICARD, Professeur, Université de Bretagne Occidentale Président D. PRIEUR, Professeur, Université de Bretagne Occidentale Examinateur A.L. REYSENBACH, Professeur, Université de Portland, USA Examinatrice Remerciements Le travail présenté dans ce mémoire a été réalisé avec le soutien financier de la Communauté Urbaine de Brest Je remercie tout d'abord Daniel Prieur pour m'avoir accueilli dans son équipe de recherche, d'abords en DEA puis en thèse et de m'avoir fait confiance tout au long de ces années. Mes remerciements s'adressent tout particulièrement à Christian Jeanthon pour son soutien, sa patience et sa disponibilité. Je tiens à lui exprimer ici ma reconnaissance pour toutes les discussions enrichissantes, les conseils avisés et les directives salutaires qu'il a su m'indiquer tout au long de ce travail. Merci profondément pour le temps consacré à la correction des articles. Je remercie également Anna-Louise Reysenbach pour m'avoir accueilli dans son laboratoire à Rutgers et de m'avoir permis de travailler sur les échantillons de Vent Cap. Son expérience en biologie moléculaire, ses conseils en phylogénie m'ont été grandement utiles. Je la remercie également de m'avoir fait partager durant mon séjour à Rutgers sa passion pour les chevaux et pour le trafic de fromages et de vins français. Je remercie également les membres du jury : Pierre Caumette, Philippe Normand, Michel Magot et Bertrand Picard d'avoir accepté de juger mon travail de thèse. Je tiens à adresser mes plus profonds remerciements à l'ensemble des étudiants avec qui j'ai eu la joie de travailler : Cédric Hobel (qui a essuyé les plâtres), la brillante Anne Postec, l'inénarrable mais néanmoins charmante Catherine Charpentier et enfin le précieux Laurent Toffin avec qui j'ai passé une année de travail formidable. Une grande partie des résultats présentés dans ce manuscrit sont le fruit de leur acharnement et de leur persévérance. J'adresse un remerciement tout particulier à Tristan Barbeyron pour m'avoir formé aux techniques moléculaires, pour sa disponibilité et son savoir intemporel sur tout ce qui à trait la biologie moléculaire, la microbiologie, la radioactivité, Serge Lama et les 24 heures du Mans. Merci également à Hermie Harmsen pour ses conseils avisés à la paillasse comme à l'e-mail. Je remercie tout particulièrement Olivier Collin, Stéphan Jouannic et Jean Ranc pour les heures qu'ils ont accepté de passer à lire et corriger ce manuscrit. Spéciale dédicace à Stéphane et Olivier pour leur travail de pionniers dans la jungle des fautes d'orthographes et les marais fangeux des accords mal à propos. Je remercie également tous les membres du laboratoire. En particulier Stéphane et Edmond pour leur amitié, les foulées et la sueur que nous avons partagé (enfin surtout eux) sur de nombreux kilomètres. Jenny pour m'avoir supporté et pour nous être épaulé mutuellement dans la dernière (longue, très longue) ligne droite. Marc, Patrick, Olivier, Jean-Louis, Laurent, Viggo, Soizick et Valérie pour avoir survécu dans les bureaux que nous avons partagés. Enfin je remercie les membres de la Station Biologique de Roscoff pour leur accueil, leurs compétences et la formidable ambiance qu'ils savent entretenir dans leur laboratoire et dans les couloirs de la station. Merci à Odile, Claire, Catherine's, Jeff's, Didier, Vincent et Lolo, à la famille Collin à 2 jambes ou 4 pattes pour leur soutient moral et véliplanchesque ainsi que pour leur accueil tout au long de mon travail. Merci à l'ensemble de ma famille, à Juliette de m'avoir aiguillé vers la Station Biologique, à mes parents, "beaux"-parents et grands-parents de leur soutien. Merci enfin à Anne d'avoir su supporter au quotidien mes sautes d'humeurs, mes absences et d'avoir finalement, plus qu'aucune autre personne, contribué à ce que je mène à bien ce travail. Introduction générale La microbiologie de l'environnement est une discipline jeune, qui a pour vocation d'étudier les relations qu'entretiennent les micro-organismes avec leur biotope. Si l'adage populaire n'accorde que peu d'influence à ce qui est invisible à l'oeil et si la présence de micro-organismes est souvent associée à une nuisance plutôt qu'à un bienfait, le rôle des micro-organismes est pourtant prépondérant dans le fonctionnement de nombreux écosystèmes naturels et anthropiques. L'étude des micro-organismes a longtemps été liée à la capacité des microbiologistes à recréer in vitro des conditions favorables à la croissance des communautés microbiennes. Or, des écosystèmes complexes, soumis à des contraintes physico-chimiques extrêmes telles que, de fortes températures, de fortes pressions ou l'absence d'oxygène, ou à des fluctuations de sources de nutriments ou d'énergie, sont particulièrement difficiles à simuler au laboratoire. Aussi, dans ces écosystèmes ,comme dans d'autres étudiés déjà depuis fort longtemps tels que le sol où l'eau de mer, un grand nombre de micro-organismes ont échappé à toute méthode de culture. Le développement des techniques de biologie moléculaire et l'utilisation de la molécule d'ARN ribosomique comme marqueur évolutif a permis de s'affranchir des étapes de culture et a permis d'asseoir sur des bases phylogénétiques les relations taxinomiques s'établissant entre les différents organismes vivants. Le travail présenté dans ce mémoire a pour objet l'étude de la diversité microbienne de deux écosystèmes considérés comme extrêmes : les sources hydrothermales profondes et les réservoirs pétroliers. Les techniques mises en oeuvre sont fondées soit sur l'analyse des ARN ribosomiques (ARNr) sans étape préalable de culture, par séquençage ou hybridation in situ, soit sur le positionnement phylogénétique de souches isolées de ces environnements. Avant de présenter les résultats de ce travail sous la forme d'articles en préparation, soumis ou publiés, je me propose : - de présenter une revue des techniques d'écologie microbienne classiques et moléculaires couramment utilisées pour accéder à la diversité des écosystèmes microbiens, de présenter des moyens d'accéder à la diversité fonctionnelle de ces écosystèmes et d'insister sur les biais inhérents à chacune de ces techniques, - d'effectuer un tour d'horizon des différents habitats microbiens qui ont été étudiés ces dernières années et de présenter pour chacun de ces habitats les apports des techniques moléculaires dans la description de la diversité et la découverte de nouveaux groupes phylogénétiques, et enfin - de présenter et de discuter les différentes techniques que nous avons mises en oeuvre pour étudier la diversité microbienne d'un écosystème pétrolier continental et d'un site hydrothermal profond. ii Abréviations IPTG : isopropylthio-ß-D-galactoside ISRT : In Situ Reverse Transcription A : Adénosine Knuc : distance phylogénétique ADN: Acide désoxyribonucléique Kpb : Kilo paires de bases ADNc : ADN cloné LB (milieu liquide ou gélose) : Luria-Bertani ADNr 16S : ADN codant pour la sous unité 16S de l'ARN ribosomal LFRFA : Low Frequency Restriction Fragment Analysis ADNr : ADN codant pout l'ARN ribosomal LMW RNA: ARN de faible poids moléculaire AFLP : Amplified Fragment Length Polymorphism LPS : Lipopolysaccharides APS : Ammonium Persulfate LUCA : Last Unversal Common Ancestor ARDRA : Amplified rDNA Restriction Analysis. MAR : Middle Atlantic Ride ARN : Acide ribonucléique MPN : Most probable number ARNm : ARN messagé NPP : Nombre le plus probable ARNr 16S : sous unité 16S de l'ARN ribosomal OTU : Operational Taxonomic Unit ARNr : ARN ribosomale p/v : (masse à volume) ATP : Adénosine tri-phosphate pb : paire de base BrdU : bromodéoxyuridine. PCB : PolyChlorinated Biphenyl BSA : Bovine Serum Albumin PCR : Polymerase Chain Reaction
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