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Composition Et Fonctionnement D'une Communauté Microbienne

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Composition Et Fonctionnement D'une Communauté Microbienne Thèse présentée pour obtenir le grade de docteur de l’Université de Strasbourg Discipline : Sciences du Vivant Mention : Aspects moléculaires et cellulaires de la biologie Composition et fonctionnement d’une communauté microbienne au sein d’un drainage minier acide : Approches culturales et fonctionnelles Présentée par : François Delavat Soutenue publiquement le 05/10/2012 Composition du jury Mme Valérie Geoffroy (Université de Strasbourg) Rapporteur interne M. Alain Dufour (Université de Bretagne Sud) Rapporteur externe M. Pascal Simonet (Université de Lyon) Rapporteur externe Mme Marie‐Claire Lett (Université de Strasbourg) Directrice de thèse M. Didier Lièvremont (Université de Strasbourg) Membre invité Équipe Écophysiologie Moléculaire des Micro‐organismes Laboratoire de Génétique Moléculaire, Génomique et Microbiologie UMR7156 CNRS et Université de Strasbourg 28, rue Goethe, 67000 Strasbourg (France) REMERCIEMENTS Me voici à l’embouchure d’un long fleuve tortueux, celui qui a mené à écrire ce manuscrit. Le long du cours d’eau, de nombreuses personnes m’ont aidé, soutenu, et ont navigué avec moi. Au bout du fleuve, la mer, d’innombrables chemins, autant de destinations. Aujourd’hui, je tiens en ces quelques lignes à exprimer ma reconnaissance envers ceux qui, de près ou de loin, ont contribué à ce périple. Merci aux commandants de bord, Serge Potier et Philippe Bertin, qui m’ont permis de monter à bord du navire GMGM, équipage EM2. Merci à tous les membres d’équipage (les deux Florence, Frédéric, Sandrine, Audrey, Jessica, Marie, David et Jérémy, mais aussi l’ensemble des autres membres du navire, notamment M. Kammerer) pour leur aide sur le ponton. À Vincent Phalip et Anne Forster, mais aussi aux stagiaires (Kévin, Lisa et Daphné) qui sont ponctuellement montés à bord pour m’aider aux manœuvres. À Lucie, plus qu’un mousse, une amie qui m’a soutenu. Un grand merci aux capitaines Didier et Marie‐Claire, qui ont tenu la barre lors d’avis de tempête, mais qui m’ont laissé prendre le cap durant ces 3 dernières années. Un énorme MERCI à mes parents, qui m’ont donné la chance de naviguer sur le fleuve que j’ai choisi, mais qui surtout avaient toujours un œil attentif et bienveillant sur la direction prise et sur la santé du petit matelot. Un merci tout particulier à Aurélie, pour m’avoir essuyé le front, avoir pansé mes blessures et tiré la corde avec moi en toutes circonstances. Enfin, merci aux membres du jury pour avoir accepté l’évaluation de ce travail de thèse, dernière écluse avant de se jeter dans la mer TABLE DES MATIERES TABLE DES MATIERES PARTIE 1 : PRÉSENTATION DU SUJET .................................................................................................................................... 15 A. Étude d’une communauté microbienne : les grandes avancées ................................................... 21 I. Historique de l’écologie microbienne .............................................................................................................................. 21 a. Diversité et impact des micro‐organismes .......................................................................................................................... 21 b. Découverte des micro‐organismes ......................................................................................................................................... 22 c. Débuts de l’écologie microbienne ............................................................................................................................................ 23 d. Découverte des limites des approches culturales ............................................................................................................ 25 II. Avènement de l’ère moléculaire en laboratoire ....................................................................................................... 27 a. Classification phylogénétique par analyse du gène codant l’ARNr 16S ................................................................... 27 b. Approches globales sur des bactéries cultivées ................................................................................................................ 27 III. De la structure au fonctionnement in situ d’une communauté ....................................................................... 28 a. Métagénomique descriptive ciblée .......................................................................................................................................... 28 b. Métagénomique fonctionnelle ciblée ..................................................................................................................................... 30 c. Métagénomique descriptive globale ....................................................................................................................................... 31 d. Méta –omique fonctionnelle ...................................................................................................................................................... 33 IV. Conclusion ................................................................................................................................................................................ 34 B. Limites des techniques moléculaires ....................................................................................................... 36 I. Introduction ............................................................................................................................................................................... 36 II. Extraction du matériel d’étude ........................................................................................................................................ 36 a. Stabilité de l’ADN exogène au sein d’une matrice ............................................................................................................. 36 b. Influence du protocole de lyse cellulaire .............................................................................................................................. 37 c. Impact sur la compréhension de la structure et du fonctionnement d’une communauté ............................... 39 III. Biais liés à l’amplification par PCR ............................................................................................................................... 39 a. Introduction ...................................................................................................................................................................................... 39 b. Amorces « universelles » ............................................................................................................................................................. 40 c. ADN « difficile » ................................................................................................................................................................................ 41 d. Quantité d’ADN matrice ............................................................................................................................................................... 41 7 TABLE DES MATIERES IV. Conclusion ................................................................................................................................................................................ 42 C. Nouvelles techniques de culture ................................................................................................................ 44 I. Introduction ............................................................................................................................................................................... 44 II. Approches « classiques » améliorées ............................................................................................................................. 45 a. Introduction ...................................................................................................................................................................................... 45 b. Modification de la concentration en nutriments ............................................................................................................... 46 c. Définition du milieu en fonction des conditions in situ .................................................................................................. 47 d. Augmentation du temps d’incubation ................................................................................................................................... 47 e. Ajout de molécules de signal ...................................................................................................................................................... 48 III. Co­culture ................................................................................................................................................................................. 49 a. Introduction ...................................................................................................................................................................................... 49 b. Efficacité de la co‐culture ............................................................................................................................................................ 49 IV. Techniques « récentes » ...................................................................................................................................................... 52 a. Introduction .....................................................................................................................................................................................
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