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Peptides Antimicrobiens Des Entérobactériesetude De La Voie De Peptides antimicrobiens des entérobactériesEtude de la voie de maturation et du mécanisme d’import de la microcine J25, peptide antimicrobien inhibiteur de l’ARN polymérase Sophie Duquesne To cite this version: Sophie Duquesne. Peptides antimicrobiens des entérobactériesEtude de la voie de maturation et du mécanisme d’import de la microcine J25, peptide antimicrobien inhibiteur de l’ARN polymérase. Biochimie [q-bio.BM]. Université Pierre et Marie Curie - Paris VI, 2007. Français. tel-00193192 HAL Id: tel-00193192 https://tel.archives-ouvertes.fr/tel-00193192 Submitted on 1 Dec 2007 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. THESE DE DOCTORAT DE L’UNIVERSITE PARIS 6 Spécialité Biochimie Présentée par Sophie DUQUESNE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITE PARIS 6 Peptides antimicrobiens des entérobactéries Etude de la voie de maturation et du mécanisme d’import de la microcine J25, peptide antimicrobien inhibiteur de l’ARN polymérase Antimicrobial peptides from enterobacteria Maturation process of the antibacterial RNA polymerase inhibitor microcin J25, and study of its bacterial uptake mechanism Soutenue le 14 mai 2007, devant le jury composé de : Directeur de Recherche CNRS Rapporteur Jean-Michel Camadro (Institut Jacques Monod, Paris) Professeur Rapporteur Jean-Marie Frère (Institut de Chimie, Liège, Belgique) Professeur Examinateur Solange Lavielle (Université Paris VI) Professeur Examinateur Mohamed Marahiel (Philipps Universität, Marburg, Allemagne) Président du Muséum National d'Histoire Examinateur André Ménez Naturelle, Paris (Président) Chargée de recherche CNRS Directeur de thèse Delphine Destoumieux-Garzόn (Ifremer, Montpellier) Professeur Directeur de thèse Sylvie Rebuffat (Muséum National d'Histoire Naturelle, Paris) A mes grands-parents et à mes parents, A Julie, A mon amour Jean, A tous ceux qui m’ont épaulée ces dernières années. Remerciements Cette étude a été effectuée dans l’Unité de Chimie et Biochimie des Substances Naturelles du Muséum National d’Histoire Naturelle, UMR 5154 CNRS-MNHN, dirigée par Monsieur le Professeur Bernard Bodo. Je tiens à lui adresser mes sincères remerciements pour m’avoir permis de réaliser ces travaux au sein de son unité. Je tiens tout d’abord à remercier Monsieur Jean Michel Camadro, Directeur de Recherche au CNRS (Laboratoire d’Ingénierie des Protéines et Contrôle Métabolique, Institut Jacques Monod, Paris) et Monsieur Jean Marie Frère, Professeur (Institut de Chimie, Université de Liège, Belgique) de m’avoir fait l’honneur d’évaluer ce travail et d’en être les rapporteurs. Je remercie de même Madame Solange Lavielle, Professeur (Laboratoire de Synthèse, Structure et Fonction de Molécules Bioactives, Université Paris VI), et Messieurs Mohamed Marahiel, Professeur (Laboratoire de Chimie et Biochimie, Philipps Universität, Marburg, Allemagne) et André Menez, Président du Muséum National d’Histoire Naturelle (Paris), d’avoir accepté de participer à ce jury de thèse. Je tiens à remercier tout particulièrement ma directrice de thèse, Madame Sylvie Rebuffat, Professeur au MNHN dans l’Unité de Chimie et Biochimie des Substances Naturelles, pour les discussions scientifiques que nous avons pu avoir, son encadrement et sa franchise. Je lui exprime ici toute ma reconnaissance pour m’avoir permis de réaliser ce travail de thèse et pour m’avoir soutenue tout au long de ce travail. Je tiens à remercier très vivement Madame Delphine Destoumieux-Garzón pour avoir confirmé mon goût pour la recherche, grâce à son encadrement patient et son optimisme quotidien. Je lui suis très reconnaissante pour tout l’intérêt qu’elle a continué à me porter malgré son départ de l’Unité, et toutes les connaissances théoriques et pratiques qu’elle m’a transmises. Je souhaite aussi remercier Monsieur Jean Péduzzi pour son soutien dans les moments difficiles, sa sincérité, ainsi que pour m’avoir fait part de sa grande expérience dans le domaine de la biochimie. Je remercie sincèrement Mademoiselle Séverine Zirah, pour sa grande contribution à ces travaux de recherche, et notamment aux expériences de spectrométrie de masse, pour sa confiance et ses conseils scientifiques et personnels précieux. Je tiens à exprimer mes remerciements à Messieurs Lionel Dubost et Arul Marie pour la réalisation des spectres de masse. Mes plus vifs remerciements s’adressent cependant à Monsieur Christophe Goulard pour son aide journalière et surtout efficace, ainsi qu’à Monsieur Gérard Gastine et Mademoiselle Manon Vandervennet pour leur aide très appréciée à la préparation des milieux bactériologiques, ainsi que pour le partage de leur joie de vivre dans des moments parfois délicats. Mes remerciements vont également à toutes les personnes avec lesquelles j’ai eu la chance de pouvoir collaborer au cours de cette étude, et en particulier Mesdames Lucienne Letellier et Pascale Boulanger pour m’avoir accueillie au Laboratoire de Transport Membranaire de Macromolécules de l’Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC, UMR 9619 CNRS, Orsay) et m’avoir ainsi permis de réaliser les expériences de chromatographie d’exclusion en présence de radioactivité, ainsi que Monsieur Michel Desmadril et Madame Magali Niçaise, du Laboratoire de Modélisation et d’Ingénierie des Protéines (IBBMC, UMR 9619 CNRS, Orsay) pour avoir dirigé les expériences de microcalorimétrie et Madame Geneviève Auger, du laboratoire Enveloppes Bactériennes et Antibiotiques (IBBMC, UMR 9619 CNRS, Orsay) pour la réalisation des composition en acides aminés en présence de radioactivité. Je souhaite aussi remercier Messieurs Jean-Claude Tabet, du Laboratoire de Chimie Structurale Organique et Biologique (UMR 7613, Université Paris VI), Jean Michel Camadro, du Laboratoire d’Ingénierie des Protéines et Contrôle Métabolique (Institut Jacques Monod, Paris) et Michael C. Marden de l’Unité INSERM U779 du Kremlin-Bicêtre, pour les accès respectifs au spectromètre de masse à trappe d’ion, à la plateforme de spectrométrie de masse, et au spectropolarimètre de dichroïsme circulaire. Je remercie aussi Monsieur Philippe Grellier et Madame Nathalie Dogna, du Laboratoire de Biologie Fonctionnelle des Protozoaires (USM 0504, Muséum National d’Histoire Naturelle), pour avoir supervisé la production d’anticorps chez la souris. Enfin, je remercie Mohamed Marahiel, du Laboratoire de Chimie et Biochimie (Philipps Universität, Marburg, Allemagne) pour m’avoir accueillie dans son laboratoire dans le cadre du projet PROCOPE. Enfin, je tiens à remercier très vivement tous les membres de l’Unité pour leur bonne humeur et en particulier Mesdames Catherine Pougault, Christelle Edmond, sans qui rien n’aurait pu se faire, ainsi que Mesdames Gaelle Vassiliadis, Fabienne François-Vadrot, Vanessa Petit, Julie N’Guyen-Pouplin, Séverine Amand et Christine Bailly, et encore Messieurs Bastien Nay, Laurent Evanno et Alexandre Deville pour leur grand sens de l’humour et leur soutien. C’est grâce à eux tous que ce travail a été non seulement une véritable expérience professionnelle mais aussi une merveilleuse expérience humaine. Sommaire INTRODUCTION .................................................................................................... 1 RAPPELS BIBLIOGRAPHIQUES: LES MICROCINES..................................... 9 I. Les Microcines : peptides antibactériens des entérobacteries synthétisés selon la voie ribosomique .................................................Erreur ! Signet non défini. Publication n°1 : Microcins, gene-encoded antibacterial peptides from enterobacteria....................................................................Erreur ! Signet non défini. II. Les microcines E492, J25 et C7/C51 : diversités structurelle et fonctionnelle des microcines......................................................................................................... 38 Publication n°2: Structural and functional diversity of microcins, gene-encoded antibacterial peptides from enterobacteria ............................................................ 39 MATERIELS ET METHODES............................................................................. 49 I. Souches bactériennes, plasmides et milieux................................................. 49 I.1. Souches bactériennes et plasmides .............................................................................................. 49 I.2. Milieux de culture........................................................................................................................ 53 II. Clonage et transformation.............................................................................. 56 II.1. Purification et dosage d’ADN ....................................................................................................56 II.2. Obtention d’inserts d’ADN ........................................................................................................ 56 II.3. Réactions enzymatiques ............................................................................................................. 58 II.4. Transformation
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