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Mycoplasma Mycoides Subsp N° d’ordre :……………… THESE Présentée par Woubit SALAH Pour obtenir LE TITRE DE DOCTEUR DE L’INSTITUT NATIONAL POLYTECHNIQUE DE TOULOUSE PhD UTILISATION D’UNE SEQUENCE COMPLETE DU GENOME D’UNE SOUCHE DE MYCOPLASMA MYCOIDES SUBSP. MYCOIDES LC POUR LA MISE AU POINT DE TESTS DE DIAGNOSTIC, APPLICATION A L’EXPRESSION DE GENES HETEROLOGUES Ecole doctorale: SEVAB Spécialité: SCIENCE VETERINAIRE Soutenance le 25 Janvier 2008, devant le jury composé de: Henri Dabernat Président Colette Saillard Rapporteur Dominique Le Grand Rapporteur Xavier Berthelot Directeur de thèse François Poumarat Membre Alain Blanchard Membre Christine Citti Membre François Thiaucourt Membre H. Dabernat : Laboratoire de Bactériologie Hygiène, Institut Fédératif de Biologie 330 avenue de Grande Bretagne - TSA 30021 ; 31059 Toulouse Cédex 9 (F) Tél. +33 (0)5 67 69 04 17; Fax +33 (0)5 67 69 04 83 ; E.mail : [email protected] UTH C. Saillard : Laboratoire de Biologie Cellulaire et Moléculaire, UMR 1090 IBVM, INRA BP 81, 71, Avenue Edouard Bourlaux, 33883 Villenave d’Ornon Cedex, France Tel: + 33 (0)5 57 12 23 62; E-mail: [email protected] D. Le Grand : UMR Mycoplasmoses des Ruminants AFSSA-ENVL ; Pathologie du Bétail ; Ecole Nationale Vétérinaire de Lyon ; 1, Avenue Bourgelat - 69280 Marcy l'Etoile Tel: +33 (0)4.78.87.26.05 / Fax: +33 (0)4.78.87.26.06 ; E-Mail: [email protected] UTH X. Berthelot : Departement Elevage et Produits et Unite Mixte de Recherches INRA- ENVT 1225 "Interactions Hôtes-Agents Pathogènes" (IHAP) ; Ecole Nationale Veterinaire, 23 chemin des Capelles, B.P. 87614, F-31076 Toulouse cedex 3 Tel +33 (0)5 61 19 38 57 ; Fax +33 (0)5 61 19 32 73; E.mail : [email protected] UTH F. Poumarat : Unité "Mycoplasmologie" AFSSA site de Lyon ; 31 av. Tony-Garnier 69364 LYON cedex 07 ; Tel : +33 (0)4 78 69 68 31 ; Fax : 33 (0)4 78 61 91 45 E.mail : [email protected] UTH A. Blanchard : UMR 1090 GDPP ; INRA, IBVM, Centre de Recherche de Bordeaux 71 avenue Edouard Bourlaux BP 81 33883 Villenave D'Ornon Cedex France Tel +33 (0)5 57 12 23 93; Fax: +33 (0)5 57 12 23 69; E.mail : [email protected] UTH C. Citti : UMR 1225 INRA-ENVT Ecole Nationale Vétérinaire 23 Chemin des Capelles BP 87614 ; 31076 Toulouse Cedex 3 France Tel : +33 (0)5 61193856; Fax : +33 (0)5 61193273 ; E.mail: [email protected] UTH F. Thiaucourt :CIRAD, UPR 15 « Contrôle des maladies » TA A-15/G Campus de Baillarguet 34398 Montpellier cedex 5 Tel : +33 (0)467593723 ; Fax : +33 (0)467593798 ; E.mail : [email protected] UTH TABLE OF CONTENTS ACKNOWLEDGEMENT…...............................................................................................................................I LIST OF PUBLICATIONS…..........................................................................................................................III RESUME…………………..……………………………………………………………………......................IV SUMMARY………………………….………………………………………………………………………...VI LIST OF FIGURES…....................................................................................................................................VIII LIST OF TABLES…………………………………..………………………………………………………...XI LIST OF ABBREVIATIONS…......................................................................................................................XII GENERAL INTRODUCTION.................................................................................................1 CHAPTER I Introduction to Mycoplasma, the M. mycoides cluster and Genome sequencing 1.1 The Mycoplasmas..........................................................................................................................4 1.1.1 General description and habitat ...............................................................................................4 1.1.2 Genome characteristics and phylogeny....................................................................................5 1.1.3 Pathogenicity in Mycoplasmas ................................................................................................7 1.1.3.1 Adhesion to host cells.......................................................................................................7 1.1.3.2 Invasion ............................................................................................................................7 1.1.3.3 Antigenic variation ...........................................................................................................7 1.2 The Mycoplasma mycoides cluster ..............................................................................................8 1.2.1 Diseases associated with the M. mycoides cluster .................................................................11 1.2.1.1 CBPP and CCPP.............................................................................................................11 1.2.1.2 Contagious agalactia.......................................................................................................11 1.2.1.3 Other disease conditions.................................................................................................13 1.2.2 Pathogenicity of members of the M. mycoides cluster...........................................................13 1.2.2.1 Variable surface protein..................................................................................................13 1.2.2.2 Metabolic byproduct.......................................................................................................14 1.2.2.3 Capsular polysaccharide.................................................................................................14 1.3 Genome sequencing approach in Mycoplasmas.......................................................................15 1.3.1 Genome sequencing...............................................................................................................15 1.3.1.1 Genome sequencing strategies........................................................................................16 1.3.1.2 Sequencing techniques ...................................................................................................18 1.3.1.3 Genomic sequence analysis............................................................................................20 1.4 Genome sequencing of M. mycoides subsp. mycoides LC.........................................................21 1.5 Annotation of M. mycoides subsp. mycoides LC genome sequence.........................................21 CHAPTER II Genome size estimation of MmmLC strain 95010‐C1 and analysis of presence of repeated genes 1 INTRODUCTION ..............................................................................................................23 1.1 Genome size estimation...............................................................................................................23 1.2 DNA Hybridization .....................................................................................................................24 2 MATERIALS AND METHODS .........................................................................................26 2.1 Strains...........................................................................................................................................26 2.2 DNA plug preparation ................................................................................................................26 2.3 Restriction Enzyme Analysis......................................................................................................26 2.3.1 Choice of restriction enzymes and digestion for PFGE.........................................................26 2.3.2 Choice of restriction enzyme and digestion for Southern blotting ........................................27 2.4 PFGE Electrophoresis conditions..............................................................................................27 2.5 DNA probes for Southern blot hybridization test ....................................................................27 2.6 Southern blotting.........................................................................................................................28 3 RESULTS AND DISCUSSION.........................................................................................30 3.1 Genome size estimation of strain 95010-C1 ..............................................................................30 3.1.1 Mycoplasma strains analyzed ................................................................................................32 3.1.1 1 Mcc, M. bovis and M. agalactiae ...................................................................................32 3.1.1.2 MmmSC strains..............................................................................................................32 3.1.1.3 MmmLC strains..............................................................................................................34 3.2 Assessment of the presence of repeated gene sequences ..........................................................35 3.2.1 Gene lppQ (ApalI digest).......................................................................................................35 3.2.2 Gene mgtA (XbaI digest)...........................................................................................................35 T 3.2.2.1 mgtA gene in strain PG1P P ...............................................................................................35 3.2.2.2 mgtA gene in strain 8740-Rita........................................................................................36
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