Thèse De DOCTORAT En SCIENCES Option : BIOTECHNOLOGIE

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Thèse De DOCTORAT En SCIENCES Option : BIOTECHNOLOGIE REPUBLIQUE ALGERIENNE DEMOCRATIQUE ET POPULAIRE MINISTERE DE L’ENSEIGNEMENT SUPERIEUR ET DE LA RECHERCHE SCIENTIFIQUE UNIVERSITE D’ORAN 1 AHMED BEN BELLA FACULTE DES SCIENCES DE LA NATURE ET DE LA VIE DEPARTEMENT DE BIOTECHNOLOGIE N° d’ordre : Thèse de DOCTORAT en SCIENCES Option : BIOTECHNOLOGIE Spécialité : Intérêt des microorganismes en Agriculture et en Agroalimentaire Caractérisation des molécules bioactives produites par des souches d’actinobactériés isolées des sols arides et semi arides d’Algérie. Présentée par Mr. Mohamed HARIR Soutenue le 09/04/2018 Devant le Jury composé de : Présidente Pr. Fortas Zohra Université d’Oran 1 Examinateur Pr. Aoues Abdelkader Université d’Oran 1 Examinateur Dr. Dib Soulef Université d’Oran 1 Examinateur Pr. Setti Benali Université de Chlef Examinateur Pr. Djibaoui Rachid Université de Mostaganem Directeur de thèse Pr. Bellahcene Miloud CU d’Ain Témouchent Invité 2017/2018 Dédicace A ma Mère A mon Père A mes frères et mes sœurs A mes nièces et mes neveux A tous mes chers Collègues et ami (e) s A la mémoire de ma collègue Madame Tlemcani Mokhtaria ALLAH yarhamha HARIR Mohamed I Remerciement Merci au Bon dieu de m’avoir aidé à réaliser ce travail Je tiens à remercier particulièrement mon directeur de thèse, Pr. BELLAHCENE Miloud qui m’a aidé à faire mon chemin du Magister à la thèse. Son rôle dans ma formation, sa patience, ses précieux conseils, sa confiance et l’autonomie qu’il m’a accordée ont été autant d’éléments qui ont contribués au bon déroulement de ce travail de thèse. Je voudrais également remercier Professeur FORTAS Zohra, pour son soutien, ses conseils et son aide. Merci beaucoup à tout ce que vous avez fait pour moi. Vous resterez à mes yeux comme une personne entière et pleine de talents aussi bien dans la recherche que dans les relations humaines. Je tiens à remercier vivement les membres du Jury de thèse qui ont accepté avec beaucoup d’amabilité d’évaluer mon travail de thèse : Madame Fortas Zohra, Professeur à l’Université d’Ahmed Ben Bella Oran 1, d’avoir accepté de présider le Jury, Monsieur Aoues Abdelkader, Professeur à l’Université d’Ahmed Ben Bella Oran 1, Madame Dib Soulef Maître de Conférences A à l’Université d’Ahmed Ben Bella Oran1 Monsieur Setti Benali, Professeur à l’Université de Chlef, Monsieur Djibaoui Rachid, Professeur à l’Université de Mostaganem, Que tous les membres trouvent ici l’expression de mes plus vifs remerciements pour avoir bien voulu être les évaluateurs de mon travail de thèse malgré leurs nombreuses occupations. Ce travail est le fruit d’une collaboration scientifique multiple et n’aurait pu être réalisé sans la contribution des Professeurs et chercheurs Rebecca Pogni, Stephania Butini, Staphano Mangani, Docteurs Simona Pollini et Camilla Brratto du Département de Biotechnologie, de Chimie et de Pharmacie - Université de Siena, Italie. Je vous exprime mes sincères remerciements pour m’avoir accueilli au sein du laboratoire et pour m’avoir donné la possibilité de réaliser une partie de la thèse dans de très bonnes conditions. Merci aussi pour votre grande hospitalité, votre disponibilité et votre soutien. Mes remerciements s’adressent également à Madame le Professeur Susana Rodríguez-Couto Directrice du Laboratoire de Division Eau & Santé Ceit-IK4 (Centre des études et de technique de recherche), Donostia-San Sebastian, Espagne, pour m’avoir si bien accueillie II au sein de son équipe et de m’avoir donné la possibilité de réaliser une partie de l’étude expérimentale de cette thèse. Je n’oublie pas de remercier Madame Carol Verheecke Docteur à l’Université de Cranfiled – Angleterre, d’avoir accepté d’évaluer mon travail de thèse. Je n’oublie pas non plus de remercier Mr. Nadhem Aissani Docteur à l’institut supérieur de biotechnologie Béja –Tunisie et Mr. Abbassi Mohamed salah Docteur à l’institut de recherche en Vétérinaire, Bab Sadoun, Tunis 1006. pour leur soutien, leur aide et leur disponibilité. Enfin j’aimerais exprimer mes sincères remerciements à mes parents qui m’ont donnée leur amour et leur confiance. Je remercie mes frères et sœurs pour leur soutien moral, leurs conseils et leurs encouragements. Je n’oublie pas non plus de remercier tous mes collègues du laboratoire LBMB. Je souhaite aussi remercier mes collègues pour leur aide et leur disponibilité à savoir, RABBES K ; BOUNAR R ; DAHDOUH F ; BENDIF H ; GHADBANE M ; HAICHOUR R, BELAOUNI HA, MIARA M.D ; TOUMATIA O, AOUICHE A, BELGHIT S. MADAH B. Merci à tous ceux qui ont contribué à la réalisation de cette étude ! III Table des Matières REMERCIMENTS AVANT PROPOS TABLE DES MATIÈRES ……………………………………………………………………… IV LISTE DES ABRÉVIATIONS…………………………………………………………………. VIII LISTE DES FIGURES………………………………………………………………………….. X LISTE DES TABLEAUX………………………………………………………………………. XII LISTE DES CHROMATOGRAMMES……………………………………………………….. XIV LISTE DES PUBLICATIONS ET COMMUNICATIONS………………………………….. XV ملخــــــص ABSTRACT RÉSUMÉ INTRODUCTION ………………………………………………………………………………. 01 Chapitre I : Revue Bibliographique I. Les Actinobactéries…………………………………………………………………………….. 04 1. Historique…………………………………………………………………………………….. 04 2. Définition des actinobactéries …………………………………………………………. 04 3. Caractéristique générale d'actinobactéries ……………………………… ………….. 05 II. Taxonomie et critères de classification des actinobactéries…………………………………… 06 1. Systématique des actinobactéries……………………………………………………… 06 2. Evolution des critères d’identification ………………………………………………... 06 3. Critères actuels d’identification ………………………………………………………. 08 3.1. Caractères morphologiques………………………………………………………………… 08 3.1.1. Caractéristiques culturales ou macromorphologiques………………………. 08 3.1.1.1. Mycélium aérien …………………………………………………. .. 0 9 3.1.1.2. Mycélium de substrat ……………………………………………… .. 09 3.1.2. Caractéristiques micromorphologiques…………………………………………………… 09 3.2. Caractères chimiotaxonomiques (chimiotaxonomie)………………………………………… 12 3.2.1. Constituants pariétaux: isomères de l’acide diaminopimélique et acides aminés….. 12 3.2.2. Glucides…………………………………………………………………………… 12 3.2.3. Constituants membranaires et pariétaux: lipides…………………………………… 13 3.2.3.1. Les phospholipides……………………………………………………………….. 13 3.2.3.2. Les ménaquinones…………………………………………………....................... 14 3.2.3.3. Acides gras……………………………………………………………………….. 14 3.2.3.4. Les acides mycoliques…………………………………………………………….. 15 3.3. Critère physiologique et taxonomie numérique ……………………………………………... 15 3.4. Critères moléculaires……………………………………………………………................... 17 3.4.1. Séquençage de l’ADN ribosomique 16S et phylogénie……………………………… 17 3.4.2. Hybridation ADN-ADN et détermination du pourcentage de guanine-cytosine…….. 17 3.4.3. Taux de GC………………………………………………………………................... 18 3.4.4. Séquençages de nouvelle génération (NGS)………………………………………… 18 3.4.4.1. Préparation des banques ou librairies……………………………….. 20 3.4.4.2. PCR en émulsion……………………………………………………. 20 3.4.4.3. Bridge PCR ………………………………………………………… 21 3.4.4.4. Séquençage Illumina ………………………………………............... 21 3.4.4.5. Annotation des génomes……………………………………………. 23 III. Cycle de développement d'actinobactérie…………………………………………………….. 25 1. Formation des spores par les actinobactéries ………………………………………...... 25 IV 1.1. Les Exo-spores …………………………………………………………………….. 27 1.2. Endospores………………………………………………………………………….. 27 IV. Habitat d'actinobactéries……………………………………………………………………... 28 1. Environnement terrestres ……………………………………………………………….. 29 2. Eaux douces et marines …………………………………………………………………. 29 3. l’Air ……………………………………………………………………………………... 30 4. les Composts …………………………………………………………………………….. 30 5. les Végétaux, les animaux et l’homme…………………………………………………... 30 V. Applications des actinobactéries …………………………………. ………………… ………. 30 1. Production des métabolites antimicrobiens (Les Antibiotiques)………………………… 31 2. Production d’enzymes……………………………………………………….................... 32 3. Production des Bioherbicides……………………………………………………………. 33 4. Production de Vitamines………………………………………………………………… 34 5. Production de Pigments………………………………………………………………….. 35 6. Bioremédiation…………………………………………………………………………... 35 7. Utilisation des actinobactéries en lutte biologique………………………………………. 36 VI. Famille des Streptomycétacées……………………………………………………………………….. 37 1. Le genre Streptomycès…………………………………………………………………… 37 1.1. Classification du genre Streptomyces (Bergy’s manual of systematic bacteriology, 38 2006)……………………………………………………………………………………….. 38 1.2. Cycle de développement………………………………………………………………. 39 1.3. Caractéristiques du genre Streptomyces ………………………………………………. 39 1.3.1. génétique des Streptomyces……………………………………………………......... 40 1.4. Identification moléculaire (ARN ribosomique 16S) des Streptomyces………………... 40 1.4.1. limites de la PCR universelle 16S en tant qu’outil de définition de nouvelles 41 espèces bactériennes……………………………………………………………………………… 41 1.5. L’Hybridation ADN-ADN : …………………………………………………………... 42 VII. Les Antibiotiques……………………………………………………………………………. 42 1. Les cibles bactériennes des antibiotiques……………………………………………….. 45 2. Les antibiotiques produits par le genre Streptomyces…………………………………… 45 3. Effet de la composition de milieux de culture sur la production des antibiotiques……… 45 4. Stratégie moléculaires de lutte contre la résistance bactérienne……………………......... 46 47 Chapitre II : Matériel et Méthodes I. Origine des échantillons de sol et isolement des actinobacteries. ……………... 50 1. Les échantillons de sols……………………………………………………………………… 50 2. Isolement des actinobactéries………………………………………………………………... 51 2.1. Prétraitement des échantillons de sols……………………………………………… 51 2.2. Les milieux de cultures
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