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Chez Azotobacter Vinelandii En Présence De Matières Organique Et Minérale

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Chez Azotobacter Vinelandii En Présence De Matières Organique Et Minérale HOMEOSTASIE DES COFACTEURS METALLIQUES DE LA NITROGENASE (MOLYBDÈNE ET VANADIUM) CHEZ AZOTOBACTER VINELANDII EN PRÉSENCE DE MATIÈRES ORGANIQUE ET MINÉRALE par Christelle Jouogo Noumsi thèse présentée au Département de biologie en vue de l'obtention du grade de docteur ès sciences (Ph.D.) FACULTÉ DES SCIENCES UNIVERSITÉ DE SHERBROOKE Sherbrooke, Québec, Canada, Décembre 2016 Le 09 Décembre 2016 le jury a accepté la thèse de madame Christelle Jouogo Noumsi dans sa version finale Membres du jury Professeur Jean-Philippe Bellenger Directeur de recherche Département de chimie, Université de Sherbrooke Professeur Vincent Burrus Co-directeur de recherche Département de biologie, Université de Sherbrooke Professeur Marc Amyot Évaluateur externe Département de sciences biologiques, Université de Montréal Professeur Pascale Beauregard Évaluateur interne Département de biologie, Université de Sherbrooke Professeur Sébastien Rodrigue Président-rapporteur Département de biologie, Université de Sherbrooke SOMMAIRE La fixation biologique du diazote (N2) constitue une étape importante du cycle biogéochimique de l’azote (N). Cette réaction permet la réduction du N2 atmosphérique, non biodisponible, en ammonium (NH3) bioassimilable par tous les organismes vivants. Cette réaction est limitée par différents facteurs, menant à une limitation de la production primaire dans de nombreux écosystèmes, particulièrement nordiques. En effet, la fixation biologique d’azote dans tous les écosystèmes dépend d’un nombre réduit de procaryotes appelés diazotrophes qui sont capables de rompre la triple liaison N-N pour former l’ammoniac. Cette réaction qui est catalysée pas une métalloenzyme, la nitrogénase, requiert beaucoup d’énergie sous forme d’ATP, compte tenue de l’énergie de liaison liant les atomes d’azote. La nitrogénase existe sous trois isoformes, que l’on différencie principalement par la nature du métal cofacteur du site actif : les nitrogénases au molybdène (Mo), au vanadium (V) et au fer (Fe). La répartition des isoformes de la nitrogénase n’est pas homogène chez les diazotrophes. Alors que tous possèdent la nitrogénase au Mo, seulement quelques-uns ont en supplément la nitrogénase au V et/ou celle au Fe. De plus, les isoformes de la nitrogénase ne sont pas équivalentes en termes d’efficacité de réduction de N2. La Mo-nitrogénase est la plus efficace à température ambiante car elle utilise le moins d’ATP pour réduire une mole de diazote. Elle est suivie de l’isoforme au V, et enfin celle au Fe. Chez les diazotrophes possédant plus d’une nitrogénase (ex. Azotobacter vinelandii), l’utilisation de celles-ci suit une hiérarchie reflétant ces efficacités relatives; la Mo-nitrogénase est exprimée par défaut, la V- nitrogénase est exprimée en absence de Mo et celle au Fer en absence de Mo et de V. Du fait de leur moindre efficacité, de leur présence chez seulement certains diazotrophes, en supplément de la Mo-nitrogénase, et de leur régulation sous contrôle du Mo, la fixation d’azote est considérée comme dépendant principalement du Mo. Pourtant depuis quelques années, plusieurs évidences remettent en cause cette prédominance de la Mo- nitrogénase: (i) la conservation des nitrogénases alternatives au fil de l’évolution suggère qu’elles ont un rôle important; (ii) la limitation de la fixation par le Mo de nombreux écosystèmes; (iii) l’omniprésence des gènes des nitrogénases alternatives dans de iv nombreux écosystèmes, limités en Mo ou non. Malgré leur importance potentielle, notre compréhension de la contribution des nitrogénases alternatives à la fixation biologique de diazote en milieu naturel reste très limitée. Plusieurs facteurs peuvent influencer l’utilisation des nitrogénases, tels que la température et l’accès aux métaux essentiels. L’acquisition des métaux cofacteurs constitue un paramètre d’intérêt majeur, car l’activation des gènes des nitrogénases est fortement dépendante de la disponibilité en métaux cofacteurs. Dans les sols le 2- molybdène et le vanadium, présents essentiellement sous forme de molybdate (MoO4 ) et 3- vanadate (VO4 ), sont fortement complexés à la matrice (matière organique et oxydes) ce qui peut limiter leur disponibilité pour les fixateurs d’azote. Chez certains diazotrophes (ex. Azotobacter vinelandii) il a été démontré que l’acquisition des métaux cofacteurs (Fe, Mo et V) est assurée par des métallophores, petits ligands organiques produits par les êtres vivants pour chélater les métaux. Leur grande affinité pour les métaux permet aux diazotrophes de pouvoir acquérir sélectivement les métaux pour la fixation biologique d’azote. En milieu naturel les interactions entre complexes naturels (matière organique, oxydes) et métallophores jouent sans doute un rôle important dans la biodisponibilité des métaux cofacteurs de la nitrogénase et donc leur utilisation. Cependant, les études sur l’acquisition des métaux et leur utilisation pour la fixation d’azote restent souvent limitées à des conditions de laboratoire peu représentatives de la réalité (métaux cofacteurs non complexés). L’hypothèse de ce projet doctoral était que la présence d’agents naturels complexant les métaux cofacteurs de la nitrogénase (Mo et V) pouvait influencer significativement la stratégie d’acquisition et d’utilisation de ceux-ci, menant à une contribution accrue des nitrogénases alternatives. Cette hypothèse a été testée en conditions de laboratoire en utilisant Azotobacter vinelandii comme bactérie modèle ainsi que l’acide tannique et des oxydes de fer comme agents complexants naturels modèles. Ces travaux ont montré que la présence d’agents complexants (acide tannique et oxydes de fer) entraîne des changements majeurs dans la gestion des métaux cofacteurs (Mo et v V) chez A. vinelandii. Les stratégies d’acquisition des métaux cofacteurs sont fortement modifiées en présence de complexants naturels avec (i) un changement important de la quantité des metallophores produits ainsi que (ii) une acquisition simultanée de Mo et V dans des conditions traditionnellement considérée comme non limitantes en Mo. Ceci se traduit par un changement important dans l’utilisation des nitrogénases; les niveaux de transcrits élevés des gènes nifD et vnfD, spécifiques des nitrogénases au Mo et au V respectivement, suggèrent une utilisation simultanée de ces isoenzymes pour assurer la fixation d’azote. Ce projet a permis de mettre en évidence que face à un stress métallique, l’utilisation des isoformes de la nitrogénase par A. vinelandii est un processus plus versatile que précédemment décrit et que le coût d’acquisition des métaux dans ces conditions serait un facteur important de la régulation de l’activité des nitrogénases. Ces travaux démontrent que la hiérarchie des nitrogénases établie sur la base d’expériences de laboratoire ne s’applique sans doute pas aux milieux naturels. Ceci suggère que les nitrogénases alternatives pourraient contribuer à la fixation d’azote de manière plus importante que présentement admis. Mots clés : fixation biologique d’azote, nitrogénase, métaux essentiels, métallophores, homéostasie, dynamique d’acquisition et d’utilisation, Azotobacter vinelandii vi REMERCIEMENTS J’aimerais tout d’abord remercier grandement Jean-Philippe et Vincent pour avoir permis que tout ce travail soit possible. Grâce à eux j’ai beaucoup appris durant ces quatre années, et j’ai aussi appris à apprendre. Ce fût un véritable challenge et j’ai eu un accompagnement des plus formidables. Rigueur, persévérance, autonomie, éthique, sont les maîtres mots qui décrivent le mieux l’encadrement qu’ils m’ont apporté. Je ne pouvais espérer mieux. Je remercie toutes les sources de financement ayant permis ce doctorat (les chaires de recherche du Canada détenues par JPB et VB et le soutien facultaire au recrutement aux études supérieures). Les membres de mon comité de conseillers Ryszard, Pascale et Sébastien, je vous remercie pour l’encadrement administratif et pour les conseils que vous m’avez donné durant mon doctorat. Mes partenaires de laboratoire, présents et passés. J’ai eu la chance de faire partie de deux équipes aussi différentes que formidables. Ce fût tellement agréable de travailler avec vous, j’ai beaucoup appris grâce à votre aide. J’ai en plus eu la chance de trouver en vous de fantastiques amis avec qui la vie continuait hors laboratoire. Nicolas, Dominic, Geneviève, Éric, (Mireille), Nina, Romain Da., Marion, Lorène, Jefferson, Augustin, Romain Du., Fanny, je n’ai pas assez de place ici pour vous exprimer toute ma gratitude, mais vous le savez, je n’en pense pas moins. À mes amis (formidablement géniaux), Suzy, Nil, Sarah (choupinette), Liliane, Thomas, Hugo, Svilena, Hervé, Loïc, pour ne citer qu’eux, un énorme merci pour le soutien quotidien. Ma famille, loin des yeux, près du cœur. Vous m’accompagnez depuis le début, merci pour le soutien, merci pour l’amour dont vous m’abreuvez. À ma maman. À ma sœur Mireille. À mon frère Guy Alain... vii TABLE DES MATIÈRES SOMMAIRE ............................................................................................................................ iv REMERCIEMENTS ............................................................................................................... vii LISTE DES ABRÉVIATIONS ................................................................................................ xi LISTE DES TABLEAUX ......................................................................................................xiii LISTE DES FIGURES ..........................................................................................................
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