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Anaerobic respiration may be an essential trait in lifestyle, environment colonization and survival. Until now, the only confirmed anaerobic respiration in Agrobacterium spp. is denitrification. Interestingly, this pathway is unequally widespread among Agrobacteria. These observations led me to my hypothesis which is anaerobic respiration and notably denitrification could explain the coexistence of Agrobacteria and their distribution in specific niches in the rhizosphere. My thesis was undertaken to explore the anaerobic respiration strategies of Agrobacterium spp. and to relate them to niche adaptation. The objectives of my thesis were to (1) characterize all the genes involved in denitrification in A. fabrum C58 in vitro, (2) explore the genes of denitrification that are needed during maize root colonization and (3) discover new anaerobic respirations that occur during maize root colonization (Figure 16). Mutational analysis is the classic way to determine the involvement of a gene in specific pathway. However, this method implies an a priori view and solid knowledge on target genes and cannot be applied for every situation. We have to develop a more adapted method to identify essential genes involved in growth in specific anaerobic conditions. -Denitrification genes in A. fabrum C58 in vitro. To complete denitrification pathway in A. fabrum C58 and identify all the genes and regulators involved in the denitrification function, we adopted two strategies: Firstly, an a priori view to (1) identify the nitrate reductase involved in the first step of denitrification and (2) validate the role of a non-coding RNA in denitrification control. To do so, we constructed a mutant of napA of A. fabrum C58 and a mutant of the non-coding RNA NopR and we evaluated their growth and capacity to produce N2O under anoxic conditions. Secondly, to identify all the genes involved in denitrification, we constructed a mutant transposon library of C58 and tested its growth under denitrification conditions in vitro in the presence of either nitrate or nitrite. -Role of A. fabrum C58 denitrifying genes in the root colonization of maize. It is well known that Transposon-sequencing (Tn-Seq) is a very powerful method to determine genes required for bacterial growth in the presence of their host. To determine denitrifying genes involved in root colonization under anaerobic conditions, we used the library constructed in C58 and performed in planta assays. The mutant library was inoculated on maize plants grown on fertile-ground and cultured under flooded conditions miming anaerobic conditions. Sequencing the recovered A. fabrum C58 cells will evidence the genes involved in this anaerobically specific niche colonization. -Discovery of new anaerobic respiration pathways in A. fabrum C58. To discover new anaerobic respiration pathways, we set-up growth assays of C58 under anoxic conditions in the presence C and N sources as terminal electrons acceptors. Interestingly, by culturing WT and NapA-deficient strains in contact with maize root under anoxic conditions (Chapter 1), we showed growth of both strains, suggesting that root exudates serve as terminal electrons acceptors for anaerobic growth of C58. To determine which maize exuded compounds can serve as TEAs, primary metabolites were identified by HPLC and some were tested as TEAs under the set-up conditions, La respiration anaérobie peut être un trait essentiel dans le mode de vie, la colonisation de l'environnement et la survie. Jusqu'à présent, la seule respiration anaérobie confirmée chez Agrobacterium spp. est la dénitrification. De façon intéressante, cette voie est inégalement répandue chez les agrobactéries. Ces observations m'ont amené à mon hypothèse, à savoir la respiration anaérobie et notamment la dénitrification pourraient expliquer la coexistence d'agrobactéries et leur distribution dans des niches spécifiques de la rhizosphère. Ma thèse visait à explorer les stratégies de respiration anaérobie d’Agrobacterium spp. et de les relier à l'adaptation de niche écologiques différentes. Les objectifs de ma thèse étaient (1) de caractériser tous les gènes impliqués dans la dénitrification chez A. fabrum C58 in vitro, (2) d'explorer les gènes de la dénitrification nécessaires à la colonisation des racines du maïs et (3) de découvrir de nouvelles respirations anaérobies pendant la colonisation racinaire du maïs (Figure 16). Réaliser des mutants et les tester dans des conditions particulières est le moyen classique de déterminer l'implication d'un gène dans une voie spécifique. Cependant, cette méthode implique une vision à priori et des connaissances solides sur les gènes cibles et ne peut pas être appliquée à toutes les situations. Nous avons alors dû développer une méthode plus adaptée pour identifier les gènes essentiels impliqués dans la croissance dans des conditions anaérobies spécifiques. -Gènes de dénitrification chez A. fabrum C58 in vitro. Pour compléter la voie de dénitrification chez A. fabrum C58 et identifier tous les gènes et régulateurs impliqués dans la dénitrification, nous avons adopté deux stratégies : Premièrement, une vision à priori pour (1) identifier la nitrate réductase impliquée dans la première étape de la dénitrification et (2) valider le rôle d'un ARN non codant dans le contrôle de la dénitrification. Pour ce faire, nous avons construit un mutant napA de A. fabrum C58 et un mutant de l'ARN non codant NopR et nous avons évalué leur croissance et leur capacité à produire du N2O dans des conditions anoxiques. Deuxièmement, pour identifier tous les gènes impliqués dans la dénitrification, nous avons construit une banque de transposons mutants de C58 et testé sa croissance dans des conditions de dénitrification in vitro en présence de nitrate ou de nitrite. -Rôle des gènes de la denitrification de A. fabrum C58 dans la colonisation racinaire du maïs. Il est bien connu que le séquençage de transposons (Tn-Seq) est une méthode très puissante pour déterminer les gènes nécessaires à la croissance bactérienne en présence de leur hôte. Pour déterminer les gènes de dénitrification impliqués dans la colonisation des racines en anoxie, nous avons utilisé la banque construite chez C58 et l’avons inoculée sur les plants de maï cultivées sur un sol fertile et cultivées dans des conditions inondées mimant des conditions anaérobies. Le séquençage des cellules d ‘A. fabrum C58 récupérées mettra en évidence les gènes impliqués dans la colonisation anaérobie de cette niche spécifique. -Découverte de nouvelles voies de respiration anaérobie chez A. fabrum C58. Pour découvrir de nouvelles voies de respiration anaérobie, nous avons mis en place des tests de croissance de C58 dans des conditions anoxiques en présence de sources de C et de N en tant qu'accepteurs terminaux d'électrons. De façon interéssante, en cultivant des souches WT et mutée dans le gène napA au contact de la racine de maïs dans des conditions anoxiques (chapitre 1), nous avons montré une croissance des deux souches. Ce résultats suggère que les exsudats de racine servent d'accepteurs d'électrons terminaux pour la croissance anaérobie de C58. Pour déterminer quels composés exsudés du maïs peuvent servir de TEA, les principaux métabolites ont été identifiés par HPLC et certains ont été testés en tant que TEA dans des conditions anoxiques

The rhizosphere is the interface between plant roots and soil where intense, varied interactions between plants and microbes influence plants 'health and growth through their influence on biochemical cycles, such as the carbon, nitrogen, and iron cycles. The rhizosphere is also a changing environment where oxygen can be rapidly limited and anaerobic zones can be established. Microorganisms successfully colonize the rhizosphere when they possess specific traits referred to as rhizosphere competence. Anaerobic respiration flexibility contributes to the rhizosphere competence of microbes. Indeed, a wide range of compounds that are available in the rhizosphere can serve as alternative terminal electron acceptors during anaerobic respiration such as nitrates, iron, carbon compounds, sulfur, metalloids, and radionuclides. In the presence of multiple terminal electron acceptors in a complex environment such as the rhizosphere and in the absence of O-2, microorganisms will first use the most energetic option to sustain growth. Anaerobic respiration has been deeply studied, and the genes involved in anaerobic respiration have been identified. However, aqueous environment and paddy soils are the most studied environments for anaerobic respiration, even if we provide evidence in this review that anaerobic respiration also occurs in the plant rhizosphere. Indeed, we provide evidence by performing a BLAST analysis on metatranscriptomic data that genes involved in iron, sulfur, arsenate and selenate anaerobic respiration are expressed in the rhizosphere, underscoring that the rhizosphere environment is suitable for the establishment of anaerobic respiration. We thus focus this review on current research concerning the different types of anaerobic respiration that occur in the rhizosphere. We also discuss the flexibility of anaerobic respiration as a fundamental trait for the microbial colonization of roots, environmental and ecological adaptation, persistence and bioremediation in the rhizosphere. Anaerobic respiration appears to be a key process for the functioning of an ecosystem and interactions between plants and microbes.

The rhizosphere is the interface between plant roots and soil where intense, varied interactions between plants and microbes influence plants 'health and growth through their influence on biochemical cycles, such as the carbon, nitrogen, and iron cycles. The rhizosphere is also a changing environment where oxygen can be rapidly limited and anaerobic zones can be established. Microorganisms successfully colonize the rhizosphere when they possess specific traits referred to as rhizosphere competence. Anaerobic respiration flexibility contributes to the rhizosphere competence of microbes. Indeed, a wide range of compounds that are available in the rhizosphere can serve as alternative terminal electron acceptors during anaerobic respiration such as nitrates, iron, carbon compounds, sulfur, metalloids, and radionuclides. In the presence of multiple terminal electron acceptors in a complex environment such as the rhizosphere and in the absence of O-2, microorganisms will first use the most energetic option to sustain growth. Anaerobic respiration has been deeply studied, and the genes involved in anaerobic respiration have been identified. However, aqueous environment and paddy soils are the most studied environments for anaerobic respiration, even if we provide evidence in this review that anaerobic respiration also occurs in the plant rhizosphere. Indeed, we provide evidence by performing a BLAST analysis on metatranscriptomic data that genes involved in iron, sulfur, arsenate and selenate anaerobic respiration are expressed in the rhizosphere, underscoring that the rhizosphere environment is suitable for the establishment of anaerobic respiration. We thus focus this review on current research concerning the different types of anaerobic respiration that occur in the rhizosphere. We also discuss the flexibility of anaerobic respiration as a fundamental trait for the microbial colonization of roots, environmental and ecological adaptation, persistence and bioremediation in the rhizosphere. Anaerobic respiration appears to be a key process for the functioning of an ecosystem and interactions between plants and microbes.