Sinorhizobium meliloti est une bactérie du sol Gram- capable d'induire la formation denodosités fixatrices d'azote lors d'une interaction symbiotique avec les plantes de la familledes légumineuses. L'importance de la balance redox au cours de cette interaction a été miseen évidence. Ainsi, des mutants bactériens déficients dans la production du glutathion (GSH),présentent un phénotype altéré d'infection et de fixation de l'azote atmosphérique.Le premier objectif a donc été de déterminer si les phénotypes observés chez les mutants de lavoie de biosynthèse du GSH étaient liés à l'activité des glutarédoxines (GRX). Une analysebioinformatique a révélé la présence de trois gènes codant des GRX chez S. meliloti. Lesmutants, Smgrx1, Smgrx2 et Smgrx3, déficients pour chacune des GRX, ne produisent pasdes phénotypes similaires à ceux observés avec les mutants GSH. Si Smgrx2 présente unphénotype moins marqué, Smgrx1 est plus sévèrement affecté puisqu'il n'est plus capable dese différencier en bactéroïde. L'implication de SmGrx2 dans la régulation du métabolisme dufer et la mise en place des centres Fe-S a, par ailleurs, été mise en évidence.Le second objectif a été de définir s'il existait, chez S. meliloti, une redondance fonctionnelleentre les GRX et les thiorédoxines (TRX). Ainsi, le mutant SmtrxB, dépourvu de thiorédoxineréductase, présente la particularité d'induire la formation d'un plus grand nombre de nodulesque la souche sauvage. Le système TRX de S. meliloti apparaît donc comme un régulateurnégatif de la nodulation. D'autre part, les nodosités formées par ce mutant SmtrxB, ont uneactivité fixatrice d'azote significativement diminuée. Les rôles des TRX et des GRXapparaissent donc, au moins partiellement, distincts.Les résultats obtenus ici apportent des éléments nouveaux sur l'implication du GSH, des GRXet des TRX dans la mise en place d'une nodosité fonctionnelle, et ouvrent de nouvellesperspectives d'études sur les rôles de ces molécules dans le processus de fixation d'azote. / Sinorhizobium meliloti is a soil bacterium Gram- able to induce the formation of nitrogenfixingnodules during a symbiotic interaction with plants of the legume family. Theimportance of redox balance during this interaction has been demonstrated. In this way,bacterial mutants deficient in the production of glutathione (GSH), exhibit an alteredphenotype of infection and fixation of atmospheric nitrogen.The first objective was therefore to determine whether the phenotypes observed in mutants ofthe GSH biosynthesis pathway were related to the activity of glutaredoxins (GRX). Abioinformatic analysis revealed the presence of three genes encoding GRX in S. meliloti. Themutants, Smgrx1, Smgrx2 and Smgrx3, deficient for each of the GRX, do not producephenotypes similar to those observed with the GSH mutants. If Smgrx2 presents a less severephenotype, Smgrx1 is more severely affected since it is incapable of differentiating intobacteroïd. The involvement of SmGrx2 in the regulation of iron metabolism and theestablishment of Fe-S cluster has also been demonstrated.The second objective was to determine if there was, in S. meliloti, a functional redundancybetween GRX and thioredoxin (TRX). Thus, the SmtrxB mutant, devoid of thioredoxinreductase, has the distinctive feature of inducing the formation of more nodules than the wildtype strain. The TRX system of S. meliloti appears to be a negative regulator of nodulation.On the other hand, the nodules formed by this SmtrxB mutant have a significantly decreasednitrogen-fixing activity. Hence, the roles of TRX and GRX appear to be at least partiallydistinct.The results obtained here provide new evidence on the involvement of GSH, the GRX andTRX in the establishment of a functional nodule, and open new perspectives for studying onthe roles of these molecules in the process of nitrogen fixation.

Le monoxyde d'azote (NO) est une molécule gazeuse impliquée dans de nombreux processus biologiques chez les plantes, de la germination de la graine à la mise en place de réponses à des stress abiotiques et biotiques. Dans les interactions plante/ pathogène, le NO fait partie de l'arsenal de défenses de l'hôte. En réponse, les pathogènes ont développé des mécanismes pour contrer les effets du NO. Dans la symbiose fixatrice d'azote entre la légumineuse modèle Medicago truncatula et la bactérie Sinorhizobium meliloti, du NO a été détecté durant toutes les phases de l'interaction. L'équipe avait précédemment montré que la réponse de S. meliloti au NO est nécessaire au maintien de la symbiose puisque des nodules formés par une souche mutée dans le gène hmp (le gène hmp est induit par le NO et code pour une protéine qui dégrade le NO) sénescent prématurément. Au cours de cette thèse, nous avons étudié 3 nouveaux gènes de S. meliloti induits par le NO : nnrS1, nnrS2 et norB. nnrS1 et nnrS2 codent pour deux protéines de fonction inconnue et norB code pour une NO réductase qui dégrade le NO. Nous avons montré que ces 3 protéines participent d'une part à la résistance des bactéries au NO en culture et d'autre part, au maintien de l'interaction symbiotique. Par ailleurs, nous avons montré que ces 3 protéines sont impliquées directement ou indirectement dans la dégradation du NO et des résultats préliminaires suggèrent que NnrS1 présente une activité NO réductase. De plus, nous avons montré que NnrS1 et Hmp n'agissent pas seulement sur les bactéries mais aussi sur les protéines végétales. Il était connu que dans les nodules de M. truncatula, la glutamine synthétase (GS) végétale, une enzyme clé de la symbiose, est inhibée par tyrosine nitration, une modification post-traductionnelle dépendante du NO. Nous avons montré que NnrS1 et Hmp, en modulant le niveau de NO dans les nodules, contrôlent l'activité de la GS. Enfin des expériences préliminaires montrent que d'autres protéines (bactériennes et/ou végétales) pourraient être tyrosine nitratées. / Nitric oxide (NO) is a gaseous molecule involved in a large range of biological processes in plants from the seed germination to abiotic and biotic stress responses. In plant-pathogen interactions, NO is part of the defense systems. In response, pathogens have developed mechanisms in order to counteract the NO effects. In the nitrogen fixing symbiosis between the model leguminous plant Medicago truncatula and the bacterium Sinorhizobium meliloti, NO has been detected at all stages of the symbiosis. The team had previously shown that the S. meliloti response to NO is necessary to maintain the symbiotic interaction since nodules elicited by a strain mutated in the hmp gene (hmp is induced by NO and codes for a flavohemoglobine that degrades NO) senesce prematurely. During this thesis, we have studied 3 new genes of S. meliloti whose expression is induced by NO: nnrS1, nnrS2 and norB. nnrS1 and nnrS2 encode two proteins of unknown function and norB codes for a NO reductase which degrades NO. We have shown that these 3 proteins participate on one hand in bacterial NO resistance in culture and on the other hand in maintaining the symbiotic interaction. Furthermore, we have shown that these 3 proteins are involved, directly or indirectly, in NO degradation and preliminary results suggest that NnrS1 displays a NO reductase activity. Moreover, we have shown that NnrS1 and Hmp are not only dedicated to protect bacteria against NO but also play a role on plant proteins. It was already known that, in M. truncatula nodules, the plant glutamine synthétase (GS), a key enzyme of the symbiosis is inhibited by tyrosine nitration, a NO post-translational modification. We have shown that NnrS1 and Hmp, by modulating NO levels in nodules, control the GS activity. Finally, preliminary experiments suggest that other proteins (from bacterial and/or plant origin), could be tyrosine nitrated.

Les Légumineuses ont développé une interaction symbiotique avec des bactéries du sol, les rhizobia, qui fixent l’azote atmosphérique et le transfèrent à la plante sous forme assimilable.Cette interaction a lieu, au sein des nodosités, des organes racinaires où les bactéries intracellulaires se différencient en bactéroïdes. Chez Medicago truncatula, ces bactéroïdes correspondent à un stade de différentiation terminale corrélée à une endoréplication de leur génome, une augmentation de la taille des cellules, une modification des membranes et une faible capacité à se propager. Cette différentiation est induite par des facteurs de la plante appelés NCR (Nodule-specific Cysteine Rich). Les peptides NCRs ressemblent à des défensines, des peptides antimicrobiens ayant une activité antimicrobienne in vitro, tuant des bactéries. Ainsi, un élément clef dans la différenciation des bactéroïdes est la protéine bactérienne BacA, un transporteur membranaire qui confère une résistance contre l’activité antimicrobienne des peptides. Dans le cadre de ce travail de thèse, j’ai montré que l'expression des NCR est soumise à une régulation stricte et qu’ils sont activés dans trois vagues dans les cellules symbiotiques polyploïdes.Les mécanismes de contrôle par la plante sur les rhizobia intracellulaires demeurent à ce jourpeu connus et le seul modèle étudié, au début de ce travail de thèse, restait l'interaction entre M. truncatula et S. meliloti. Je me suis donc intéressée à la symbiose de certaines Légumineuses tropicales du genre Aeschynomene appartenant au clade des Dalbergoïdes où jemontre qu’ils utilisent une classe différente de peptides riches en cystéine (NCR-like) pour induire la différenciation des bactéroïdes. Ce mécanisme est analogue à celui décrit précédemment chez Medicago qui était jusqu'à présent supposé être limitée aux légumineuses appartenant au clade des IRLC. J’ai également montré que Bradyrhizobium, symbionte d’Aeschynomene possèdent un transporteur de type ABC homologues à BacA de Sinorhizobium nommé BclA. Ce gène permet l'importation d'une variété de peptides comprenant des peptides NCR. En l'absence de ce transporteur, les rhizobiums sont incapables de se différencier et de fixer l'azote.Cette étude a permis d'élargir nos connaissances sur l'évolution de la symbiose en montrant qu’au cours de l’évolution, deux clades de Légumineuses relativement éloignés (IRLC et Dalbergoïdes) aient convergé vers l’utilisation de peptides de l’immunité innée afin de contrôler leur symbionte bactérien et d’en tirer un bénéfice maximal au cours de l’interaction symbiotique. / The ability of legumes to acquire sufficient nitrogen from the symbiosis with Rhizobium relies on the intimate contact between the endosymbiotic, intracellular rhizobia, called bacteroids, and their host cells, the symbiotic nodule cells. A well-studied example is the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti, which nodulates the legume Medicago truncatula. Nodules of M. truncatula produce an enormous diversity of peptides called NCRs which are similar to antimicrobial peptides (AMPs) of innate immune systems. These NCRs are involved in maintaining the homeostasis between the host cells in the nodules and the large bacterial population they contain. Although many NCRs are genuine AMPs which kill microbes in vitro, in nodule cells they do not kill the bacteria but induce them into the terminally differentiated bacteroid state involving cell elongation, genome amplification, membrane fragilization and loss of cell division capacity. Protection against the antimicrobial action of NCRs by the bacterial BacA protein is critical for bacteroid survival in the symbiotic cells and thus for symbiosis. As a part of my PhD thesis, I have shown that the differentiation of the symbiotic cells in M. truncatula is associated with a tremendous transcriptional reprogramming involving hundreds of genes, mainly NCR genes, which are only expressed in these cells. Although the extensive work on the model M. truncatula/S. meliloti, little is known how the plant controls its intracellular population and imposes its differentiation into a functional form, the bacteroids in other symbiotic systems.In my PhD work, I provide several independent pieces of evidence to show that tropical legumes of the Aeschynomene genus which belong to the Dalbergoid legume clade use a different class of cysteine rich peptides (NCR-like) to govern bacteroid differentiation. This mechanism is similar to the one previously described in Medicago which was up to now assumed to be restricted to the advanced IRLC legume clade, to which it belongs. I have also shown that the Bradyrhizobium symbionts of Aeschynomene legumes possess a multidrug transporter, named BclA, which mediates the import of a diversity of peptides including NCR peptides. In the absence of this transporter, the rhizobia do not differentiate and do not fix nitrogen. BclA has a transmembrane domain of the same family as the transmembrane domain of the BacA transporter of Rhizobium and Sinorhizobium species which is known to be required in these rhizobia to respond to the NCR peptides of IRLC legumes. Again this is a mechanism which is analogous to the one described in S. meliloti the symbiont of Medicago.This study broaden our knowledge on the evolution of symbiosis by showing that the modus operandi involving peptides derived from innate immunity used by some legumes to keep their intracellular bacterial population under control is more widespread and ancient than previously thought and has been invented by evolution several times.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

The effect of Ascophyllum nodosum extracts on the nodulation and growth of alfalfa was investigated. Plant growth assay revealed that alfalfa treated with 2 g L-1 ANE exhibited a significant increase in leaf area. Under salt stress, alfalfa treated with 0.5 g L-1 ANE exhibited a significant increase in total length compared to controls. A root hair deformation assay indicated that ANE 0.5 g L-1 stimulated the synthesis of Nod factors secreted by rhizobia thus accelerate root hair deformation of alfalfa. Similarly, ANE 0.5 g L-1 caused an increase in nodC gene expression suggesting that ANE may act similarly to flavonoids in the rhizobium-legume symbiosis. Under field conditions, ANE increased the total number of functional nodules, total root length and total leaf area. Taken together, the results suggest that ANE may contain compound(s) that promote specific metabolic pathway both in alfalfa and bacterium thus enhance the symbiotic relationship.

Les cassures double-brin (CDBs) de l'ADN sont décrites comme étant les lésions de l'ADN les plus délétères puisqu'elles conduisent systématiquement à la mort de la cellule si elles ne sont pas réparées. Les CDBs peuvent être réparées par différents mécanismes et notamment par Non-Homologous End-Joining (NHEJ). Chez les eucaryotes, les protéines centrales de la NHEJ, Ku70 et Ku80, forment un hétérodimère capable de se lier aux extrémités de l'ADN générées par la cassure. Par la suite, Ku70 et Ku80 recrutent de nombreuses autres protéines permettant la modification des extrémités et la réparation de la CDB par ligation. La NHEJ a également été caractérisée chez un nombre limité de bactéries chez qui le mécanisme semble moins complexe que chez les eucaryotes. Chez les bactéries, la NHEJ nécessite seulement deux protéines : un homodimère de Ku, et la protéine multifonctionnelle LigD capable de modifier les extrémités et d'effectuer la ligation. La majorité des études faites sur la NHEJ ont été menées chez des bactéries ne possédant qu'une seule paire des gènes ku/ligD. Cependant, de nombreux autres génomes bactériens possèdent plusieurs copies de ces deux gènes et le fonctionnement de la NHEJ chez ces organismes est inconnu. Le génome de la bactérie symbiotique Sinorhizobium meliloti code quatre Ku putatives (ku1-4) et quatre LigD putatives (ligD1-4). A ce jour, une seule étude a été menée chez ce modèle bactérien montrant que chacun des simples mutants ku est plus sensible que la souche sauvage à un traitement aux rayonnements ionisants, suggérant que chacune des Ku joue un rôle dans la réparation des CDBs par NHEJ. Par l'utilisation de différentes approches in vivo, nous avons mené une caractérisation génétique de la NHEJ chez S. meliloti permettant de clarifier les contributions relatives des gènes ku et ligD dans le mécanisme. Pour la première fois chez une bactérie, nous avons pu obtenir des résultats montrant la présence de plusieurs systèmes indépendants de NHEJ chez S. meliloti, et suggérant l'existence d'un possible hétérodimère de Ku. Nous avons également mis en évidence que la NHEJ est activée dans différentes conditions de stress, telles que le stress thermique et la carence nutritive, et qu'une partie de cette réparation est sous le contrôle du régulateur central de la réponse générale au stress RpoE2. Par ailleurs, nous avons montré que la NHEJ, et plus généralement les mécanismes de réparation des CDBs sont impliqués dans la résistance à la dessiccation chez S. meliloti. Enfin, nous avons généré la première preuve expérimentale d'une implication de la NHEJ dans le transfert horizontal de gène chez les bactéries. Dans leur ensemble, ces travaux enrichissent nos connaissances sur les mécanismes de réparation des CDBs chez les bactéries possédant plusieurs orthologues de Ku et LigD. Ils suggèrent également que la NHEJ pourrait contribuer à l'évolution des génomes, en particulier en condition de stress, non seulement en raison du caractère mutagène de ce type de réparation mais également en participant à l'acquisition d'ADN exogène originaire de bactéries distantes. / DNA double-strand breaks (DSBs) are described as the most deleterious DNA damages as they can lead to cell death if they are not repaired. DSBs can be repaired through several mechanisms, including Non-Homologous End-Joining (NHEJ). In eukaryotes, the main NHEJ proteins, Ku70 and Ku80, bind DNA ends as a heterodimer, and then recruit several additional proteins including enzymes which catalyze the processing and ligation of DNA ends. NHEJ has also been characterized in a limited number of bacteria, where the repair mechanism appears to be less complex than in eukaryotes. Indeed, only two proteins are required: a homodimeric Ku protein, and a multifunctional LigD enzyme able to process and ligate the DNA ends. However, most studies were performed on bacterial species encoding a single pair of ku/ligD. Actually, many bacterial species encode multiple copies of these genes, whose relative contributions to NHEJ in vivo are so far unknown. The Sinorhizobium meliloti genome encodes four putative Ku (ku1-4) and four putative LigD (ligD1-4). To date, a single study conducted on this model bacterium showed that every ku single mutant is more sensitive than the wild type strain to ionizing radiations showing that all ku genes are involved in NHEJ repair of DSBs in this organism. Here, using several in vivo approaches, we performed a comprehensive genetic characterization of NHEJ repair in S. meliloti, and clarified the respective contributions of the various ku and ligD genes. For the first time in bacteria, we obtained results showing the presence of several independent NHEJ systems in S. meliloti and suggesting the existence of a putative heterodimeric form of Ku. We also demonstrated that NHEJ repair is activated under various stress conditions, including heat and nutrient starvation, and that part of this repair is under the control of the general stress response regulator RpoE2. We showed that NHEJ and more generally DSB repair mechanisms are involved in desiccation resistance in S. meliloti. Finally, for the first time in bacteria, we provided evidence that NHEJ not only repairs DSBs, but can also erroneously integrate heterologous DNA molecules into the breaks. Altogether, our data provide new insights into the mechanisms of DSB repair in bacteria which encode multiple Ku and LigD orthologues. It also suggest that NHEJ might contribute to the evolution of bacterial genomes under adverse environmental conditions not only through error-prone repair of DSB by its mutagenesis repair characteristic but also by participating in the acquisition of foreign DNA from distantly related organisms during horizontal gene transfer events.

Microorganisms interact frequently with each other and with higher organisms. This contact and communication takes place at the molecular level. Microbial interactions with eukaryotes can be pathogenic or mutualistic. One of the best-studied symbioses is the complex interaction between nitrogen-fixing soil bacteria, termed rhizobia, and legumes. This symbiosis culminates in the elaboration of a new organ, the root nodule. Many of the molecular signals exchanged between the host plant and the invading rhizobia have been deduced, but there is still much that remains to be discovered. The molecular determinant of host range at the genus level of the plant host has been determined to be lipochitooligomers called Nod factors. The molecular determinants of host range at the species and cultivar level are less well-defined. Part of my work has been to identify and characterize accessory plasmids that disrupt the normal progression of symbiosis between legumes of the genus Medicago and their rhizobial symbiont, Sinorhizobium meliloti. A cre--loxP-based system capable of making large, defined deletions was developed for the analysis of these plasmids. This system is also being employed to cure the laboratory strain, S. meliloti Rm1021 of its two megaplasmids-a loss of nearly half of its genome. I have also done work to determine whether locally-collected sinorhizobia are native, invasive, or native with symbiosis genes acquired horizontally from invasive sinorhizobia. Finally, I have studied Sinorhizobium meliloti as a host by identifying an outer membrane porin that several bacteriophages use to adsorb to the S. meliloti cell surface.

<p> This thesis focused on the development of comprehensive, rapid and simple methodologies for the analysis of fatty acids by gas chromatography mass spectrometry (GC/MS) and intact lipids by electrospray ionization tandem mass spectrometry (ESIIMS/MS). The methodologies were applied as a tool for functional genomics in the soil bacterium Sinorhizobium meliloti. The effects of inorganic phosphate (Pi)-starvation and acidity on lipid composition were studied. </p> <p> A micro-scale, one-vial method for the analysis of fatty acids as their fatty acid methyl esters by GC/MS was developed. The method required small sample sizes, involved minimum handling and avoided tedious extraction steps, which increased sample throughput. A series of quality controls were included to measure losses due to handling, derivatization efficiencies and the extent of side reactions. The method was suitable for the analysis of sensitive bacterial fatty acids such as cyclopropane fatty acids. </p> <p> A shotgun lipidomics approach was developed for the analysis of intact lipids by ESIIMS/MS. Fatty acid distributions were obtained for eight lipid classes and up to 58 individual lipids were identified in crude lipid extracts without sample cleanup or chromatography. For the first time, fatty acid distributions were provided for non-phosphorus containing lipids using shotgun lipidomics. Fatty acid distributions within lipid classes suggested that phospholipids and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine lipids (TMHSs) were both synthesized from phosphatidic acid while sulfoquinovosyldiacylglycerol (SLs) had a different biosynthetic origin. </p> <p> The methodologies were applied to study knockout mutants of five genes thought to participate in lipid metabolism in S. meliloti. It was demonstrated that: (1) cfa2 gene coded for the main cyclopropane fatty acyl synthase; (2) the plsC gene coded for a fatty acy 1 transferase specific for C 16 fatty acids in the sn-2 position of phospholipids; (3) a metabolic phenotype was revealed for knockout mutants of dme and tme genes (DME and TME, malic enzymes) when succinate was the carbon source. </p> / Thesis / Doctor of Philosophy (PhD)

<p>The <em>Sinorhizobium meliloti</em> genome consists of 6204 predicted protein-coding regions of which approximately 2000 are proteins of unknown function (PUFs). To identify functions of <em>S. meliloti</em> PUFs, we employed the FRT/Flp recombination system to delete large gene clusters and then screened for phenotypes. Large-scale deletions have been mainly used to define minimal gene sets that contain only those genes that are essential and sufficient to sustain a functioning cell. To adapt FRT/Flp for use in <em>S. meliloti</em>, we used an already constructed pTH1522-derived integration gene library of the <em>S. meliloti</em> genome (pTH1522 carries a single FRT site). A second FRT site was inserted at defined locations in the genome through integration of a second plasmid (pTH1937) that also carries a single FRT site. Here we outline how this Flp/FRT system was used to delete defined regions and hence generate multiple gene knock-out mutants. This system was used to delete 32 and 56 defined regions from the 1340 Kb pSymA and 1678 Kb pSymB megaplasmid, respectively. The structures of the resulting megaplasmid deletion mutants were confirmed by PCR analysis. Carbohydrate and nitrogen utilization phenotypes were associated with the deletion of specific regions. Deleting large, regions of the genome helped us to identify phenotypes such as inability to grow on minimal media with fucose, maltotriose, maltitol, trehalose, palatinose, lactulose and galactosamine as sole carbon source. For several FRT-flanked regions, few or no recombinants were recovered which suggested the presence of essential genes. Through this strategy, two essential genes <em>tRNA^arg </em>and<em> engA</em> located on the pSymB and three toxin/antitoxin-like systems, <em>sma0471</em>/<em>sma0473</em>, <em>sma2105</em> and <em>sma2230</em>/<em>sma2231</em> on pSymA megaplasmid were identified.</p> / Doctor of Philosophy (PhD)