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>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)

<p> Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate with the simultaneous reduction of a nicotinamide cofactor. It was previously reported that the nitrogen-fixing bacterium, Sinorhizobium meliloti, has two malic enzymes, a diphosphopyridine-dependent malic enzyme (DME) and a triphosphopyridine-dependent malic enzyme (TME). The dme gene is essential for symbiotic nitrogen-fixation in alfalfa root nodules and this symbiotic requirement cannot be met through increased expression of tme. In order to determine if a metabolic difference exists between the dme and tme mutants which might explain the symbiotic phenotypes, we conducted an analysis of intracellular and extracellular polar metabolomes. Differences noted between the intracellular profiles of the dme and tme mutant strains hinted at osmotic stress or a disturbance in central carbon metabolism. Extracellular studies indicated that dme mutant cells excreted at least 10-fold greater concentrations of both malate and fumarate. When considered together, the metabolic data implies that the DME enzyme is primarily responsible for the conversion of malate to pyruvate to generate acetyl-CoA whereas the TME enzyme must serve a secondary function within the cell.</p> <p> While the C-terminal 320 amino acid regions from both DME and TME are similar in sequence to phosphotransacetylase enzymes, enzyme assays with DME and TME proteins have failed to detect PTA activity. Here we report that the chimeric malic enzyme structure is conserved among various gram negative bacteria including Agrobacterium tumefaciens, Escherichia coli, Bradyrhizobium japonicum and Porphyromonas gingivalis. Moreover these chimeric proteins are also present in the archaebacteria. Halobacterium salinarum and Haloarcula marismortui. To further our understanding of the functions of DME and TME in S. meliloti, we have fused protein domains from DME to an affinity tag consisting of strepII and a calmodulin binding peptide. To identify proteins interacting with this fusion, we expressed these protein fusion constructs in S. meliloti, prepared extracts containing the soluble proteins and passed these through tandem affinity chromatography columns. All proteins that coeluted with the fusion proteins appeared to be interacting with antibodies specific for the DME protein and so may have been aggregates or break-down products of DME.</p> / Thesis / Master of Science (MSc)

In this thesis, an analytical platform was designed and applied to various in vitro bacterial and eukaryotic cell cultures. An extraction and an analytical protocol were developed for comprehensive and simultaneous analysis of both lipid and polar metabolites for intra- and extracellular metabolomics using HILIC-LC-TOF-MS. This analytical platform was applied to four diverse research questions such as the effect of oxygen environment on growth, the interplay between gene expression and metabolism, metabolic changes that occur with age, and PAH toxicity. Specifically: (i) the effect of oxygen on the growth, physiology and metabolism of the Gram positive Streptococcus intermedius were investigated by comprehensive intra- and extracellular metabolomes and transcriptome. (ii) Metabolic insights into the role of the multipartite genome of the Gram negative bacteria Sinorhizobium meliloti and its metabolic preferences in a nutritionally complex environment. (iii) Age-associated metabolic dysregulation in murine bone marrow-derived macrophages during bacterial lipopolysaccharide-induced inflammation. (iv) Comprehensive intracellular metabolomic profiles of Sinorhizobium meliloti to sub-lethal exposure of individual or mixtures of polycyclic aromatic hydrocarbon revealed additive and dose-dependent effects. This thesis has demonstrated the versatility of the designed analytical platform and its use for diverse research in cell biology. / Thesis / Doctor of Philosophy (PhD)

No description available.

Des génomes entiers de bactéries sont séquencés en nombre croissant. Parallèlement sont mis en place des programmes d'analyse systématique de l'expression des gènes et des protéines dans différentes conditions. La compréhension du fonctionnement d'un organisme nécessite une annotation des fonctions des gènes et l'intégration de ces données dans des schémas fonctionnels. Les voies métaboliques constituent une classe de fonctions permettant d'aborder ce problème d'intégration, elles sont bien répertoriées chez de nombreux organismes et sont accessibles à l'expérimentation.<br />Dans un premier temps, nous avons développé une méthode automatique de prédiction de fonction spécifique des enzymes. Cette méthode nommée PRIAM (PRofils pour l'Identification Automatique du Métabolisme) repose sur la nomenclature des enzymes et sur la construction automatique d'un jeu de profils spécifiques des fonctions enzymatiques. Puis, cette méthode permet d'identifier les enzymes dans un génome complet et de visualiser les résultats obtenus sur les graphes des voies métaboliques de la base de données KEGG. <br />Dans un second temps, cette méthode a été appliquée sur le génome de la bactérie fixatrice d'azote Sinorhizobium meliloti et nous a permis l'analyse des voies métaboliques spécifiques de cet organisme symbiote.

Contrary to the predictions of numerous theoretical models, variation in partner quality continues to persist in mutualisms, including in the symbiosis between legumes and rhizobia. One potential explanation for the maintenance of this genetic diversity is genotype × genotype interactions, however it is unknown which genetic regions might underlie these interactions. To investigate this question, I performed a quantitative trait loci mapping experiment with two different rhizobium strains to locate potential regions of the genome influencing genotype × genotype interactions between the legume Medicago truncatula and its symbiont Sinorhizobium meliloti. I found no evidence for genotype × genotype or QTL × rhizobium interactions, however some of the QTLs colocalized with genes involved in the symbiosis signaling pathway, suggesting variation in these genes could potentially affect plant performance and fitness traits. These findings have important implications for the evolutionary interactions between legumes and rhizobia, and the genetic architecture of Medicago truncatula.

Contrary to the predictions of numerous theoretical models, variation in partner quality continues to persist in mutualisms, including in the symbiosis between legumes and rhizobia. One potential explanation for the maintenance of this genetic diversity is genotype × genotype interactions, however it is unknown which genetic regions might underlie these interactions. To investigate this question, I performed a quantitative trait loci mapping experiment with two different rhizobium strains to locate potential regions of the genome influencing genotype × genotype interactions between the legume Medicago truncatula and its symbiont Sinorhizobium meliloti. I found no evidence for genotype × genotype or QTL × rhizobium interactions, however some of the QTLs colocalized with genes involved in the symbiosis signaling pathway, suggesting variation in these genes could potentially affect plant performance and fitness traits. These findings have important implications for the evolutionary interactions between legumes and rhizobia, and the genetic architecture of Medicago truncatula.

Many bacteria accumulate carbon stores as poly-3-hydroxybutyrate (PHB) when growth is limited but carbon availability is not. This stored carbon can then be utilized during conditions of limited carbon availability. The net PHB accumulation in the cell is dependent on the balance between PHB synthesis and degradation. Sinorhizobium meliloti accumulates PHB in the free-living stage but not in the symbiotic stage. The physiological role of the PHB cycle in S. meliloti is unknown. As a first step to understand the genetics of PHB degradation, transposon-generated mutants that were not able to use PHB degradation intermediates, such as 3-hydroxybutyrate and acetoacetate, as a sole carbon source, were isolated. Genetic mapping revealed that there were at least three chromosomal loci involved in acetoacetate metabolism. Identification of these three loci determined that in S. meliloti: (1) acetoacetyl-CoA synthetase (AcsA), encoded by acsA2 gene, rather than the enzyme acetoacetate:succinyl-CoA transferase, is the enzyme that catalyzes activation of acetoacetate to acetoacetyl-CoA; (2) PHB synthase, encoded by phbC, is required for acetoacetate utilization; (3) a putative transporter protein encoding gene, aau-3, may also be involved in acetoacetate metabolism. acsA2 and aau-3 were 78% linked in co-transduction, while phbC was mapped to somewhere else on the chromosome. Biochemical analysis revealed that acsA2::Tn5 mutants lacked AcsA activity but not acetoacetate:succinyl-CoA transferase activity, while phbC::Tn5 maintained similar level of AcsA activity as wild type in vitro. PHB was absent in the phbC mutant. / One transposon-generated mutant, age-1, showed enhanced growth rate on acetoacetate medium. Genetic mapping and transductional analysis indicated that the location of the mutation in age-1 is tightly linked to acsA2. Fine mapping with PCR and DNA sequence techniques showed that Tn5 in age-1 was located at 132 by upstream of the putative translation start site of acsA2. Gene expression analysis indicated that age-1 insertion results in elevated transcription of acsA2. Thus enhanced growth rate on acetoacetate was due to the increased gene expression. acsA2 transcription was induced by acetoacetate and 3-hydroxybutyrate, and repressed by glucose and acetate. / All mutants formed root nodules that fixed nitrogen with varying decrease of impairment. Acetoacetate metabolism and the PHB degradation are not essential for symbiosis.