Genes involved in purine degradation in Sinorhizobium meliloti to date remain largely uncharacterized. Analysis of the bdhAxdhA2xdhB2 operon established a link between the degradation of purines and the carbon storage compound poly-3-hydroxybutyrate (PHB). This operon contains genes (xdhA2xdhB2) that encode xanthine oxidase / xanthine dehy- drogenase, an enzyme involved in the conversion of hypoxanthine and xanthine to uric acid. The bdhA gene located in the same operon encodes 3-hydroxybutyrate dehydrogenase, an enzyme responsible for catalyzing the second step in PHB degradation. This linkage be- tween the degradation of PHB (a carbon source) and purines (a nitrogen source) suggests a possible means by which Sinorhizobium meliloti obtains sufficient carbon and nitrogen to allow it to successfully colonize a host plant. Purine degradation genes in S. meliloti have also been studied by the phenotypic char- acterization of Tn5 mutants unable to utilize hypoxanthine. Mutations resulting in these phenotypes were found in three different genes, SMc03849 (ccmC), a cytochrome c bio- genesis mutant, SMb20684, a gene coding for a hypothetical protein possibly involved in the utilization of glyoxylate and SMb2192, a gene coding for a membrane spanning protein possibly involved in purine transport. In this study we further characterized these mutants by examining their ability to establish a symbiosis with Medicago sativa (alfalfa) and to fix atmospheric nitrogen. It was demonstrated that in the case of all the mutant strains there was a competitive deficiency in terms of gaining entry to root nodules relative to the wild-type strain. It was shown that this deficiency occurred even in strains capable of fixing atmospheric nitrogen suggesting that the inability to utilize hypoxanthine impairs the ability of S. meliloti to colonize the host plant. Of all of these genes studied thus far only one (SMb21292) is located in the region of the genome containing the greatest number of genes potentially involved in purine degra- dation. In this study we used transcriptional fusions to confirm the activation of genes in this genomic region when grown in media containing purines as carbon and nitrogen sources. These genes include xdhA1, SMb21284 and guaD1. Genes from the genome re- gion containing the mixed function operon including xdhA2 and guaD2 were also studied. In addition we were able to demonstrate the requirement of xdhC in producing a func- tional oxidase / xanthine dehydrogenase as well as the ability to grow on hypoxanthine or xanthine as a carbon and nitrogen source. This work was also able to demonstrate the critical nature of the LysR transcription regulator (SMb21291) in purine degradation in S. meliloti. Mutating this gene resulted in an inability to grow on hypoxanthine or xanthine as well as an alteration in levels of xanthine oxidase / xanthine dehydrogenase activity. By transducing the gene fusions into the LysR mutant background it was demonstrated that the protein coded for by SMb21291 acts to regulate or influence the expression of genes involved in the purine degradation cycle such as SMb21284 and xdhA1. In addition we were able to characterize strains with mutations in purine degradation genes in terms of their growth on different purines.

Genes involved in purine degradation in Sinorhizobium meliloti to date remain largely uncharacterized. Analysis of the bdhAxdhA2xdhB2 operon established a link between the degradation of purines and the carbon storage compound poly-3-hydroxybutyrate (PHB). This operon contains genes (xdhA2xdhB2) that encode xanthine oxidase / xanthine dehy- drogenase, an enzyme involved in the conversion of hypoxanthine and xanthine to uric acid. The bdhA gene located in the same operon encodes 3-hydroxybutyrate dehydrogenase, an enzyme responsible for catalyzing the second step in PHB degradation. This linkage be- tween the degradation of PHB (a carbon source) and purines (a nitrogen source) suggests a possible means by which Sinorhizobium meliloti obtains sufficient carbon and nitrogen to allow it to successfully colonize a host plant. Purine degradation genes in S. meliloti have also been studied by the phenotypic char- acterization of Tn5 mutants unable to utilize hypoxanthine. Mutations resulting in these phenotypes were found in three different genes, SMc03849 (ccmC), a cytochrome c bio- genesis mutant, SMb20684, a gene coding for a hypothetical protein possibly involved in the utilization of glyoxylate and SMb2192, a gene coding for a membrane spanning protein possibly involved in purine transport. In this study we further characterized these mutants by examining their ability to establish a symbiosis with Medicago sativa (alfalfa) and to fix atmospheric nitrogen. It was demonstrated that in the case of all the mutant strains there was a competitive deficiency in terms of gaining entry to root nodules relative to the wild-type strain. It was shown that this deficiency occurred even in strains capable of fixing atmospheric nitrogen suggesting that the inability to utilize hypoxanthine impairs the ability of S. meliloti to colonize the host plant. Of all of these genes studied thus far only one (SMb21292) is located in the region of the genome containing the greatest number of genes potentially involved in purine degra- dation. In this study we used transcriptional fusions to confirm the activation of genes in this genomic region when grown in media containing purines as carbon and nitrogen sources. These genes include xdhA1, SMb21284 and guaD1. Genes from the genome re- gion containing the mixed function operon including xdhA2 and guaD2 were also studied. In addition we were able to demonstrate the requirement of xdhC in producing a func- tional oxidase / xanthine dehydrogenase as well as the ability to grow on hypoxanthine or xanthine as a carbon and nitrogen source. This work was also able to demonstrate the critical nature of the LysR transcription regulator (SMb21291) in purine degradation in S. meliloti. Mutating this gene resulted in an inability to grow on hypoxanthine or xanthine as well as an alteration in levels of xanthine oxidase / xanthine dehydrogenase activity. By transducing the gene fusions into the LysR mutant background it was demonstrated that the protein coded for by SMb21291 acts to regulate or influence the expression of genes involved in the purine degradation cycle such as SMb21284 and xdhA1. In addition we were able to characterize strains with mutations in purine degradation genes in terms of their growth on different purines.

Thèse (M.Sc.)--Université Laval, 2006. / Bibliogr.: f. [66]-73. Publié aussi en version électronique.

The goal of this thesis is to determine if Sinorhizobium meliloti can be useful as a sentinel soil microorganism for assessing the impacts of contaminant stressors on the metabolome of a microorganism. Not only is a good deal known about this organism, but it is an important organism in agriculture. Moreover, the currently available gene array and a large library of gene fusion can be used as facile pathways to explore genetic and genomic impacts in addition to metabolomic impacts of contaminants, should such studies be deemed worthwhile. In this study, the polar metabolome of the soil microorganism, Sinorhizobium meliloti, has been analyzed by LC-ESI-MS using a HILIC column coupled to a medium mass resolution time-of-flight mass spectrometer. This approach has resulted in the retention (k' > 0.7) of over 300 polar metabolites as detected in both positive ion and negative ion modes. These data do not include ions corresponding to adduct ions, isotopic features or ions resulting from in-source decay processes. The retained peaks showed excellent linear responses and did not suffer from ion suppression, a common problem in flow-injection ESI analysis. This methodology has been applied to the analysis of S. meliloti exposed to fluorene, a common PAH contaminant, and to a coal tar fraction containing low molecular mass PAHs. Multiple cultures of S. meliloti were grown on M9 glucose minimal medium in the absence and presence of fluorene (0.14 mg/L and 1.4 mg/L) and a PAH mixture (total PAH concentrations of 0.14 mg/L and 1.4 mg/L). Analyses of biological replicates were performed in pentuplicate. The retention times of the resulting chromatograms were aligned, peak areas determined and the resulting data processed using PCA and OPLS-DA methods. The retention time reproducibilities of peaks were within ± 10 seconds and the biological variabilities of over 700 components averaged 23% ± 15% (n=25) . The impacts of fluorene exposures and PAH mixture exposures on the S. meliloti metabolomes (polar) caused significant changes in the metabolome. The lower concentration exposures had less of an impact than the higher dosages. Low dosages of both fluorene and the PAH mixture produced a similar metabolic response in S. meliloti, while at higher dosages the responses were more specific to each toxin. The use of SUS plots coupled with S-plots of the OPLS-DA analysis were particularly advantageous for the identification of metabolites of interest. Changes were seen in the levels of adenine, adenosine, glutamate, and aspartate, among others. In the future, the profiles of the non-polar metabolites of each of sample will be analyzed using a previously developed 'shotgun lipidomics' method. / Thesis / Master of Science (MS)

Phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step of gluconeogenesis, and the gene which encodes this enzyme (pckA) is transcriptionally regulated. High pckA expression is observed in succinate-grown cells, while little expression is observed in glucose-grown cells. pckA regulatory mutants have previously been isolated (Osteras et al. 1997) and pckR, a gene encoding a Lacl-GaIR DNA-binding transcriptional regulator, has been implicated in the regulation of pckA transcription. Here we shew that pckR insertion mutations result in a dramatic decrease in pckA expression even in succinate-grown cells. We demonstrate that the previously identified rpk-9 mutation is tightly linked to pckR. The rpk-9 mutation results in constitutive pckA expression, and we show that plasmids carrying the pckR gene complement the rpk-9 mutation in glucose-grown cells. A putative Lacl-GaIR operator binding site has been identified in the pckA promoter, however no evidence of an interaction between this site and the pckR gene product could be found. / Thesis / Master of Science (MS)

L'interaction symbiotique entre la bactérie du sol Sinorhizobium meliloti et la plante de la famille des légumineuses Medicago sp. conduit au développement d’un nouvel organe racinaire: la nodosité. Au sein de cet organe, les bactéries différenciées en bactéroïdes, réduisant l’azote atmosphérique en ammoniac directement assimilable par la plante, favorisant ainsi sa nutrition azotée. En échange, la plante, grâce à son activité photosynthétique, fournit aux bactéroïdes des composés carbonés. Cette association à bénéfice mutuel n’est toutefois pas permanente. En effet, quelques semaines seulement après l'établissement de la symbiose, une sénescence définie par une dégradation des bactéroïdes puis des cellules végétales, est observée. Cette étape du développement nodositaire est aujourd’hui encore peu étudiée et mal comprise.L’objectif premier de ce travail était donc d’analyser le rôle du bactéroïde dans cette rupture symbiotique. Pour cela, nous nous sommes plus particulièrement intéressés au rôle des systèmes Toxine Antitoxine (TA) de type VapBC de S. meliloti. En effet, ces opérons sont, dans la littérature, connus pour être impliqués dans la réponse aux stress, la persistance et/ou la mort bactérienne ainsi que la survie de la bactérie au sein de la cellule hôte. Dans un premier temps, nous avons développé une analyse globale du rôle des 11 systèmes VapBC chromosomiques de S. meliloti dans l’interaction symbiotique par des analyses in silico et de phénotypes de mutants d'invalidation du gène de la toxine en interactions avec Medicago sp. Deux études ont été réalisés de façon plus détaillées sur deux modules vapBC (VapBC5 et VapBC7). / The symbiotic interaction between the soil bacterium Sinorhizobium meliloti and the legumes plant Medicago sp. led to the development of a new root organ: the nodule. In this nodule differenciated bacteria into bacteroids, reducing atmospheric nitrogen into ammonia directly assimilated by the plant, thus promoting its nitrogen nutrition. In exchange, the plant, thanks to its photosynthetic activity, provides carbon compounds to the bacteroids. This mutual benefit association is however not permanent. Indeed, just weeks after the establishment of the symbiosis, senescence defined by a degradation of Bacteroides and plant cells, is observed. This stage of development is poorly understood in particularly about bacterial signal.The primary objective of this study was therefore to analyze the role of bacteroids in this symbiotic rupture. For this, we are particularly interested in the role of VapBC toxin antitoxin systems (TA) of S. meliloti. Indeed, in the literature, they are known to be involved in the stress response, persistence and / or bacterial death and the survival of the bacteria within the host cell. At first, we developed a global analysis of the role of 11 VapBC chromosomal systems in S. meliloti symbiotic interaction. After an in silico study, we studied the symbiotic phenotype with Medicago sp., Of each of the bacterial toxin mutants invalidation. Given the results, we, as a second step, developed a detail analysis of phenotypes obtained with two of these mutants: vapC5- and vapC7-.

The rhizobium – legume symbiosis is a complex process that involves genetic cooperation from both bacteria and plants. Previously, our lab described naturally occurring accessory plasmids in rhizobia that inhibit this cooperation. A transposon mutagenesis was performed on the plasmids to detect the genetic factor that blocked nitrogen fixation. Several of the plasmids were found to possess a replication operon that when disrupted by transposon insertion, restored symbiotic function. This study describes an in-depth investigation into one of those plasmids, pHRC377, and into its replication operon. The operon, which we have called repA2C2, comes from the repABC family of replication and partitioning systems commonly found in alphaproteobacteria. In this study we show that this operon is not necessary for pHRC377 replication in LB culture or free living cells, but is necessary for plasmid amplification in the plant, specifically during rhizobial differentiation into nitrogen fixing bacteroids. We also show how the other repABC type operons on pHRC377 function in relation to plasmid maintenance and copy number during endoreduplication and how they do not have the same phenotypic effect as repA2C2.

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium. Initial investigation into this enzyme categorized it as a methyltransferase. Further investigation revealed that this was incorrect, and that MosA is actually a dihydrodipicolinate synthase, part of the N-acetylneuraminate lyase superfamily. One of the characteristics of enzyme superfamilies is their low sequence identity, but relatively high structural similarity. The structural investigation reported here confirms the high structural similarity between MosA and other superfamily members. <p>Investigation of MosA was carried out by means of x-ray crystallography. It was believed that detailed structural information may shed light into not only the enzymatic mechanism, but also the inhibition of MosA by lysine, the final product of the enzymatic pathway. Insight into enzyme mechanism and inhibition may ultimately prove useful in herbicide or insecticide development, as other dihydrodipicolinate synthases from harmful fungi, bacteria, or plants, make attractive targets for inhibition. Lysine is an essential amino acid for humans, meaning that there is no endogenous lysine production to block the use of these hypothetical inhibitors. Specific inhibitors based on crystal structures have proven to be effective in the past and hopefully, will continue to be useful in the future. <p>Here we report the structure of MosA, solved to 1.95 Å resolution with lysine 161 forming a Schiff-base adduct with pyruvate. This adduct is consistent with the currently accepted dihydrodipicolinate synthase enzyme mechanism.

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium. Initial investigation into this enzyme categorized it as a methyltransferase. Further investigation revealed that this was incorrect, and that MosA is actually a dihydrodipicolinate synthase, part of the N-acetylneuraminate lyase superfamily. One of the characteristics of enzyme superfamilies is their low sequence identity, but relatively high structural similarity. The structural investigation reported here confirms the high structural similarity between MosA and other superfamily members. <p>Investigation of MosA was carried out by means of x-ray crystallography. It was believed that detailed structural information may shed light into not only the enzymatic mechanism, but also the inhibition of MosA by lysine, the final product of the enzymatic pathway. Insight into enzyme mechanism and inhibition may ultimately prove useful in herbicide or insecticide development, as other dihydrodipicolinate synthases from harmful fungi, bacteria, or plants, make attractive targets for inhibition. Lysine is an essential amino acid for humans, meaning that there is no endogenous lysine production to block the use of these hypothetical inhibitors. Specific inhibitors based on crystal structures have proven to be effective in the past and hopefully, will continue to be useful in the future. <p>Here we report the structure of MosA, solved to 1.95 Å resolution with lysine 161 forming a Schiff-base adduct with pyruvate. This adduct is consistent with the currently accepted dihydrodipicolinate synthase enzyme mechanism.

No description available.