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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)

Rhizobium aspartate transcarbamoylase (ATCase; EC 2.1.3.2) was previously believed to be similar to the Pseudomonas ATCase which has been studied extensively. To facilitate the study of the Rhizobium ATCase a pyrimidine-requiring mutant of R. meliloti was isolated and used in the purification of the enzyme.

Fifteen mutants of Sinorhizobium (Rhizobium ) meliloti unable to utilize acetate as a sale carbon source (Ace-) were characterized in this study. Merodiploid complementation tests showed that nine of these mutations were in loci distinct from previously described gluconeogenic loci. The chromosomal locations of the mutations were determined, and complementing clones were isolated from the cosmid library of S. meliloti genomic DNA. The mutants were placed into four groups (I--IV) based on genetic linkage in phage co-transduction. None of the mutations were in glyoxylate shunt enzyme-encoding genes. Nucleotide sequence analysis of ace mutants from Groups III and IV showed mutations in genes encoding acetyl-CoA synthetase ( acsB) and anaerobic coproporphyrinogen III oxidase (hemN ) respectively. Cell extracts of the hemN mutant exhibited double the isocitrate lyase levels of the wild type. The acsB mutant lacked acetyl-CoA synthetase activity and had an interesting growth phenotype; it was able to grow on low concentrations of acetate only. (Abstract shortened by UMI.)

This study was undertaken to characterize the role and pathway for assimilation of the intracellular carbon storage compound, poly-beta-hydroxybutyrate (PHB), in Rhizobium (Sinorhizobium) meliloti. Mutants unable to utilize the degradation intermediates, 3-hydroxybutyrate (HB) and/or acetoacetate (AA) were characterized. A mutant unable to utilize HB (Hbu-) while retaining the ability to utilize AA was found to be deficient in 3-hydroxybutyrate dehydrogenase (Bdh) activity. The bdhA mutant showed no symbiotic defects in association with alfalfa plants. However, when co-inoculated with the wild type, the mutant showed significantly reduced competitiveness. A more severe competition defect was observed for a PHB synthesis mutant (phaC). Both these mutants also showed reduced competitiveness when subjected to multiple cycles of subculturing through alternating carbon-rich and carbon-poor media, with the phaC mutant showing a greater loss in competitiveness. The results indicate that the ability to efficiently deposit and utilize cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability. / The gene encoding Bdh (bdhA) was isolated and sequenced. Two transcription start sites, S1 and S2 were identified but no known consensus promoter sequences were identified upstream of either start site. A sigma 54 consensus binding sequence was found to be located between S1 and S2 but no corresponding transcript was detected. Transcriptional bdhA-lacZ fusion studies indicated that gene expression was growth-phase associated. The bdhA gene from Rhizobium sp. NGR234 was also isolated and characterized and found to be highly homologous to the R. meliloti bdhA sequence. Unlike R. meliloti , NGR234 is able to accumulate PHB during symbiosis. An NGR234 bdhA mutant showed symbiotic defects on Leucaena but not on Tephrosia, Macroptilium or Vigna host plants, indicating that the phenotype was host-dependent. / Mutations that suppress the Hbu- phenotype without restoring Bdh activity were identified, indicating the existence of a Bdh-independent pathway for HB utilization. These mutations mapped to the age-1 locus, which causes enhanced growth rate on HB and AA minimal media. Introduction of plasmid-borne multiple copies of a gene encoding acetoacetyl-CoA synthetase (acsA) into the bdhA mutant also results in suppression of the Hbu- phenotype. A possible mechanism of suppression involving direct activation of HB to 3-hydroxybutyryl-CoA, followed by reduction to acetoacetyl-CoA by the NADP-acetoacetyl-CoA reductase (encoded by phaB) was investigated. A strain carrying the triple mutations, age-1::Tn5-Tp, bdhA ::Tn5 and phaB::OSmSp retained the ability to utilize HB, indicating that the bypass mechanism does not involve NADP-acetoacetyl-CoA reductase. / The phaB mutant does not accumulate PHB or utilize HB or AA. Furthermore, colonies of the phaB and phaC mutants exhibit non-mucoid phenotype on yeast extract mannitol agar. The observation that a R. meliloti exoS null mutant is also Hbu- provides further support for a link between PHB and exopolysaccharide synthesis. Since ExoS is a positive regulator of succinoglycan biosynthesis it is hypothesized that regulation of succinoglycan synthesis by ExoS requires PHB synthesis.

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-.

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