Objetivo general. Abordar la caracterización molecular y funcional de un sistema binario de plásmidos crípticos involucrados en la transferencia horizontal de genes por conjugación en la bacteria fijadora de nitrógeno y simbionte de alfalfa, Sinorhizobium meliloti. Objetivos específicos. En el marco del objetivo precedente abordaremos el estudio de las funciones que hacen a la transferencia conjugativa y a la replicación del plásmido críptico modelo pSmeLPU88b de S. meliloti descripto previamente por Pistorio et al., (2003) según el siguiente esquema: - Identificación y caracterización de elementos estructurales y genes involucrados en la movilización del plásmido modelo pSmeLPU88b. - Caracterización del complejo Dtr (DNA transfer and replication). - Clonado, y caracterización de la región génica del plásmido pSmeLPU88b asociada al módulo Dtr. Búsqueda y caracterización del gen de la relaxasa y de su producto de traducción como una de las proteínas centrales de la transferencia de ADN via conjugativa. - Identificación y caracterización funcional del origen de transferencia (oriT). - Evaluación con herramientas moleculares del grado de ubicuidad de funciones de movilización (Dtr) en S. meliloti. - Identificación y caracterización de los módulos de replicación del plásmido pSmeLPU88b. - Clonado de los módulos de replicación, secuenciamiento, estudios de incompatibilidad. - Evaluación de la transmisibilidad del plásmido pSmeLPU88b en el medio suelo, en condiciones de laboratorio y de campo.

Phosphorous (P) is an essential nutrient, playing a central role in the life of a bacterial cell. It is involved in cellular metabolic pathways, cell signaling and is a component of many of the cell’s macromolecules. Since a majority of the biosphere’s microorganisms have not yet been cultured, much more can be learned about the biochemical and genetic mechanisms that govern bacterial P metabolism. The function-driven approach to metagenomics was applied to study P metabolism in the bacterial communities present in pulp and municipal wastewater treatment plant activated sludge and soil, leading to the isolation and identification of three new phosphatases, genes involved in P transport, regulation of P related functions and additional genes which may be important for the bacterial cell’s adaptation to the above communities. The identification of two new nonspecific acid phosphatases (NSAPs) phoNACX6.13 and phoNBCX4.10 and an alkaline phosphatase, phoAACX6.71, belonging to the nucleotide pyrophosphatase phosphodiesterase (NPP) family is reported here. The genes for the three phosphatases were cloned, sequenced, and analysed for upstream regulatory sequences in addition to biochemical characterization of their protein products. PhoB-binding sites were found upstream to phoAACX6.71 and NSAP phoNACX6.13, suggesting these genes are governed by the mechanisms of the previously described “pho” regulon. The two NSAPs have pH optima in the acidic neutral range while the alkaline phosphatase has an optimal pH at 9.5. The three phosphatases appear to be distantly related to known bacterial phosphatase enzymes. Phylogenetic analysis shows the newly identified NSAPs appear on a separate clade from known bacterial NSAPs. Key amino acid residues involved in the catalytic site of these NSAPs were identified in PhoNACX6.13 and PhoNBCX4.10.In PhoAACX6.71, key amino acid residues involved in catalysis and metal cofactor coordination were identified. The roles of these residues were confirmed based on the predicted molecular structure of these proteins. The structures indicate the three proteins are globular with folding patterns suitable for catalytic residues to bind and cleave the P substrate. This is the first report of functional characterization of phosphatases from uncultured bacteria. In addition to exploring the hydrolysis of phosphate esters, the transport and metabolism of other P compounds was also investigated. By phenotypic complementation of phosphonate growth deficient mutants of the legume symbiont, Sinorhizobium meliloti and large scale sequencing of selected metagenomic clones, 92 ORFs were isolated. As expected, about 25% of these ORFs are P transport proteins and P related regulators. Genes involved in other regulatory functions made up about 12% of the total while genes related to Nitrogen metabolism and assimilation account for about 8% of the newly identified ORFs. About 30% of the ORFs encoded general cellular functions or hypothetical proteins of unknown function. The results of this investigation demonstrate the effectiveness of functional metagenomics in studying genetic diversity of bacteria inhabiting complex microbial communities and in identifying new proteins of interest.

Phosphorous (P) is an essential nutrient, playing a central role in the life of a bacterial cell. It is involved in cellular metabolic pathways, cell signaling and is a component of many of the cell’s macromolecules. Since a majority of the biosphere’s microorganisms have not yet been cultured, much more can be learned about the biochemical and genetic mechanisms that govern bacterial P metabolism. The function-driven approach to metagenomics was applied to study P metabolism in the bacterial communities present in pulp and municipal wastewater treatment plant activated sludge and soil, leading to the isolation and identification of three new phosphatases, genes involved in P transport, regulation of P related functions and additional genes which may be important for the bacterial cell’s adaptation to the above communities. The identification of two new nonspecific acid phosphatases (NSAPs) phoNACX6.13 and phoNBCX4.10 and an alkaline phosphatase, phoAACX6.71, belonging to the nucleotide pyrophosphatase phosphodiesterase (NPP) family is reported here. The genes for the three phosphatases were cloned, sequenced, and analysed for upstream regulatory sequences in addition to biochemical characterization of their protein products. PhoB-binding sites were found upstream to phoAACX6.71 and NSAP phoNACX6.13, suggesting these genes are governed by the mechanisms of the previously described “pho” regulon. The two NSAPs have pH optima in the acidic neutral range while the alkaline phosphatase has an optimal pH at 9.5. The three phosphatases appear to be distantly related to known bacterial phosphatase enzymes. Phylogenetic analysis shows the newly identified NSAPs appear on a separate clade from known bacterial NSAPs. Key amino acid residues involved in the catalytic site of these NSAPs were identified in PhoNACX6.13 and PhoNBCX4.10.In PhoAACX6.71, key amino acid residues involved in catalysis and metal cofactor coordination were identified. The roles of these residues were confirmed based on the predicted molecular structure of these proteins. The structures indicate the three proteins are globular with folding patterns suitable for catalytic residues to bind and cleave the P substrate. This is the first report of functional characterization of phosphatases from uncultured bacteria. In addition to exploring the hydrolysis of phosphate esters, the transport and metabolism of other P compounds was also investigated. By phenotypic complementation of phosphonate growth deficient mutants of the legume symbiont, Sinorhizobium meliloti and large scale sequencing of selected metagenomic clones, 92 ORFs were isolated. As expected, about 25% of these ORFs are P transport proteins and P related regulators. Genes involved in other regulatory functions made up about 12% of the total while genes related to Nitrogen metabolism and assimilation account for about 8% of the newly identified ORFs. About 30% of the ORFs encoded general cellular functions or hypothetical proteins of unknown function. The results of this investigation demonstrate the effectiveness of functional metagenomics in studying genetic diversity of bacteria inhabiting complex microbial communities and in identifying new proteins of interest.

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.

A Sinorhizobium meliloti (S. meliloti ) mutant, Rm30O49, deficient in malate dehydrogenase (MDH) activity was isolated via random Tn5tac1 mutagenesis. DNA sequence analyses revealed 60 the inaction is within the mdh gene. Rm30049 lacks MDH activity under all growth conditions, but shows increased or decreased activities of the TCA cycle enzymes 2-oxoglutarate dehydrogenase and succinate dehydrogenase in the presence or absence, respectively, of IPTG (isopropyl beta-D-thiogalactoside). The symbiotic phenotype of the mutant is an inability to fix nitrogen. Alfalfa seedlings inoculated with Rm30049 produced small white root nodules, but were chlorotic and failed to reach a wild-type shoot dry weight. Cosmid clone pDS15 was isolated by heterologous complementation of a Rhizobium leguminosarum sucD mutant by the S. meliloti pLAFR1 clone bank. This cosmid also restored MDH activity to Rm30049, and complemented the mutant growth and symbiotic phenotypes. Three Tn5 insertions isolated in pDS15 within sucA failed to complement Rm30049. DNA sequence analyses indicate that the mdh gene is part of the TCA cycle operon with sucCD, and that downstream and upstream of this, are operons encoding sucAB and sdhCDAB, respectively.

Transcriptional fusion mutants and Tn5-B20 transposon mutants were isolated where the only genes affected are believed to either be involved in the hypothetical GABA shunt or code for subunits of the alpha-ketoglutarate dehydrogenase enzyme complex of Sinorhizobium meliloti. The growth phenotypes of Rm30222 (gabT) and eight mutants in gabD1, 2, 3, and 4 on minimal media were comparable to that of the wild-type. Compared to wild-type, Rm30222 (gabT) lacked alpha-ketoglutarate-dependent gamma-aminobutyrate transaminase activity showed high induction of gabT on GABA but produced green plants indicative of being Fix+. Mutants in gabD alleles maintained wild-type levels of succinic semialdehyde dehydrogenase activity and could fix nitrogen as well as the wild-type in symbiosis. / Mutation of sucB encoding a subunit of a-ketoglutarate dehydrogenase produced a mutant, Rm30230, that initially had difficulty growing on minimal media supplemented with either arabinose or glutamate. In symbiosis with alfalfa, Rm30230 had a fix- phenotype and was also devoid of alpha-ketoglutarate dehydrogenase activity. The ability of Rm30230 to grow on arabinose or glutamate, without alpha-ketoglutarate dehydrogenase activity, strengthens the hypothesis that S. meliloti has a functional GABA shunt allowing it to circumvent the forward-direction TCA cycle from alpha-ketoglutarate to succinate. Mutation of the second potential dihydrolipoamide succinyltransferase component (E2) of alpha-ketoglutarate dehydrogenase yielded Rm30267 (SMb20019) with wild-type growth on minimal media and a Fix+ phenotype in plants. The introduction or a sucB mutation into the SMb20019 mutant background (Rm30275) was comparable to the sole sucB mutation. This finding shows that the locus SMb20019 cannot be substituted for sucB in the alpha-ketoglutarate dehydrogenase enzyme complex.

The transcriptional promoter sequences for the Rhizobium meliloti nitrogen fixation genes nifA and nifB were cloned to a β-galactosidase gene fusion plasmid vector and transferred by homologous recombination to a specialized transducing phage. The promoter fusions were then transduced to a recombination deficient strain of Escherichia coli as single-copy lysogens and analysed under defined aerobic and anaerobic conditions. The lysogenic strains contained plasmids encoding either of two transcriptional activator proteins, NifA or FixJ, produced from a constitutive plasmid promoter. The expression of the nifA and the nifB promoters was found to be sensitively regulated by the carbon source used for anaerobic fermentation or anaerobic respiration, the redox potential of the terminal electron acceptor used for anaerobic respiration, and the growth phase of anaerobic cultures. The repression of nit promoter expression by oxygen respiration was specifically compared to anaerobic respiration of alternative electron acceptors. Both nifA and nifB promoter expression decreased exponentially as the reduction potential of the terminal respiration reaction increased. The repressive effect of oxygen appears to be due soley to the exponential relationship between nit promoter expression and the redox potential of oxygen respiration. In addition to separate fusions of the nifA and nifB promoters to β-galactosidase, a single-copy fusion of the entire nifA-nifB region was constructed. In this construct, plasmid-encoded FixJ protein stimulated the expression of a chromosomal nifA gene to produce the NifA protein, which then stimulated the expression of the nitB promoter. This strain produced 20-fold lower activity than a strain in which nifB promoter expression was stimulated by plasmid-encoded NifA protein. Finally, the nifA locus was found to contain a transcriptionally active element, oriented opposite to the nifA promoter. / Graduation date: 1992

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

Techn. Universiẗat, Diss., 2005--Berlin.