<p>Phosphate is of central importance in cellular metabolism and since bacteria are often exposed to various concentration of phosphorous in their environment, they have acquired various Pi transport systems for its uptake. <em>Sinorhizobium</em> <em>meliloti</em> has three Pi-uptake systems: a low affinity system encoded by <em>pap</em>-<em>pit</em> and two ABC type systems encoded by the <em>phnCDET</em> and <em>pstSCAB</em> operons. It is currently known that PstSCAB₂, a high affinity, high velocity transporter is induced under Pi limiting conditions and its transcription is controlled mainly in a PhoB-P dependent manner. During excess phosphate conditions, the negative regulation of the Pho regulon seems to involve PstSCAB₂ and PhoU. PhoU appears to be a negative regulator of the Pho regulon however; the mechanism by which PhoU accomplishes this task is currently unknown. In <em>Escherichia</em> <em>coli</em> and some other bacteria, mutations in <em>phoU</em> result in constitutive Pho regulon expression as do mutations in the <em>pstSCAB</em> genes. In order to address the function of PhoU in <em>Sinorhizobium</em> <em>meliloti</em>, we report the creation of a <em>Sinorhizobium</em> <em>meliloti</em> <em>ΔphoU</em> mutant strain. Results from the analysis of the <em>S. meliloti ΔphoU</em> strain suggest that this mutant behaves similarly to <em>E. coli phoU </em>mutant where one observes constitutive expression of the Pho regulon.</p> / Master of Science (MSc)

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.

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)

The conversion of atmospheric dinitrogen (N2) into plant available nitrogen (N), by legumes and their prokaryotic microsymbionts, is an integral component of sustainable farming. A key constraint to increasing the amount of N2 fixed in agricultural systems is the prevalence of symbioses which fix little or no N. The biotic factors leading to this suboptimal N2 fixation have not been extensively analysed. Using the widely studied and cultivated perennial legume Medicago sativa and the model indeterminate annual legume Medicago truncatula with the sequenced bacterial microsymbiont Sinorhizobium meliloti 1021 (Sm1021) as a basis, the work presented in this thesis examined the effectiveness of N2 fixation in these associations and in other comparable systems and investigated factors which lead to the establishment of suboptimally effective symbioses. The ability of Sm1021, S. medicae WSM419 and the uncharacterised Sinorhizobium sp. WSM1022 to fix N with M. truncatula A17, M. sativa cv. Sceptre and a range of other Medicago spp. was evaluated in N-limited conditions. As measured by plant shoot dry weights and N-content, Sm1021 was partially effective with M. truncatula A17 whereas WSM1022 and WSM419 were both effective with this host in comparison to nitrogen-fed (N-fed) control plants. In contrast, Sm1021 and WSM1022 were effective with M. sativa while WSM419 was only partially effective. Nodules induced by Sm1021 on M. truncatula A17 were more numerous, paler, smaller in size and more widely distributed over the entire root system than in the two effective symbioses with this host. On the contrary, nodule number, size and distribution did not differ between these three strains on M. sativa. WSM1022 was effective on M. littoralis, M. tornata and two other cultivars of M. truncatula (Jemalong and Caliph) but Sm1021 was only partially effective on these hosts. These data indicate that the model indeterminate legume symbiosis between M. truncatula and Sm1021 is not optimally matched for N2 fixation and that Sm1021 possesses broader symbiotic deficiencies. In addition, the interaction of WSM1022 with M. polymorpha (small white nodules but does not fix N), M. murex (does not nodulate), M. arabica (partially effective N2 fixation) and M. sphaeorcarpus (partially effective N2 fixation), and the sequence of the 16S rDNA, are all consistent with this isolate belonging to the species S. meliloti. The colony morphology of TY, half-LA and YMA agar plate cultures of Sm1021, WSM419 and WSM1022 suggested differences in EPS profiles between these strains. Sm1021 is very dry, compared to the mucoid WSM419 and extremely mucoid WSM1022. Sm1021 is known to carry an insertion in expR rendering the gene non-functional and resulting in the dry colony phenotype. WSM419 has an intact copy of expR, while the expR status of WSM1022 is not known. Rm8530, a spontaneous mucoid derivative of Sm1021 with an intact expR, was significantly less effective with M. truncatula than Sm1021, but there was no difference in effectiveness between these strains on M. sativa. The effectiveness of Sm1021, when complemented with a plasmid-borne copy of expR from Rm8530, was significantly reduced on M. truncatula but not M. sativa, implicating a functional expR as being the cause of reduced N2 fixation observed with Rm8530 on M. truncatula. ExpR could reduce the effectiveness of Rm8530 by acting as a negative regulator of genes essential for symbiosis with M. truncatula, or by altering the quantity or structure of succinoglycan and/or galactoglucan produced. These data support the emerging view of ExpR being a central regulator of numerous cellular processes. The timing of nodulation between Sm1021 and WSM419 on M. truncatula and M. sativa was investigated. Compared to the other symbioses analysed, the appearance of nodule initials and nodules was delayed when M. truncatula was inoculated with Sm1021 by 3 and 4 days, respectively. To explore whether events during early symbiotic signalling exchange could account for these observed delays, leading to the establishment of a suboptimal N2-fixing symbiosis, a novel system was developed to compare the response of the Sm1021 transcriptome to roots and root exudates of M. truncatula A17 and M. sativa cv. Sceptre. This system consisted of a sealed 1 L polycarbonate chamber containing a stainless steel tripod with a wire mesh platform on which surface-sterilised seeds could be placed and allowed to germinate through the mesh, into a hydroponic medium below. After germination, Sm1021 cells were inoculated into the hydroponic solution, exposed to the roots and root exudates for 16 h, harvested and their RNA extracted. Comparison of Sm1021 mRNA from systems exposed to M. truncatula or M. sativa revealed marked differences in gene expression between the two. Compared to the no plant control, when M. sativa was the host plant, 23 up-regulated and 40 down-regulated Sm1021 genes were detected, while 28 up-regulated and 45 down-regulated genes were detected with M. truncatula as the host. Of these, 12 were up-regulated and 28 were down-regulated independent of whether M. truncatula or M. sativa was the host. Genes expressed differently when exposed to either M. truncatula or M. sativa included nex18, exoK, rpoE1 and a number of other genes coding for either hypothetical proteins or proteins with putative functions including electron transporters and ABC transporters. Characterisation of these differentially expressed genes along with a better understanding of the composition of M. truncatula root exudates would yield a clearer insight into the contribution of early signal exchange to N2 fixation. Comparison of the regulation of nodule number between Sm1021 and WSM419 on M. truncatula and M. sativa revealed nodule initials at 42 days post-inoculation (dpi) on M. truncatula inoculated with Sm1021. In contrast, no new nodule initials were present 21 dpi on any of the other interactions examined. Moreover, analysis of nodule sections revealed that the number of infected cells in M. truncatula-Sm1021 nodules was less than for comparable symbioses. These data suggest that nodule number is not tightly controlled in the M. truncatula-Sm1021 association, probably due to N2 fixation being insufficient to trigger the down regulation of nodulation. Quantification of N2 fixation activity in this and other more effective symbioses is required. The poor effectiveness of the M. truncatula-Sm1021 symbiosis makes these organisms unsuitable as the model indeterminate interaction and the implications for legume research are discussed. The recently sequenced WSM419 strain, revealed here to fix N2 more effectively with M. truncatula than Sm1021, may be a better model microsymbiont, although WSM419 is only partially effective for N2 fixation with M. sativa. The sequencing of S. meliloti WSM1022, a highly effective strain with both M. truncatula and M. sativa, would provide a valuable resource in indentifying factors which preclude the establishment of effective symbioses.