Thesis (Ph. D.)--University of Texas at Dallas, 2007. / Includes vita. Includes bibliographical references (leaves 191-192)

Hydroxyproline (trans-4-hydroxy-L-proline (4-L-Hyp)) can be used by certain microorganisms as a source of carbon and nitrogen. The nitrogen fixing bacterium, Sinorhizobium meliloti carries a cluster (hyp cluster) of 14 genes responsible for the transport and degradation of this amino acid in the cell. The biological functions of several gene products in the hyp cluster are still unknown. So far, it is known that the conversion of trans-4-hydroxy-proline to α-ketoglutarate, one of the intermediate of the TCA cycle, occurs in four enzymatic reactions. The whole hyp cluster is up regulated in the presence of 4-hydroxy-proline in the media. Previous studies have shown several other 4-hydroxy-proline-inducible genes. One of these genes, smc04388, has been annotated as a putative omega amino transaminase. The role of this omega transaminase in the main catabolic pathway of 4-hydroxy-proline has not been investigated. In order to address this, two mutant strains; a single smc04388 mutant and a double smc04388 hypD mutant were created. Growth curves of these mutants in minimal media showed that the Smc04388 protein is not required for the growth of S. meliloti in the presence of trans-4-hydroxy-L-proline as the sole carbon and nitrogen source. The Smc04388 protein was overexpressed as a Strep-tagged and purified from S. meliloti. The purified enzyme showed amino transaminase activity with pyruvate and α-methylbenzylamine. In addition, an enzymatic reaction using the product of the second enzyme of the 4-hydroxy-proline pathway, Δ1-pyrroline-4-hydroxy-2-carboxylate, was carried out to test the activity of Smc04388 with this compound. Mass spectrometry analysis of this reaction mixture revealed the formation of L-alanine from pyruvate and Δ1-pyrroline-4-hydroxy-2-carboxylate, suggesting the utilization of this compound as an amino donor by the Smc04388 transaminase. It was also shown that transamination activity in cell extracts increase in the absence of Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase, the enzyme that catalyzes the conversion of this compound in the 4-hydroxy-proline pathway. These results confirmed the hypothesis that the Smc04388 omega amino transaminase is involved in a secondary pathway related to the known catabolic pathway of 4-hydroxy-proline in bacteria. Further understanding of this secondary pathway will contribute to the study of the metabolism of 4-hydroxy-proline in bacteria. In addition to this, the complete characterization of the Smc04388 omega amino transaminase could have practical application in the pharmaceutical industry. / Thesis / Master of Science (MSc)

The genome of the a-proteobacterium Sinorhizobium meliloti has been completely sequenced and annotated. providing a wealth of information about this endosymbiotic N2 fixing organism. Although the structure of the genome, consisting of a circular chromosome and two smaller pSymA and pSymB replicons, has long been known, only a small portion of ORFs have previously been characterized. Sequence analysis of pSymB has revealed that a large portion of the 1570 ORFs code for solute uptake systems and polysaccharide biosynthesis. The pSymB replicon been referred to as a "megaplasmid," implying that pSymB is non-essential for viability of the organism. However, coded on pSymB are several essential genes, including a tRNA ArgCCG gene and the minCDE genes, which are not found elsewhere in the genome. Replication of pSymB is controlled by repABC genes, a typical property of plasmids among Rhizobiaceae. Therefore, the genome signature, a compositional analysis that allows comparison of whole replicons rather than focusing on particular genes, was used to provide support for designation of pSymB as a second chromosome in S. mdiloti. It was found that among a-proteobacteria, plasmids and chromosomes have distinctive patterns of dinucleotide biases, and in this respect, pSymB is chromosome-like while pSymA is plasmid-like. This brings into question how the pSymB replicon came to acquire chromosome-like properties while appearing to be maintained as a plasmid in the genome. Whole-genome nearest neighbor analysis shows that the linear chromosome of Agrobacterium tumefaciens and pSymB may have a common origin. Despite conservation of gene order within small groups of genes, it is evident that rearrangements, duplications, and horizontal transfer of genes since the divergence of these species have contributed to the mosaic nature of pSymB. Since synteny between the S. meliloti chromosome and A. tumefaciens circular chromosome is highly conserved, it appears that the instability of pSy mB has played a key role in the adaptation and evolution of S. meliloti. / Thesis / Master of Science (MS)

DNA replication initiates at a precise location on the bacterial chromosome, the origin of replication (oriC). This work has localized the origin of DNA replication on the Sinorhizobium meliloti chromosome to a region spanning the hemE gene. A genetic dissection of the locus revealed that a much larger fragment of DNA (1802 bp) is required for a functional oriC than that of the other characterized alpha-proteobacterial chromosome origin from Caulobacter crescentus. Site-directed mutations of predicted DnaA binding sites has identified several essential elements for replication of the plasmid borne oriC. Mutations in these DnaA boxes also reduce transcription of hemE and thus it is likely that transcription of hemE and replication of the S. meliloti chromosome are coupled. The ColEl plasmid pUCP30T can autonomously replicate when the S. meliloti oriC is cloned into the suicide vector (pTH838) and can be efficiently mobilized out of S. meliloti into E. coli. The pTH838 oriC plasmid when transferred into S. meliloti results in both small and large colonies and both of these transconjugant classes take longer to form than the S. meliloti recA::Tn5 recipient. We attributed this phenotype to the very low copy number of the pTH838 plasmid which was determined to be 0.053 - 0.135 copies per chromosome. The DnaA protein responsible for replication initiation in many bacteria has been purified and used in electrophoretic mobility shift assays. The DnaA protein interacts specifically with sequences in the hemE - Y02793 intergenic region and upstream of the repA2 gene on the pSymA megaplasmid. The DnaA protein has also been implicated as a link between DNA replication and cell division in S. meliloti as overexpression of DnaA in both E. coll and S. meliloti results in filamentation. / Thesis / Master of Science (MSc)

<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)

Soils contain a complex mixture of compounds many of which can be transported and metabolized by microorganisms. 𝘚𝘪𝘯𝘰𝘳𝘩𝘪𝘻𝘰𝘣𝘪𝘶𝘮 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 is a soil bacterium whose gene sequence was recently determined. The diversity of the carbon and nitrogen sources than can be utilized by this organism is reflected in the large number of annotated ATP-binding cassette transporters in its genome. Although many of these genes are not necessary for survival, it is hypothesized that they arid in the competitive fitness of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 in the field. Many of these transporters remain uncharacterized. In this study a high throughput screen was developed to measure β-glucuronidase activity in a 96 well microtitre plate format to quantify expression of many reporter gene fusions under a variety of conditions. This system was used to analyze the expression of putative small molecule ABC transporters in 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪. 45 𝘨𝘶𝘴𝘈 reporter gene transcriptional fusions to these transport genes were generated and recombined into the genome. These strains were grown in 96 well plates in minimal media containing a large number of carbon sources and carious legume root and seed exudates to be tested as inducers of transporter gene expression. Two transport systems were found to be induced by glucosamine and galactosamine and others were found to be induced by various sugars including mannose, arabinose, xylose and palatinose as well as protocatechuate and hydroxybenzoate. The bacteria-plant symbiosis of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 and alfalfa plays an important role in agriculture. To further understand the role of ABC transporters in the competition of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 the 𝘨𝘶𝘴𝘈 reporter fusions strains will also e inoculated onto alfalfa roots and nodules will be assayed for GusA activity to give a more complete picture of the role of ABC transporters in the competition and symbiosis of 𝘚. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪. Two substrates, galactitol and hydroxyproline found to induce transport systems were studied in depth in order to more fully understand the transport, metabolism and regulation of these compounds. / Thesis / Master of Science (MS)

Previous studies in this laboratory have identified two phosphate transport systems in Sinorhizobium meliloti encoded by the phoCDET and orfA-pit genes respectively. The PhoB regulatory protein is required for transcriptional activation of the phoCDET genes but repress the transcription of orfA-pit. Determination of the DNA sequence upstream of phoU-phoB revealed the presence of genes homologous to the pstA-pstB genes, which encode components of an ABC-type high affinity Pi specific transport system in E. coli. Further analysis of sequence from the S. meliloti genome project (unpublished) revealed the phoR-pstS-pstC genes upstream of pstA-pstB. Using an R-prime approach, we cloned a 7.5 kb Hindlll gene fragment which included the above phoR-pstS-pstC-pstA-pstB and partial phoU genes. Using Tn5-B20 and lacz-aacc1 cassette gene disruption/fusions, we mutated pstA, pstB and phoR gene respectively. We found that: a) pstA-pstB-phoU-phoB are in one operon, b) pstB expression is not regulated by the media phosphate concentration and is independent of phoB, c) in free-living cells, pstB mutants, like phoU or phoB mutants, exhibit alkaline phosphatase negative phenotypes, d) in plant tests, a pstB mutant had normal nitrogen fixation ability and like phoB mutations, the pstB mutation suppressed the Fix- phenotype of phoCDET mutants, e) phoB expression is neither regulated by phosphate concentration nor does its expression appear to be auto-regulated, and f) a phoR mutant exhibited an alkaline phosphatase negative phenotype. Sequence analysis showed that there is no pho box in the upstream of pstA-pstB-phoU-phoB operon and the phoR, but pstS gene has one putative pho box in its promoter region. Also discussion and some ideas for future study were presented. / Thesis / Master of Science (MS)

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.