Page i was not in the other copies of this thesis. -Digitization Centre / Tn5 mutagenesis was used to generate four independent mutants of Rhizobium meliloti that were unable to grow on protocatechuate (Pca-). Two of the Pca- mutations were mapped to a region of the second symbiotic megaplasmid (pRmeSU47b) previously shown to be required for growth on protocatechuate. This pca locus was shown to consist of the first five structural genes of the protocatechuate branch of the B-ketoadipate pathway, in the order pcaDCHGB. This gene order is the same as determined for Agrobacterium tumefaciens. An additional reading frame with homology to LysR-type regulators was found to be upstream of, and transcribed divergently to the pcaDCGHB operon. This is likely to fulfil the same role as the regulatory gene, pcaQ, of A. tumefaciens. A cosmid plasmid which carried these pea genes failed to complement the Pca- phenotype of a strain carrying a 300 kb megaplasmid deletion encompassing this pea locus. This implies that another pca locus, perhaps pcaIJ, is present within the deleted region of the rnegaplasmid. Two Pca- Tn5 insertions which did not map to the megaplasmid locus were isolated. One of these insertions appears to be in a catalase gene. / Thesis / Master of Science (MS)

Genetic mapping of pDS15 revealed that this cosmid clone carries the Sinorhizobium meliloti TCA cycle genes mdh, sucCDAB, sdhAB and part of lpdA. Three genes (mdh, sucC , and sucA) were completely sequenced and submitted to GenBank. The nucleotide and amino acid sequences of the TCA cycle genes encoded on pDS15 were aligned and found to be highly homologous with other closely related rhizobial species. S. meliloti cells grown in LBmc express the mdh-sucCDAB operon as one transcript, based on RT-PCR results. Alternative sigma factor sigma54 was not found to have a role in mdh-sucCDAB expression. Despite considerable effort, we have not been able to isolate sucA mutants via random transposon Tn5tac1 mutagenesis to date. Homologous recombination between a plasmid-borne sucA::Tn5 and wild-type S. meliloti sucA failed to generate a bona fide mutant, as revealed by Southern blot analysis. Plasmid pDS15 was mutagenized with transposons Tn5, Tn5tac1, and Tn5-B20. Three Tn5-B20 insertions were mapped to mdh, sucD, and sucA respectively, and preliminary gene expression studies were done.

No description available.

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. coli and S. meliloti results in filamentation. / Thesis / Master of Science (MSc)

Thesis (Ph.D.) -- McMaster University, 1997. / Includes bibliographical references (leaves 237-260). Also available via World Wide Web.

Techn. Universiẗat, Diss., 2000--Berlin.

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)