Sinorhizobium meliloti maintains a complex lifestyle, including saprotrophy, rhizophere colonization and root hair infection leading to the formation of root nodules in which the plant provides sustenance in return for nitrogen fixation. S. meliloti cells use a variety of carbon substrates for growth; this omnivory probably contributes to competitive ability in the soil. Several candidates for contribution to the catabolic capacity are found within the family of short chain dehydrogenases /reductases (SDR), which catalyze NAD(P)(H) dependent oxidation / reduction reactions. The 6.7 Mb genome of S. meliloti contains 78 SDR-encoding genes distributed on all three replicons. In this work each of these genes were disrupted by single crossover mutagenesis. These mutants were screened for growth on 93 different compounds as carbon source, and phenotypes were found for 17 of the mutants, providing suggestions for potential substrates of the corresponding enzymes. Carbon sources for which phenotype was observed include sugar alcohols, leucine, lysine, ornithine, galactitol, rhamnose, arabinose, mono-methyl succinate and ribono-γ-lactone. In addition, one of the mutants was found to be a proline auxotroph. In several cases, the phenotypes were consistent with the phenotypes of deletion mutants in which large sections of pSymB were absent. Eight of the mutants exhibited symbiotic deficiency after inoculation of alfalfa, while viable cells of three of the mutants could not be isolated from the nodules even though nitrogen fixation occurred. The results suggest that the corresponding SDR enzymes are involved in a pathway that is required for maintenance of viability by cells throughout infection and nodule development. This work demonstrates that members of the SDR family contribute to both the catabolic capacity and the symbiotic interactions of S. meliloti. Further experiments will address the details of the biochemical pathways involved. Knowledge of the substrate specificities of these enzymes should also prove informative in the description and annotation of orthologs that are identified in other genome sequences.

Sinorhizobium meliloti maintains a complex lifestyle, including saprotrophy, rhizophere colonization and root hair infection leading to the formation of root nodules in which the plant provides sustenance in return for nitrogen fixation. S. meliloti cells use a variety of carbon substrates for growth; this omnivory probably contributes to competitive ability in the soil. Several candidates for contribution to the catabolic capacity are found within the family of short chain dehydrogenases /reductases (SDR), which catalyze NAD(P)(H) dependent oxidation / reduction reactions. The 6.7 Mb genome of S. meliloti contains 78 SDR-encoding genes distributed on all three replicons. In this work each of these genes were disrupted by single crossover mutagenesis. These mutants were screened for growth on 93 different compounds as carbon source, and phenotypes were found for 17 of the mutants, providing suggestions for potential substrates of the corresponding enzymes. Carbon sources for which phenotype was observed include sugar alcohols, leucine, lysine, ornithine, galactitol, rhamnose, arabinose, mono-methyl succinate and ribono-γ-lactone. In addition, one of the mutants was found to be a proline auxotroph. In several cases, the phenotypes were consistent with the phenotypes of deletion mutants in which large sections of pSymB were absent. Eight of the mutants exhibited symbiotic deficiency after inoculation of alfalfa, while viable cells of three of the mutants could not be isolated from the nodules even though nitrogen fixation occurred. The results suggest that the corresponding SDR enzymes are involved in a pathway that is required for maintenance of viability by cells throughout infection and nodule development. This work demonstrates that members of the SDR family contribute to both the catabolic capacity and the symbiotic interactions of S. meliloti. Further experiments will address the details of the biochemical pathways involved. Knowledge of the substrate specificities of these enzymes should also prove informative in the description and annotation of orthologs that are identified in other genome sequences.

<p>Pap-Pit is a low affinity phosphate transporter found in <em>S. meliloti</em> and many other microorganisms. Pit is the transporter and Pap is the Pit accessory protein. Pap has been shown to be required for the function of Pap-Pit system in <em>S. meliloti</em>. In this study, <em>pap-pit</em> or <em>pit</em> alone from three species of bacteria have been expressed <em>in trans</em> in the <em>E. coli</em> Pi uptake mutants to check their ability to complement the Pi uptake deficiency of the hosts. A visualization tag, SNAP-tag, has been fused to <em>S. meliloti</em> Pap to help determine the subcellular localization of Pap. Here we show that there is an optimal level of Pap-Pit in the cells, and Pap appears to modulate this level to optimize the function of the system. We also demonstrate that Pap is probably localized intracellularly along the cell membrane. In addition, a <em>S. meliloti pap-pit </em>deletion strain has been prepared and to be used as the background strain for site-directed mutagenesis in Pap. The highly conserved surface amino acids in Pap have been identified to be the candidates for the site-directed mutagenesis.</p> / Master of Science (MSc)

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)