Thesis (Ph. D.)--Ohio State University, 2002. / Title from first page of PDF file. Document formatted into pages; contains xi, 148 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Wolfgang D. Bauer, Dept. of Horticulture and Crop Science. Includes bibliographical references (p. 127-148).

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)

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

No description available.

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)

The flagellated soil-dwelling bacterium Sinorhizobium meliloti is known for its symbiotic relationship with several leguminous genera. The symbiosis between the bacterium and its host plants facilitates fixation of atmospheric nitrogen and ultimately replenishment of nitrogen to the soil. However, before nitrogen fixation can occur, the bacterial cells must actively travel to the plant's roots and successfully induce formation of a plant organ called a root nodule. To initiate the nodulation process, the bacterium needs to be in direct contact with the root hairs. This requires free living bacterial cells within the soil to sense the presence of their host plant and travel to its roots. S. meliloti is able to do this through a process called chemotaxis. Chemotaxis is the ability to respond to chemical gradients within the environment by directed movement. It is facilitated by Methyl-accepting Chemotaxis Proteins (MCPs) as part of a two-component signal transduction system. These receptor proteins are able to bind ligands and influence the state of the signal transduction system, ultimately controlling flagellar behavior. The chemotaxis system of Escherichia coli has been well characterized and serves as a useful point of comparison to that of S. melilot throughout this work. Within this work we have determined the stoichiometry of all chemotaxis proteins of S. meliloti by means of quantitative immunoblotting. Chapter 2 addresses the stoichiometry of MCPs and the histidine kinase CheA. The eight MCPs were grouped by total abundance within the cell, in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY, McpZ and McpT). The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 22.3:1, highly similar to the 23:1 ratio known for Bacilius subtiltis. Chapter 3 continues the investigation of the protein stoichiometry, expanding to all chemotaxis proteins. We compare ratios of S. meliloti chemotaxis proteins to those of E. coli and B. subtilis. We address the possible reasons for the high ratio of CheR / CheB to the total amount of receptors. Proteins again can be grouped by abundance: CheD, CheY1, and CheY2 are the most abundant. CheR and CheB appear in lower amounts, CheS and CheT appear to be auxiliary proteins, and finally CheW1 and CheW2, which directly interact with the receptors and CheA. Chapter 4 focuses on altered receptor abundance in S. meliloti due to the fusion of common epitope tags to the C-terminus. The fusion of these tags promotes greater cellular abundance of many receptors including McpU. The fusion of charged residues to the C-terminus promotes a greater increase in McpU abundance thanthe addition of single amino acid residues. Truncations of McpU were made to investigate the presence of a protease recognition site near the C-terminus. These truncations resulted in an increase in abundance similar to those resulting from epitope tag fusions. As epitope tags are widely used in protein studies to help determine protein stoichiometry, this study obviates a potential stumbling block for future experimenters. The function of CheT, a small protein (13.4 kDa) encoded by the last gene in S. meliloti's major chemotaxis operon, is the subject of chapter 5. A cheT deletion strain is chemotactically deficient compared to the S. meliloti wild-type strain. Through two separate experiments (a glutaraldehyde cross-linking assay and co-purification) we demonstrated that CheT interacts with the methyltransferase CheR. We also investigate its possible role in CheR's methylation activity through a series of methylation assays. This work contributes to our understanding of Sinorhizobium meliloti's chemotaxis signal transduction system. We have discovered evidence for new a protein-protein interaction within our system and have revealed the abundance of all chemotaxis proteins within the cell. We also showed that fusions of epitope tags to various chemotaxis proteins can dramatically influence their abundance. We shed light on the possible function of a previously uncharacterized protein, although more work is required to determine its exact role. / Doctor of Philosophy / Sinorhizboium meliloti is a bacterium that lives in the soil and forms a symbiotic relationship with many plants including alfalfa, a commonly grown cover crop. This symbiotic relationship is important because it allows for nitrogen to be replenished into the soil without the use of artificial fertilizer. However, to form this relationship the bacterial cells in the soil must be able to colonize the plant roots. The soil is a complex environment with many different kinds of chemical molecules and sources of nutrients. Like many other types of bacteria, S. meliloti uses flagella (long helical structures that rotate much like a propeller) to move through the soil. Control of the flagella falls to what is known as a chemotaxis signal transduction system, which can be thought of as a navigation system for each bacterial cell. The system has proteins that act as receptors to sense different chemical molecules. The bacterial cells can sense signals for the plant and move towards their host. This work shows the abundance of each type of receptor and other components within the cell. It also examines the function of a previously unknown protein, CheT, within the chemotaxis system.

<p>This thesis details a study of saprophytic competence in the Gram-negative bacterium Sinorhizobium meliloti, and comprises three main areas of research. The B-ketoadipate pathway is required for the catabolism of a wide range of aromatic compounds that are released into soil through the degradation of lignin. We demonstrate that S. meliloti encodes enzymes associated with the protocatechuate branch of the B-ketoadipate pathway within two operons (pcaDCHGB and pcaIJF) whose expression is regulated by the LysR-protein PcaQ and the IclR-type regulator PcaR, respectively. We show that purified PcaQ recognizes a motif with partial dyad symmetry (5' ATAACCN4-GGTTAA 3') positioned upstream of the pcaD promoter, and that this site is required for the regulated expression of pcaD in vivo. We report that PcaQ also regulates the expression of a protocatechuate-inducible ABC-type transport system that we infer is involved in the uptake of this aromatic acid, and we extend this analysis to identify PcaQbinding motifs in the genomes of a-,B-, and y-proteobacteria. </p> <p>In addition to protocatechuate, S. meliloti may utilize hydroxyproline as an energy source, as this amino acid is released into soil during the natural decay of plant tissue. We demonstrate that S. meliloti encodes a hydroxyproline-inducible ABC-type transport system that mediates the uptake of trans-4-hydrox-L-proline, as determined via growth and transport assays. </p> <p>As a more comprehensive method of examining saprophytic competence, we assayed the growth of S. meliloti upon inoculation into sterile bulk soil. We screened 40 S. meliloti strains carrying deletions within the pSymA or pSymB megaplasmids for growth in soil, and report that the majority of strains establish a stable population (greater than or equal to 10^8 cells g^(-1) soil) that persists for several weeks. In contrast, two S. meliloti strains exhibited a decreased ability to colonize soil, indicating that loci within the deleted regions play a role in saprophytic competence. </p> / Thesis / Doctor of Philosophy (PhD)

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)