Persistence in human macrophages is central to the virulence of Mycobacterium tuberculosis, which is the causative agent of tuberculosis. Although the intracellular parasitism is apparent, molecular determinants of mycobacterial virulence are not well understood. The current investigation identified virulent genes of M. tuberculosis by measuring survivability of Mycobacterium smegmatis recombinants inside a human monocytic cell line THP-1 after acquiring various virulent gene candidates of M. tuberculosis. These gene candidates included nine virulent gene candidates suggested by other studies, five genomic polymorphisms identified in hypervirulent strains of M. tuberculosis using microarray-based comparative genomic hybridization, and ten single nucleotide polymorphisms identified in the hypervirulent strains using full genome sequencing. Interestingly, only recombinants harboring a truncated Rv2820c and a known virulent gene mce1A survived significantly better than vector control after six hours of ex vivo infection. As nucleotide sequencing indicated that the truncated Rv2820c loses around 60% of gene at 3’ end, ex vivo survivability of M. smegmatis recombinants harboring the last 60% of Rv2820c as well as the intact Rv2820c was measured, but was similar to that of vector control. The 3’ truncated portion itself did not alter mycobacterial survivability ex vivo, but its presence did compromise the survival advantage gained due to the truncated Rv2820c. To determine whether the truncated and the intact Rv2820c could enhance mycobacterial virulence in vivo, these two alleles were transformed into Mycobacterium marinum and their recombinants were used to infect zebrafish. In vivo infection showed that zebrafish infected with the recombinant harboring truncated Rv2820c died significantly faster than vector control, whereas the recombinant harboring intact Rv2820c behaved similarly to vector control. Results indicated that the truncated Rv2820c, but not the intact Rv2820c, could enhance mycobacterial virulence both ex vivo and in vivo. Additional nucleotide sequencing revealed that the 3’ truncation in Rv2820c is caused by a Beijing/W-defining deletion RD207 and is commonly found in Beijing/W strains of M. tuberculosis. Non-Beijing/W strains possess the intact Rv2820c conversely. Since Beijing/W strains have proven to be more virulent than non-Beijing/W strains both ex vivo and in vivo, the truncated Rv2820c may be one of the Beijing/W-specific virulence determinants. To confirm that Rv2820c of Beijing/W strains really enhances M. tuberculosis survival in human macrophages, the truncated Rv2820c was transformed into non-Beijing/W M. tuberculosis strains and their recombinants were used to infect THP-1 cells. Ex vivo infection confirmed that the truncated Rv2820c could enhance M. tuberculosis survival inside human macrophages, but is unlikely to induce a different profile of cytokine secretion from infected macrophages. In conclusion, the current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence both ex vivo and in vivo. Enhanced phenotypic virulence, however, was not observed for the intact Rv2820c of non-Beijing/W strains. The truncated Rv2820c may be one of the Beijing/W-specific virulence determinants and collaboratively contribute to the high phenotypic virulence of this family. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Bacterial degradation is an important way to detoxify environmental pollutants haloacids, and the key enzyme involved is dehalogenase. In contrast to the well characterized dehalogenases, haloacids transporters that mediate uptake of haloacids are poorly understood. The deh4p gene in a haloacids-degrading bacterium Burkholderia species MBA4 is the first reported haloacids transporter gene. It is located downstream of the dehalogenase gene deh4a and the two forms a haloacids operon. The role of Deh4p as a haloacids transporter was confirmed by heterologous expression. It was later found that a mutant of MBA4 without functional Deh4p was still able to grow in monochloroacetic acid (MCA), and Deh4p seems to be not the only haloacids transporter in MBA4. This study aimed at gaining a deeper understanding of the haloacids transport process in MBA4, and establishing the role of a newly identified gene dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. Disruption of deh4p in MBA4 caused a 32% decrease in MCA uptake rate, confirming the role of Deh4p as a haloacids transporter, but not the only one. A gene showing homology to deh4p in MBA4 was identified and named dehp2. The role of Dehp2 as a second haloacids transporter in MBA4 was confirmed by both gene disruption and heterologous expression. Like deh4p, the expression of dehp2 is also MCA-inducible. A double mutant with both deh4p and dehp2 disrupted only retained 36% MCA uptake rate, further confirming the roles of Deh4p and Dehp2 as haloacids transporters. Dehp2 and Deh4p were also shown to be the two major haloacids transporters. Both Dehp2 and Deh4p are specific toward acetate and its halogenated derivatives, but Dehp2 has a broader spectrum of substrates than Deh4p. Deh4p was found to be a better MCA transporter than Dehp2, and also has a higher affinity for MCA. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) and pH on MCA uptake supported the symport of proton(s) and the inclusion of both Dehp2 and Deh4p in the metabolites:H+ symporter family. Orthologs of dehp2 are widely found in the Burkholderia genus, and phylogenetic analysis showed that they were conserved in the genus. The roles of dehp2 orthologs in haloacids transport in three non-pathogenic Burkholderia species were studied. The three species were engineered to gain the ability to utilize MCA as the sole carbon source, and were shown to have MCA-inducible MCA uptake activities. Expressions of the dehp2 orthologs in them are MCA-inducible, strongly suggesting their relationship with MCA uptake. Fusion analysis with lacZ as a reporter gene confirmed the presence of MCA-inducible promoter activity in the upstream non-coding region of dehp2, and the results of electrophoretic mobility shift assay (EMSA) suggested a positive regulation of dehp2. This study established Dehp2 as a second haloacids transporter in MBA4, and also confirmed dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. The presence of at least two haloacids transporters in MBA4 demonstrated the complexity of this process, and future work should figure out the transport mechanisms. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Bacterial dehalogenase is a key enzyme involved in bioremediation of halogenated organic compounds. A dehalogenase, Deh4a, was isolated from the Gram-negative bacterium Burkholderia caribensis MBA4, which can utilize haloacetic acids as carbon source. The haloacid operon in MBA4 was identified and characterized. It is composed of the structural genes forDeh4a and a transporter Deh4p. Transcription of this operon is negatively regulated, but the mechanism and the relevant regulator are still poorly understood. In this study, magnetic DNA affinity chromatography and Tn5transposon mutagenesis were employed to explore the regulatory factors that affected the expression of this haloacid operon. A process that uses lysates from glycolate-grown cells, magnetic DNA affinity chromatography and LC-MS/MS has identified a TetR family transcriptional regulator, TetR8620, which binds to the promoter region of deh4a. Disruption of the TetR8620 gene in mutant Ins8620 abolished the formation of a slow migrating complex in electrophoretic mobility shift assay (EMSA) using lysates from glycolate-grown cells. Moreover, expressions of deh4a were enhanced in bothglycolate- and MCA- grown Ins8620. The addition of recombinant histidine-tagged TetR8620 to lysates of Ins8620 resumed the formation of a retardation complex, but different from that using purified His-tagged TetR8620.This suggested that TetR8620 is responsible for formation of retardation complexes, and an additional protein might be involved. To investigate other putative factors that interact with TetR8620, purified His-tagged TetR8620 was immobilized with Ni-NTA agarose and used for isolation of interacting proteins. Chemical cross-linking of the purified fraction with BS3established that TetR8620 interacts with a proteinof30 kDa. Separation of the cross-linked complex in SDS-PAGE gel also showed that a protein with similar MW was specifically pulled down. These results suggest that TetR8620 was interacting with a ~30 kDa protein. Protein identification using mass spectrometry assay proposed that this protein is probably a universal stress protein UspA encoding by peg.3485 or acetyl-glutamate kinase (EC 2.7.2.8) encoding by peg.714 in MBA4. Tn5transposon mutagenesis was also employed to explore the factors that regulate the haloacid operon ofMBA4. A derivative of MBA4, MK06, which contains a kanamycin resistant gene (kan) with a deh4apromoter was constructed. Kanamycin resistancy of this derivative was MCA inducible. Transposon mutagenesis was conducted on this derivative, and Tn-containing mutants were isolated as tetracycline resistant colonies on pyruvate plates. These colonies were further selected on their resistance tokanamycin in pyruvate plates. Gene peg.6589 encoding a putative transcriptional regulator, DehR1, was disrupted by Tn insertion. While the production of dehalogenase was still MCA-inducible, this mutant has partially relieved the repression of the haloacid operon in media containing pyruvate. Moreover, constitutive production of DehR1 in MBA4 decreased the transcript levels of deh4ain medium containing pyruvate or MCA. This study has identified two transcription factors, TetR8620 and DehR1, which regulate the expression of Deh4a negatively. TetR8620 is a DNA-binding protein that interacts with the deh4apromoter. Results from this study imply that the regulation of the haloacid operon in MBA4 is likely to be under the control of multiple factors. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

When the DNA of Escherichia coli is damaged a set of events termed "SOS functions" occur to aid cellular survival. The recA and lexA proteins are involved in the regulation of these functions. To determine the role of the lexA protein, amber mutations, designated spr-55(amber), were isolated in the lexA-3 gene. The lack of the lexA-3 gene product abolished sensitivity to ultraviolet light and resulted in the constitutive synthesis of recA protein. Introduction of amber suppressor mutations restored the original lexA-3 phenotype. It was concluded that spr mutations inactivate lexA protein resulting in the constitutive expression of the SOS functions. These data provide evidence that the lexA protein is the repressor for the recA gene. The repair of phage lambda (λ⁺) by ultraviolet light was determined in the strains carrying alleles of the spr, uvrA, and recA genes. The survival of the phage was more in the spr-51 uvrA⁺ strain as compared to wild type. These results were not dependent on the recA genotype. Introduction of the uvrA-6 mutation into the spr-51 uvrA⁺ recA⁺ strains resulted in the same relative decrease of phage survival. These results suggest that lexA protein is involved in the regulation of uvrA-dependent excision repair and that inactivation of lexA leads to the constitutive expression of excision repair. New mutant forms of lexaA protein were isolated. The lexA⁺, lexA-3, lexA-10, and lexA-27 proteins displayed identical mobilities in the Weber and Osborn gel system. The lexA-10 and lexA-27 genes showed different phenotypes and encoded proteins of different mobilities in the Laemmli gel system. It was concluded that the differences in mobilities observed in the Laemmli gel system are due to alterations in charge or amino acid, not in size; furthermore; the molecular weight of lexA⁺ protein was determined to be 24 kilodaltons.

Graduation date: 2005

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 120-137). Also available on the World Wide Web.

Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: Justin R. Nodwell. Includes bibliographical references (leaves 126-142).