published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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

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published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

abstract / toc / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy