The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression

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dc.contributor.advisorSteyn, Lafras Men_ZA
dc.contributor.advisorZappe, Harolden_ZA
dc.contributor.authorMulder, Michelle Anneen_ZA
dc.date.accessioned2017-10-13T07:45:51Z
dc.date.available2017-10-13T07:45:51Z
dc.date.issued1998en_ZA
dc.date.updated2017-07-13T11:16:53Z
dc.description.abstractA clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms.en_ZA
dc.identifier.apacitationMulder, M. A. (1998). <i>The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Microbiology. Retrieved from http://hdl.handle.net/11427/25662en_ZA
dc.identifier.chicagocitationMulder, Michelle Anne. <i>"The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Microbiology, 1998. http://hdl.handle.net/11427/25662en_ZA
dc.identifier.citationMulder, M. 1998. The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Mulder, Michelle Anne AB - A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms. DA - 1998 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1998 T1 - The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression TI - The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression UR - http://hdl.handle.net/11427/25662 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/25662
dc.identifier.vancouvercitationMulder MA. The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Microbiology, 1998 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/25662en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Medical Microbiologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMycobacterium Tuberculosis - isolation & purification - chemistryen_ZA
dc.subject.otherGene Expression Regulation, Bacterialen_ZA
dc.titleThe Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expressionen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
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uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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