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The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression
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 gen...
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| Format: | Thesis |
| Language: | English |
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Division of Medical Microbiology
2017
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| _version_ | 1867613276984049664 |
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| access_status_str | Open Access |
| author | Mulder, Michelle Anne |
| author2 | Steyn, Lafras M |
| author_browse | Mulder, Michelle Anne Steyn, Lafras M |
| author_facet | Steyn, Lafras M Mulder, Michelle Anne |
| author_sort | Mulder, Michelle Anne |
| collection | Thesis |
| description | 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. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/25662 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:33:33.643Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Division of Medical Microbiology |
| publisherStr | Division of Medical Microbiology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/25662 The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression Mulder, Michelle Anne Steyn, Lafras M Zappe, Harold Mycobacterium Tuberculosis - isolation & purification - chemistry Gene Expression Regulation, Bacterial 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. 2017-10-13T07:45:51Z 2017-10-13T07:45:51Z 1998 2017-07-13T11:16:53Z Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/25662 eng application/pdf Division of Medical Microbiology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Mycobacterium Tuberculosis - isolation & purification - chemistry Gene Expression Regulation, Bacterial Mulder, Michelle Anne The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression |
| thesis_degree_str | Doctoral |
| title | The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression |
| title_full | The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression |
| title_fullStr | The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression |
| title_full_unstemmed | The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression |
| title_short | The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression |
| title_sort | mycobacterium tuberculosis katg gene identification of a novel function and analysis of the regulation of expression |
| topic | Mycobacterium Tuberculosis - isolation & purification - chemistry Gene Expression Regulation, Bacterial |
| url | http://hdl.handle.net/11427/25662 |
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