Dokument: cAMP dependent regulation of the TCA cycle by GlxR and oxidative stress response regulation by RosR in Corynebacterium glutamicum
| Titel: | cAMP dependent regulation of the TCA cycle by GlxR and oxidative stress response regulation by RosR in Corynebacterium glutamicum | |||||||
| Weiterer Titel: | cAMP dependent regulation of the TCA cycle by GlxR and oxidative stress response regulation by RosR in Corynebacterium glutamicum | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=13982 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20101104-151101-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Bussmann, Michael [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Bott, Michael [Gutachter] Prof. Dr. Ernst, Joachim F. [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibungen: | DNA-binding transcription factors are key components in the regulation of gene expression in all organisms. They sense changes in the intra- or extracellular environment and trigger appropriate responses by activation or repression of their target genes. In this work, two transcriptional regulators and an adenylate cyclase of the Gram-positive soil bacterium Corynebacterium glutamicum have been studied, leading to the following results:
(i) The Crp-like transcriptional regulator GlxR was found to regulate genes of the tricarboxylic acid (TCA) cycle. Expression of the succinate dehydrogenase operon sdhCAB and of the P2 transcript of the citrate synthase gene gltA is weakly repressed by GlxR by binding to distinct DNA sequence motifs in the promoter regions. On the other hand, evidence was obtained that expression of the fumarase gene fum is weakly activated by GlxR. Thus, GlxR appears to be involved in fine-tuning the expression of TCA cycle genes. (ii) DNA-binding of GlxR is strictly dependent on cAMP and consequently GlxR activity is controlled by the cellular cAMP concentration. Therefore, a mutant lacking the cyaB gene encoding the only annotated adenylate cyclase in C. glutamicum was characterized. Strain cyaB showed growth defects in the presence of acetate or propionate. This phenotype could be reversed by plasmid-borne cyaB or by extracellular cAMP. The cAMP level in the cyaB mutant was decreased compared to wild type. DNA microarray experiments revealed an altered expression of many of the known or proposed GlxR target genes in the cyaB mutant . (iii) The MarR-type transcriptional regulator RosR (Cg1324) is probably involved in the response to oxidative stress. Direct target genes of RosR were identified by transcriptome comparisons of a rosR mutant and the wild type and electrophoretic mobility shift assays (EMSA). Purified RosR was found be a homodimer that binds to a conserved 20-bp inverted repeat (5-WTTGTTGAYR-YRTCAACWAA-3’). Except for the narKGHJI operon encoding a nitrate/nitrite antiporter and dissimilatory nitrate reductase, all other identified target genes were repressed by RosR. They encode four putative monooxygenases, two putative FMN reductases, a protein of the glutathione S-transferase family, a putative polyisoprenoid-binding protein (Cg1322) and RosR itself. In vitro DNA-binding of RosR was inhibited by H2O2. The oxidation status of cysteine residues was found to be important for redox-dependent DNA-binding, as exchange of Cys64, Cys92 and Cys151 to serine led to a H2O2-insensitive RosR variant. According to these data, oxidation or another type of cysteine modification inactivates RosR and causes deactivation or derepression of its target genes. Further support for involvement of RosR in the oxidative stress response was obtained by the significantly increased H2O2-sensitivity of a mutant lacking the RosR target gene cg1322.DNA-binding transcription factors are key components in the regulation of gene expression in all organisms. They sense changes in the intra- or extracellular environment and trigger appropriate responses by activation or repression of their target genes. In this work, two transcriptional regulators and an adenylate cyclase of the Gram-positive soil bacterium Corynebacterium glutamicum have been studied, leading to the following results: (i) The Crp-like transcriptional regulator GlxR was found to regulate genes of the tricarboxylic acid (TCA) cycle. Expression of the succinate dehydrogenase operon sdhCAB and of the P2 transcript of the citrate synthase gene gltA is weakly repressed by GlxR by binding to distinct DNA sequence motifs in the promoter regions. On the other hand, evidence was obtained that expression of the fumarase gene fum is weakly activated by GlxR. Thus, GlxR appears to be involved in fine-tuning the expression of TCA cycle genes. (ii) DNA-binding of GlxR is strictly dependent on cAMP and consequently GlxR activity is controlled by the cellular cAMP concentration. Therefore, a mutant lacking the cyaB gene encoding the only annotated adenylate cyclase in C. glutamicum was characterized. Strain cyaB showed growth defects in the presence of acetate or propionate. This phenotype could be reversed by plasmid-borne cyaB or by extracellular cAMP. The cAMP level in the cyaB mutant was decreased compared to wild type. DNA microarray experiments revealed an altered expression of many of the known or proposed GlxR target genes in the cyaB mutant . (iii) The MarR-type transcriptional regulator RosR (Cg1324) is probably involved in the response to oxidative stress. Direct target genes of RosR were identified by transcriptome comparisons of a rosR mutant and the wild type and electrophoretic mobility shift assays (EMSA). Purified RosR was found be a homodimer that binds to a conserved 20-bp inverted repeat (5-WTTGTTGAYR-YRTCAACWAA-3’). Except for the narKGHJI operon encoding a nitrate/nitrite antiporter and dissimilatory nitrate reductase, all other identified target genes were repressed by RosR. They encode four putative monooxygenases, two putative FMN reductases, a protein of the glutathione S-transferase family, a putative polyisoprenoid-binding protein (Cg1322) and RosR itself. In vitro DNA-binding of RosR was inhibited by H2O2. The oxidation status of cysteine residues was found to be important for redox-dependent DNA-binding, as exchange of Cys64, Cys92 and Cys151 to serine led to a H2O2-insensitive RosR variant. According to these data, oxidation or another type of cysteine modification inactivates RosR and causes deactivation or derepression of its target genes. Further support for involvement of RosR in the oxidative stress response was obtained by the significantly increased H2O2-sensitivity of a mutant lacking the RosR target gene cg1322. | |||||||
| Rechtliche Vermerke: | Promotion erst in 6 Monaten veröffentlichen | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie | |||||||
| Dokument erstellt am: | 30.03.2010 | |||||||
| Dateien geändert am: | 21.03.2010 | |||||||
| Promotionsantrag am: | 15.10.2009 | |||||||
| Datum der Promotion: | 16.12.2009 |
