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Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262
Bibliography: pages 140-158.
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2016
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| _version_ | 1867611322287390720 |
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| access_status_str | Open Access |
| author | Lin, Fu-Pang |
| author2 | Woods, David R |
| author_browse | Lin, Fu-Pang Woods, David R |
| author_facet | Woods, David R Lin, Fu-Pang |
| author_sort | Lin, Fu-Pang |
| collection | Thesis |
| description | Bibliography: pages 140-158. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/21981 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2016 |
| publishDateRange | 2016 |
| publishDateSort | 2016 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/21981 Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 Lin, Fu-Pang Woods, David R Reid, Sharon J Microbiology Bibliography: pages 140-158. Clostridium acetobutylicum P262 is an endospore-forming Gram-positive anaerobic bacterium, and it has been used in the industrial production of acetone and butanol for many years. The aim of this study was to characterize the upstream region of the βhbd gene which is linked to the adh1 gene, and to investigate the expression of these linked genes by transcriptional analysis. The upstream region of the βhbd gene was isolated from a gene bank of C. acetobutylicum P262, constructed using the pWE15 cosmid vector. Characterization of this upstream region was done initially at the nucleotide sequence level. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1002-bp which encoded a protein of 334 amino acid residues with a calculated Mᵣ of 35,679. This protein showed significant amino acid homology to the fixB protein of Rhizobium meliloti and Azorhizobium caulinodans and the electron transport flavoproteins from humans and rats. Studies on the expression of the three linked genes fixB, βhbd and adh1 were carried out at the transcriptional level. Northern and primer extension analyses indicated that all of the three genes were independently transcribed throughout the various stages of the acetone-butanol-ethanol (ABE) fermentation in C. acetobutylicum P262. Each of the genes produced mRNA transcripts of approximately 1.4 kb. The βhbd and adh1 genes were shown to have at least two major and one minor transcriptional start sites in C. acetobutylicum P262. Transcription was initiated at the same promoter region of the fixB gene in both C. acetobutylicum P262 and Escherichia coli. the βhbd gene was shown to have a stronger promoter region than those of the fixB and adh1 genes based on the lacZ-fusion studies in E. coli. The βhbd and adh1 genes encode the 3-hydroxybutyryl-CoA dehydrogenase (BHBD) and the NADPH-dependent alcohol dehydrogenase (ADH), respectively. The BHBD enzyme is part of the central fermentation pathway and is required for acid and solvent production, whereas the ADH is part of a branched solvent pathway and is only required for solvent production. Analysis of mRNA transcription and the identification of transcription initiation sites, indicated that each of these two genes was independently and constitutively transcribed throughout the acidogenic, sol ventogenic and sporulation stages in C. acetobutylicum P262 and in exponential E. coli cells. These results suggest that the adh1 gene is not part of a branched solvent pathway which is only induced and transcribed during the solventogenic phase. 2016-09-28T19:05:23Z 2016-09-28T19:05:23Z 1992 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/21981 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Microbiology Lin, Fu-Pang Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 |
| thesis_degree_str | Doctoral |
| title | Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 |
| title_full | Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 |
| title_fullStr | Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 |
| title_full_unstemmed | Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 |
| title_short | Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 |
| title_sort | molecular characterization of three linked genes fixb βshbd and adh1 from clostridium acetobutylicum p262 |
| topic | Microbiology |
| url | http://hdl.handle.net/11427/21981 |
| work_keys_str_mv | AT linfupang molecularcharacterizationofthreelinkedgenesfixbbshbdandadh1fromclostridiumacetobutylicump262 |