Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262

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dc.contributor.advisorWoods, David Ren_ZA
dc.contributor.advisorReid, Sharon Jen_ZA
dc.contributor.authorLin, Fu-Pangen_ZA
dc.date.accessioned2016-09-28T19:05:23Z
dc.date.available2016-09-28T19:05:23Z
dc.date.issued1992en_ZA
dc.descriptionBibliography: pages 140-158.en_ZA
dc.description.abstractClostridium 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.en_ZA
dc.identifier.apacitationLin, F. (1992). <i>Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/21981en_ZA
dc.identifier.chicagocitationLin, Fu-Pang. <i>"Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1992. http://hdl.handle.net/11427/21981en_ZA
dc.identifier.citationLin, F. 1992. Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Lin, Fu-Pang AB - 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. DA - 1992 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1992 T1 - Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 TI - Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262 UR - http://hdl.handle.net/11427/21981 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/21981
dc.identifier.vancouvercitationLin F. Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1992 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/21981en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMicrobiologyen_ZA
dc.titleMolecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262en_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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