Molecular analysis of two cellulase genes from Ruminococcus flavefaciens FD-1 and their transcriptional regulation
Doctoral Thesis
1993
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University of Cape Town
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Abstract
The mesophilic Ruminococcus flavefaciens FD-1 (NCDO 2215) is a Gram-positive obligate anaerobic bacterium. The aim of this thesis was to clone, sequence and analyze a cellodextrinase gene (celA) and a carboxymethylcellulase gene (celE), and study their regulation and induction at the transcriptional level. The sequence of the celA gene from FD-1 was determined and the amino acid sequence of the CelA enzyme (336 amino acid residues) deduced. It showed 40% identity with endoglucanase C of Clostridium thermocellum and 27.4% identity with endoglucanase 3 of Fibrobacter succinogenes. These three enzymes are grouped into subfamily "A3". The ATG start codon of celA is preceded by a GAGG sequence, predicted to be a ribosome binding site. The derived amino acid sequence corresponded to a protein of Mᵣ 38686. SDS-PAGE analysis of in vitro and in vivo translational products showed that CelA has a molecular mass of ca 39 kDa and was secreted into the Escherichia coli periplasmic space. Although CelA has activity on carboxymethylcellulose, further study on the enzyme showed that it degraded cellopentaose and other cellodextrins to predominantly cellobiose. Thus CelA is a cellodextrinase. It also has high activity against p-nitrophenyl-β-D-cellobioside. A gene, expressing a protein with both carboxymethyl cellulase and xylanase activity, was cloned from R. flavefaciens FD-1 using an E. coli/Bacillus subtilis shuttle vector, pEBl. The 3.6 kb DNA insert on the plasmid pWFl, which carried the gene, celE, contained an open reading frame of 963 bp encoding 320 amino acid residues with a caculated Mᵣ of 35937. Homology analysis showed 11.6% identity and 55.3% similarity with the N-terminal catalytic region of the cellulase gene of alkalophilic Bacillus sp. strain 1139. In order to obtain expression in E. coli, the gene had to be transcribed from the lambda Pᵣ promoter. To determine whether cellulase genes of R. flavefaciens FD-1 were regulated at the level of transcription, celA and celE were used as probes against RNA isolated from R. flavefaciens FD-1 grown on cellobiose, cellulose or cellotriose. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose although not to the same extent as by cellulose. This suggests that cellotriose and possibly ether dextrins may act as key inducers to trlgger celA and eels gene expression in R. flavefaciens FD-1.
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Wang, W. 1993. Molecular analysis of two cellulase genes from Ruminococcus flavefaciens FD-1 and their transcriptional regulation. University of Cape Town.