Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum").
Northern (RNA) blot analysis of RNA from Clostridium sp. revealed induction of transcription of the celA gene when barley beta-glucan was used as carbon source, while no celA mRNA was detected after growth on cellobiose. Western blots (immunoblots), prepared by using a rabbit antiserum raised against CelA protein purified from Escherichia coli, revealed the extracellular location of CelA in Clostridium sp. Despite the absence of detectable celA mRNA, significant quantities of CelA were detected in the culture supernatant during growth on cellobiose. This finding indicated a low constitutive expression of celA. A 6.7-fold increase in the total beta-glucanase specific activity in the extracellular fraction was observed during growth on beta-glucan. The transcriptional start site of celA was mapped by extension and was found to be the same in Clostridium sp. and in E. coli expressing the cloned celA gene. A consensus E. coli -10 promoter region (AATAAT), but not a -35 promoter region, could be identified. Two direct repeats (TATTGAATTTAT) separated by 15 nucleotides flank the region where the consensus -35 promoter regions would have been. The size of the celA mRNA transcript corresponded with the size of the open reading frame. A potential stem-loop structure was found 18 nucleotides downstream of the 3' stop codon, which could be responsible for termination of transcription.
Mittendorf
V
V
Department of Microbiology, University of Cape Town, Rondebosch, South Africa.
Thomson
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics*; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D012417:Rumen / Q000382:microbiology; D017421:Sequence Analysis; D014158:Transcription, Genetic
C074775:CelA endoglucanase; D002480:Cellulase