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 indu ction of transcription of the celA gene when barley beta-glucan was us ed as carbon source, while no celA mRNA was detected after growth on c ellobiose, Western blots (immunoblots), prepared by rising a rabbit an tiserum raised against CelA protein purified from Escherichia coli, re vealed the extracellular location of CelA in Clostridium sp; Despite t he absence of detectable celA mRNA, significant quantities of CelA wer e detected in the culture supernatant during growth on cellobiose. Thi s finding indicated a low constitutive expression of celA. A 6.7-fold increase in the total beta-glucanase specific activity in the extracel lular fraction was observed during growth on beta-glucan. The transcri ptional 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 (TATTGAATTTA T) separated by 15 nucleotides flank the region where the consensus -3 5 promoter regions would have been. The size of the celA mRNA transcri pt corresponded with the size of the open reading frame. A potential s tem-loop structure was found 18 nucleotides downstream of the 3' stop codon, which could be responsible for termination of transcription.