Transcription of Clostridium cellulovorans cellulosomal cellulase and hemicellulase genes

Transcription of the cellulosomal cellulase/hemicellulase genes of Clostridium cellulovorans has been investigated by Northern blot, reverse transcriptase PCR (RT-PCR), primer extension, and S1 nuclease analysis. Northern hybridizations revealed that the cellulosomal cbpA gene cluster is transcribed as polycistronic mRNAs of 8 and 12 kb. The 8-kb mRNA coded for cbpA and exgS, and the 12-kb mRNA coded for cbpA, exgS, engH, and engK. The sizes of the mRNAs were about 3 kb for engE, 1.8 kb for manA, 2.7 kb for xynA, and 4 kb for pelA, indicating monocistronic transcription of these genes. Primer extension and S1 nuclease analysis of C. cellulovorans RNA showed that the transcriptional start sites of cbpA, engE, manA, and hbpA were located 233, 97, 64, and 61 bp upstream from the first nucleotide of each of the respective translation initiation codons. Alignment of the cbpA, engE, manA, and hbpA promoter regions provided evidence for highly conserved sequences that exhibited strong similarity to the sigma(A) consensus promoter sequences of gram-positive bacteria.