DNA sequence and transcription of an endoglucanase gene from Prevotella (Bacteroides) ruminicola AR20

The endoglucanase gene was sequenced from Prevotella ruminicola AR20, isolated as clone pJW4. The endoglucanase (BrEND) is encoded by an open reading frame (ORF1) of 501 codons, corresponding to a protein of calculated molecular weight 55.7 kDa. Analysis of proteins on SDS-PAGE revealed a protein corresponding to the calculated molecular weight of the processed BrEND. The protein showed substantial homology to members of the A4 sub-family cellulases. Primer extension studies revealed that transcription of celA is initiated at different sites in Escherichia coli and Prevotella ruminicola. E. coli sigma factor 70 recognition sequences were identified, which were located upstream from the transcription initiation site (TIS) functional in E. coli. A longer extension product was identified using RNA from P. ruminicola, indicating that the gene may normally be transcribed as part of a polycistronic message. The end of the primer extension product corresponded to a site beyond the 5' boundary of the cloned fragment, thus preventing identification of native promoter sequences. A second ORF of 110 codons (ORF2) was identified on the antisense strand, and primer extension indicated that transcription through ORF2 was initiated at an identical site in both E. coli and P. ruminicola. E. coli-like consensus sequences were located at positions -10 and -35 upstream from this site, suggesting that some promoter sequences in P. ruminicola are similar to E. coli consensus sequences, although others recognized by E. coli are non-functional in P. ruminicola. The presence of a strong potential ribosome-binding site at nucleotide positions 25-38 of the antisense transcript could indicate that ORF2 is translated in P. ruminicola; however, a protein corresponding to this ORF was not detected on SDS-PAGE. This suggests that it is more likely that ribosomal binding of the antisense transcript plays a part in translational regulation of endoglucanase expression.