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

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Abstract
The endoglucanase gene was sequenced fromPrevotella 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 ofcelA is initiated at different sites inEscherichia coli andPrevotella ruminicola. E. coli σ70 recognition sequences were identified, which were located upstream from the transcription initiation site (TIS) functional inE. coli. A longer extension product was identified using RNA fromP. 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 bothE. coli andP. ruminicola. E. coli-like consensus sequences were located at positions −10 and −35 upstream from this site, suggesting that some promoter sequences inP. ruminicola are similar toE. coli consensus sequences, although others recognized byE. coli are non-functional inP. 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 inP. 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.