DNA sequence and transcriptional characterization of a β-glucanase gene (celB) fromRuminococcus flavefaciensFD-1

Abstract

The recombinant clone pBAW101 (in pBluescript SK–) contains the celB endoglucanase gene from Ruminococcus flavefaciens FD-1. Subcloning indicated that the endoglucanase activity expressed was present within a 2.4-kb insert (pBAW104). The nucleotide sequence of the celB gene was determined, and upon analysis, revealed an open reading frame of 1943 nucleotides that encodes a polypeptide of 632 amino acids with a molecular weight of 69 414. A putative Shine–Dalgarno sequence was identified 6 bp upstream from the translation start site. The N-terminal 32 amino acid residues were typical of prokaryotic signal sequences. Hydrophobic cluster analysis (HCA) and DNA alignment of CelB to other published β-glucanase polypeptide sequences in GenBank indicate that CelB belongs in HCA cellulase family 44. Primer extension analyses were performed using RNA isolated from R. flavefaciens grown on cellulose and cellobiose, and from Escherichia coli containing the plasmid clone pBAW104. Transcription is initiated at different sites in E. coli and R. flavefaciens. In the case of R. flavefaciens transcription is initiated at a C residue (nucleotides 329), 221 bp upstream from the translation start site. There were no regions resembling E. coli σ⁷⁰-like promoter sequences present upstream from this putative transcription initiation site. In contrast, numerous transcription initiation sites were identified when RNA from E. coli was used in the primer extension analyses.Key words: Ruminococcus flavefaciens, endoglucanase, transcription, family 44 endoglucanase.