Nucleotide sequence and transcriptional analysis of the celD β-glucanase gene from Ruminococcus flavefaciens FD-1

Abstract

The nucleotide sequence of the celD gene, which encodes endoglucanase and xylanase activity, from Ruminococcus flavefaciens FD-1 was determined. The DNA sequence of celD contains an open reading frame of 1215 nucleotides that encodes a polypeptide of 405 amino acids with a molecular mass of 44 631 Da. The primary amino acid sequence of CelD was screened against the GenBank data base for similar polypeptide sequences and the analysis indicated that CelD has common features with endoglucanases from the family E cellulases. Both hydrophobic cluster and BESTFIT (Genetics Computer Group (University of Wisconsin) package) analyses confirmed this relationship. Pairwise alignments using BESTFIT revealed that CelD was most closely related to endE4 from Thermomonospora fusca over a 160 amino acid window. The histidine, aspartate, and glutamate residues identified as being essential for catalytic activity in family E cellulases are conserved in CelD. A Shine–Dalgamo-like sequence was present 5 base pairs (bp) upstream of the translation start site. Primer extension analysis indicated that different transcription initiation sites are used to initiate transcription of celD in Escherichia coli and R. flavefaciens. In the case of R. flavefaciens the transcription initiation site is at a T residue (nucleotide 273) 16 bp upstream from the translational start site. A region resembling a σ⁷⁰-like −10 promoter sequence is present upstream from the transcription initiation site but there is no apparent −35 region. In contrast, transcription in E. coli is initiated at a C residue 258 bp upstream from the translational start site and a sequence resembling a σ⁷⁰-like −10 region is present 5 bp upstream of this residue. Assuming 17 bp is the optimal distance between −10 and −35 sites for σ⁷⁰ consensus sequences, the −35 region for celD transcription initiation in E. coli would be outside the boundaries of the cloned R. flavefaciens DNA.Key words: endoglucanase, xylanase, DNA sequencing, family E cellulase.