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

P. E. Vercoe, Donn Spight, B. A. White

Research output: Contribution to journalArticle

12 Citations (Scopus)

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-Dalgarno-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.

Original languageEnglish (US)
Pages (from-to)27-34
Number of pages8
JournalCanadian Journal of Microbiology
Volume41
Issue number1
StatePublished - 1995
Externally publishedYes

Fingerprint

Ruminococcus
Transcription Initiation Site
Transcription
Base Pairing
Escherichia coli
Sequence Analysis
Cellulases
Nucleotides
Cellulase
Genes
Amino Acids
Peptides
DNA sequences
Molecular mass
Histidine
Aspartic Acid
Glutamic Acid
Catalyst activity
Nucleic Acid Databases
Consensus Sequence

Keywords

  • DNA sequencing
  • endoglucanase
  • family E cellulase
  • xylanase

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Immunology
  • Microbiology

Cite this

Nucleotide sequence and transcriptional analysis of the celD β-glucanase gene from Ruminococcus flavefaciens FD-1. / Vercoe, P. E.; Spight, Donn; White, B. A.

In: Canadian Journal of Microbiology, Vol. 41, No. 1, 1995, p. 27-34.

Research output: Contribution to journalArticle

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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-Dalgarno-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.",
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N2 - 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-Dalgarno-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.

AB - 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-Dalgarno-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.

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