A Prevotella ruminicola B₁4 Operon Encoding Extracellular Polysaccharide Hydrolases

Abstract.

When Escherichia coli XL1-Blue MRA (P2) was infected with λ DNA containing Prevotella ruminicola B₁4 chromosomal DNA, only a few plaques produced β-1,4-endoglucanase activity, and all of these had mannanase activity. Positive phage contained a 17-kb SacI DNA fragment that gave six bands after EcoRI digestion. The EcoRI fragments were ligated into pBluescript and sequenced. The order of the fragments was verified by PCR and by restriction mapping. The DNA sequence contained 6 open reading frames (ORFs). The 4th and 5th ORFs encoded two related β-1,4-endoglucanases. E. coli clones carrying ORF5 and ORF6 had β-1,4-endoglucanase and mannanase activities, while a clone carrying only ORF6 hydrolyzed mannan but not carboxymethylcellulose. The 6th ORF had three regions of homology to mannanase A from Pseudomonas fluorescens. Based on these results, ORF6 encoded the mannanase gene. The 3rd ORF had 10 regions of homology with cellulose-binding protein A from Clostridium cellulovorans. The 1st and 2nd ORFs had no significant homology to genes or amino acid sequences in GeneBank or SwissProt. All of the ORFs except 1 encoded a potential signal peptide sequence. The upstream region of ORF1 contained four direct repeats and four inverted repeat elements, but no apparent σ⁷⁰ sequence-like promoter was present. The segment of DNA containing the 6 ORFs was preceded and followed by potential transcription termination signals suggesting a single transcriptional unit.