A PREVOTELLA-RUMINICOLA B(1)4 OPERON ENCODING EXTRACELLULAR POLYSACCHARIDE HYDROLASES

When Escherichia coli XL1-Blue MRA (P2) was infected with lambda DNA c ontaining Prevotella ruminicola B(1)4 chromosomal DNA, only a few plaq ues produced beta-1,4-endoglucanase activity, and all of these had man nanase activity. Positive phage contained a 17-kb SacI DNA fragment th at gave six bands after EcoRI digestion. The EcoRI fragments were liga ted into pBluescript and sequenced. The order of the fragments was ver ified by PCR and by restriction mapping. The DNA sequence contained 6 open reading frames (ORFs). The 4th and 5th ORFs encoded two related b eta-1,4-endoglucanases. E. coli clones carrying ORF5 and ORF6 had beta -1,4-endoglucanase and mannanase activities, while a clone carrying on ly ORF6 hydrolyzed mannan but not carboxymethylcellulose. The 6th ORF had three regions of homology to mannanase A from Pseudomonas fluoresc ens. Based on these results, ORF6 encoded the mannanase gene. The 3rd ORF had 10 regions of homology with cellulose-binding protein A from C lostridium cellulovorans. The 1st and 2nd ORFs had no significant homo logy to genes or amino acid sequences in GeneBank or SwissProt. All of the ORFs except 1 encoded a potential signal peptide sequence. The up stream region of ORF1 contained four direct repeats and four inverted repeat elements, but no apparent sigma(70) sequence-like promoter was present. The segment of DNA containing the 6 ORFs was preceded and fol lowed by potential transcription termination signals suggesting a sing le transcriptional unit.