A Bacteroides ruminicola 1,4-beta-D-endoglucanase is encoded in two reading frames

Abstract

Escherichia coli transformed with a plasmid containing a Bacteroides ruminicola endoglucanase (carboxymethyl cellulase [CMCase]) gene produced three immunologically cross-reacting CMCases which had molecular weights of 40,500, 84,000, and 88,000, while B. ruminicola produced CMCases with molecular weights of 82,000 and 88,000. The two B. ruminicola enzymes (purified from culture supernatants) had different N-terminal amino acid sequences, but each enzyme was encoded by the same gene (three independent clones had the same DNA sequence). The 88,000-molecular-weight CMCase (88K CMCase) gene appeared to contain two open reading frames which overlapped for 18 bp and were -1 out of frame, and each open reading frame contained several stop codons near the overlap region. The two 88K CMCase open reading frames had enough DNA to produce a protein of 106K, but the mobility of the enzyme in sodium dodecyl sulfate gels gave a value which was 20% lower. On the basis of the -1 frame shift and the large deviation in theoretical versus actual size, it appears that an unusual event (e.g., ribosomal hopping or RNA splicing) is involved in either the translation or the transcription of the 88K B. ruminicola CMCase gene. The 82K CMCase was completely encoded in the second reading frame, and its size was in agreement with the DNA sequence.