Encoding Cellulolytic Activity in Acetobacter xylinum

Recently, it was shown that a cellulose-negative mutant (Cell) ofAcetobacterxylinum ATCC 23769 carried an insertion of an indigenous transposable element (IS1031A) about 500 bp upstream of the bcs operon, required for cellulose synthesis. Here we show that Cell can be complemented by wild-type DNA covering the insertion point. Nucleotide sequencing of this region revealed the presence of two open reading frames, ORF1 and ORF2. ORF2, which is disrupted by the IS1031A insertion in Cell, potentially encodes the complementing function. ORF1 encodes a protein (CMCax) with significant homology to previously described endoglucanases. A cloned DNA fragment containing ORF1 expressed a carboxymethyl cellulose-hydrolyzing activity in Escherichia coli. In A. xylinum, CMCax is secreted into the culture growth medium. The CMCax mature protein consists of 322 amino acids and has a molecular mass of 35.6 kDa.