Sequencing, expression, and transcription analysis of the Clostridium paraputrificum chiA gene encoding chitinase ChiA

Abstract

Immediately (17 bp) upstream of the Clostridium paraputrificum chiB gene [J. Bacteriol. 179: 7306–7314 (1997)], we found another chitinase gene chiA encoding chitinase A (ChiA). The chiA gene consists of an open reading frame of 2496 nucleotides and encodes 832 amino acids with a deduced molecular mass of 92 585 Da. The mature ChiA is a modular enzyme composed of a family-18 catalytic domain responsible for chitinase activity, two cadherin-like domains, and a chitin-binding domain. The domain organization of ChiA is fundamentally identical to that of ChiB and the overall sequence identity between them is 35.4%. ChiA was purified from the periplasm fraction of Escherichia coli harboring the chiA gene. The molecular mass of purified ChiA (89 000 Da), determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis, was in good agreement with the value (89 119 Da) calculated from the deduced amino acid sequence, excluding the signal peptide. Immunological and N-terminal amino acid sequence analyses revealed that ChiA and ChiB are major chitinases of C. paraputrificum and their production is inducible by ball-milled chitin. Northern blot analysis indicated that the chiA and chiB genes constitute a polycistronic operon. Primer-extension analysis confirmed that the transcription of this operon starts upstream of chiA.