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

Immediately (17 bp) upstream of the Clostridium paraputrificum chiB gene [J . Bacteriol. 179. 7306-7314 (1997)]. we found another chitinase gene chiA e ncoding chitinase A (ChiA). The chiA gene consists of an open reading frame of 2496 nucleotides and encodes 832 amino acids with a deduced molecular m ass 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 d omains, and a chitin-binding domain. The domain organization of ChiA is fun damentally identical to that of ChiB and the overall sequence identity betw een them is 35.4%. ChiA was purified from the periplasm fraction of Escheri chia coli harboring the chiA gene. The molecular mass of purified ChiA (89 000 Da), determined by sodium dodecyl sulfate/polyacrylamide gel electropho resis analysis, was in good agreement with the value (89 119 Da) calculated from the deduced amino acid sequence, excluding the signal peptide. Immuno logical and N-terminal amino acid sequence analyses revealed that ChiA and ChiB are major chitinases of C. paraputrificum and their production is indu cible by ball-milled chitin. Northern blot analysis indicated that the chiA and chiB genes constitute a polycistronic operon. Primer-extension analysi s confirmed that the transcription of this operon starts upstream of chiA.