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 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.
Morimoto
K
K
Faculty of Bioresources, Mie University, Tsu, Japan.
Karita
S
S
Kimura
T
T
Sakka
K
K
Ohmiya
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002686:Chitin / Q000494:pharmacology; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003013:Clostridium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*
D001426:Bacterial Proteins; D010940:Plant Proteins; D002686:Chitin; D002688:Chitinases; C094255:PLC-B protein, Phytolacca americana; C094590:chitinase ChiA