Sequencing, Expression, and Transcription Analysis of the Clostridium Paraputrificum chiA Gene Encoding Chitinase ChiA

Appl Microbiol Biotechnol. 1999 Mar;51(3):340-7. doi: 10.1007/s002530051400.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Blotting, Northern
  • Chitin / pharmacology
  • Chitinases / chemistry
  • Chitinases / genetics*
  • Chitinases / metabolism
  • Cloning, Molecular
  • Clostridium / drug effects
  • Clostridium / enzymology
  • Clostridium / genetics*
  • Genes, Bacterial*
  • Immunoblotting
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Plant Proteins / metabolism
  • Transcription, Genetic / genetics*

Substances

  • Bacterial Proteins
  • Plant Proteins
  • Chitin
  • Chitinases
  • PLC-B protein, Phytolacca americana
  • chitinase ChiA

Associated data

  • GENBANK/AB012764