Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae.
The chiA gene of Vibrio cholerae encodes a polypeptide which degrades chitin, a homopolymer of N-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans. chiA has a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa. A 52-bp region with promoter activity was found immediately upstream of the chiA open reading frame. Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity by V. cholerae required chiA. Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for beta,1-4 glycosidic bonds located between GlcNAc monomers in chitin. Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein. Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes of V. cholerae and by the closely related bacterium Aeromonas hydrophila. Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by the eps system. The eps system is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA. These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential for eps-dependent secretion.
Connell
T D
TD
Center for Microbial Pathogenesis and Department of Microbiology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA. connell@asu.buffalo.edu
Metzger
D J
DJ
Lynch
J
J
Folster
J P
JP
D016980:Aeromonas hydrophila; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002688:Chitinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011817:Rabbits; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002688:Chitinases; C094590:chitinase ChiA