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PMID:9791107
| Citation |
Connell, TD, Metzger, DJ, Lynch, J and Folster, JP (1998) Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae. J. Bacteriol. 180:5591-600 |
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| Abstract |
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. |
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| Keywords |
Aeromonas hydrophila; Alleles; Amino Acid Sequence; Animals; Antibody Formation; Bacterial Proteins/genetics; Bacterial Proteins/immunology; Bacterial Proteins/metabolism; Base Sequence; Biological Transport; Chitinase/genetics; Chitinase/immunology; Chitinase/metabolism; Chromosome Mapping; Cloning, Molecular; DNA, Bacterial; Frameshift Mutation; Molecular Sequence Data; Mutagenesis; Open Reading Frames; Promoter Regions, Genetic; Rabbits; Substrate Specificity; Vibrio cholerae/metabolism |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0005576: extracellular region |
ECO:0000314: |
C |
The section entitled "Extracellular transport of ChiA" reports that the semiquantative densitometric analysis of wild type Vibrio cholerae 569B culture was consistent with extracellular secretion of the protein. Figure 7 shows the localization distribution between the culture supernatant and the periplasmic space. A Vibrio cholerae mutant, M14, is also in Figure 7 but it is not relevant to this annotation. Additionally, immunoblot assays were performed to detect the immunoreactive ChiA polypeptides used in the semiquantative densitometric analysis. Therefore, this annotation has the evidence code IDA and not IMP. |
complete | ||||
| GO:0016798: hydrolase activity, acting on glycosyl bonds |
ECO:0000314: |
F |
The section entitled "Defining the substrate specificity ChiA" reported that the biochemical assay performed to detect chitinase activity of ChiA was consistent with the hydrolysis of glycosidic bonds. Table 2 shows the results of the assay for determining the substrate specificity of ChiA in wild type Vibrio cholerae as well as ChiA from strains of Escherichia coli that have plasmids encoding the chiA gene. |
complete | ||||
| GO:0008843: endochitinase activity |
ECO:0000315: |
F |
Figure 3 is a photograph of the growth of the Vibrio cholerae wild type strain 569B and mutant strain 569BchiA::kan. The growth medium is Luria-Bertani agar with ethylene glycol chitin as a source of chitin. The cloudy ring around the wild type strain indicates a zone of clearing due to endochitinase activity. |
complete | ||||
| GO:0004568: chitinase activity |
ECO:0000314: |
F |
Figure 1 is a photograph of the growth of the Vibrio cholerae wild type strain 569B after 24 hours of incubation. The growth medium is Luria-Bertani agar with ethylene glycol chitin as a source of chitin. The cloudy ring around the wild type strain indicates a zone of clearing due to chitinase activity. |
complete | ||||
See also
References
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