title: Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.
abstract: The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.
authors.affiliation: Department of Molecular Microbiology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Folders
authors.forename: J
authors.initials: J
authors.lastname: Algra
authors.forename: M S
authors.initials: MS
authors.lastname: Roelofs
authors.forename: L C
authors.initials: LC
authors.lastname: van Loon
authors.forename: J
authors.initials: J
authors.lastname: Tommassen
authors.forename: W
authors.initials: W
authors.lastname: Bitter
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002451:Cell Compartmentation; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002686:Chitin; D002688:Chitinases; C101644:chitinase C-1