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-␤-D-N,N,N؆-triacetylchitotriose, but not on p-nitrophenyl-␤-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorumsensing 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.Chitin, a homopolymer of ␤-1,4-N-acetyl-D-glucosamine (GlcNAc), is one of the most abundant natural polymers. This polymer is present as a structural component in the exoskeletons of insects, in the shells of crustaceans, in the cell walls of many fungi and algae, and in nematodes. Recycling of chitin from disposed materials and dead organisms results mainly from the activity of chitinolytic microorganisms. Species of the genera Serratia, Bacillus, and Vibrio have been reported to secrete several chitinolytic enzymes and chitin-binding proteins, which are thought to degrade chitin synergistically, into the extracellular environment (2, 49, 51). The production of chitinases and chitin-binding proteins is often substrate regulated. Their synthesis is repressed when the bacteria are grown in rich medium and induced when the strains are grown in minimal medium supplemented with chitin (21, 49, 51).Whereas many steps in the process from perception to catabolism of chitin by different bacteria have been elucidated (reviewed in reference 21), the transport of these metabolic proteins across the bacterial cell envelope has been studied in only a few cases. For example, the chitinase ChiA of Vibrio cholerae and the chitin-binding protein CbpD of Pseudomonas aeruginosa have been shown to be secreted into the extracellular medium via the type II secretion pathway (8, 11). Both ChiA and CbpD are synthesized with a typical N-terminal ...