One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).The opportunistic pathogen Pseudomonas aeruginosa is able to secrete many proteins, including the exoenzymes S, T, and U, exotoxin A, lipase, phospholipase C, the proteases alkaline protease and elastase (LasB), and the staphylolytic proteases LasA and LasD, into the extracellular medium. Most of these proteins contribute to the virulence of the bacteria, as they are associated with epithelial cell and tissue damage or disfunctioning of infected host cells. These proteins are secreted across the bacterial cell envelope by three entirely different mechanisms. Exoenzymes S, T, and U are secreted by a type III secretion system and are actually injected directly into eukaryotic target cells (12,51). Alkaline protease is secreted by a type I secretion machinery (8). The other proteolytic enzymes mentioned above and exotoxin A are secreted via the type II secretion pathway, encoded by the xcp genes (for a review, see reference 11). The major proteolytic enzyme, elastase, is synthesized as a preproenzyme in the cytosol. During or directly after translocation across the cytoplasmic membrane, the signal sequence is removed, and the proenzyme is folded in a process that requires the propeptide as an intramolecular chaperone (5, 35). After autoproteolytic processing, the propeptide remains noncovalently associated with the mature enzyme and inhibits enzymatic activity in the periplasm (27, 34). The propeptide dissociates from the enzyme only after translocation across the outer membrane (6). LasA, a staphylolytic protease, is synthesized as a preproenzyme of 42 kDa and is processed into a 21-kDa mature protein ...

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 ...

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.