The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

Abstract

Chitin, a highly insoluble polymer of GlcNAc, is produced in massive quantities in the marine environment. Fortunately for survival of aquatic ecosystems, chitin is rapidly catabolized by marine bacteria. Here we describe a bacterial two-component hybrid sensor/kinase (of the ArcB type) that rigorously controls expression of approximately 50 genes, many involved in chitin degradation. The sensor gene, chiS, was identified in Vibrio furnissii and Vibrio cholerae (predicted amino acid sequences, full-length: 84% identical, 93% similar). Mutants of chiS grew normally on GlcNAc but did not express extracellular chitinase, a specific chitoporin, or beta-hexosaminidases, nor did they exhibit chemotaxis, transport, or growth on chitin oligosaccharides such as (GlcNAc)(2). Expression of these systems requires three components: wild-type chiS; a periplasmic high-affinity chitin oligosaccharide, (GlcNAc)(n) (n > 1), binding protein (CBP); and the environmental signal, (GlcNAc)(n). Our data are consistent with the following model. In the uninduced state, CBP binds to the periplasmic domain of ChiS and "locks" it into the minus conformation. The environmental signal, (GlcNAc)(n), dissociates the complex by binding to CBP, releasing ChiS, yielding the plus phenotype (expression of chitinolytic genes). In V. cholerae, a cluster of 10 contiguous genes (VC0620-VC0611) apparently comprise a (GlcNAc)(2) catabolic operon. CBP is encoded by the first, VC0620, whereas VC0619-VC0616 encode a (GlcNAc)(2) ABC-type permease. Regulation of chiS requires expression of CBP but not (GlcNAc)(2) transport. (GlcNAc)(n) is suggested to be essential for signaling these cells that chitin is in the microenvironment.

Fig. 1.

Model for regulation of activity of the chitin catabolic sensor ChiS in V. furnissii and V. cholerae. (A) The minus phenotype. Three compartments, separated by two membranes are schematically illustrated: the extracellular space, the periplasmic space, and the cytoplasm. The outer membrane (OM) contains the porins, including a chitin oligosaccharide specific porin (chitoporin). The periplasmic space contains the (GlcNAc)_n high-affinity binding protein (CBP) shown in pink. ChiS, the hybrid sensor, is green, and contains the following domains: short amino terminal cytoplasmic domain, a periplasmic domain that separates two short membrane domains, and a large cytoplasmic domain comprising three subdomains: HK, the ATP-dependent, autophosphorylatable His kinase/phosphatase; RR, the Asp response regulator; and HPt, which contains a phosphorylatable His. The presumptive cognate receptor, a separate protein containing an active site Asp, is not shown. The inner membrane (IM) also contains the (GlcNAc)₂ ABC type permease. The periplasmic binding protein binds to ChiS, locking it into an inactive conformation, resulting in repression of the chitinolytic genes. (B) The plus phenotype. Extracellular chitinase(s) hydrolyze the polymer to oligosaccharides, the major product being (GlcNAc)₂. The oligosaccharides enter the periplasmic space and bind to CBP, dissociating it from ChiS, which is now transformed into the active (+) conformation. The chitinolytic genes are now expressed.

Fig. 2.

Putative V. cholerae (GlcNAc)₂ catabolic operon. The two highlighted genes, VC0620 and VC0622, are the subjects of this communication. The “operon” consists of 10 genes, VC0620–VC0611. VC0622 (chiS) must be expressed for these genes to be derepressed. The annotations of five genes are based on biochemical characterization of the proteins purified to apparent homogeneity, and of the phenotypes of mutants: VC0615 (13), VC0614 (12), VC0613 (10), and VC0612 (11). The binding protein, VC0620, has been purified to homogeneity and its binding properties have been established by equilibrium and/or flow dialysis (unpublished work). The four-gene cluster VC0619–VC0616 is an ABC-type (GlcNAc)₂ transporter based on the phenotypic behavior of mutants in any or all of the genes [no transport of the analogue MeTCB (8)]. The assignment of VC0611 as a GlcNAc-1-P mutase is speculative. The following annotations were previously assigned to these genes in the V. cholerae genomic sequence (19): VC0622, sensor; VC0620, periplasmic binding protein; VC0619–VC0616, polypeptide ABC-type transporter; VC0615, endoglucanase; VC0614, hypothetical protein; VC0613, β-N-acetylglucosaminidase; VC0612, cellobiose phosphorylase; VC0611, phosphohexose mutase. One short ORF (130 aa predicted) in the V. cholerae genome sequence, VC0621, annotated as “hypothetical protein,” is not shown because it is not found in any other known Vibrio genomic sequence or in V. furnissii.

Fig. 3.

The expression of total β-N-acetylglucosaminidase activity and its regulation [induction by (GlcNAc)₂] requires both the sensor, ChiS, and the periplasmic binding protein, CBP. Preparation of mutants, growth conditions, induction, extraction, and the β-N-acetylglucosaminidase assay technique are described in Materials and Methods. The error bars indicate the range of results obtained with a minimum of three different cell preparations. The permease deletion is a deletion of the four genes required for (GlcNAc)₂ transport, VC0619–VC0616, but not of VC0620, the gene encoding CBP. Essentially the same results were obtained when a single gene of the permease group, VC0616, was deleted.