The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor͞kinase Xibing Li and Saul Roseman* Department of Biology, Johns Hopkins University, Baltimore, MD 21218 Contributed by Saul Roseman, November 19, 2003 Chitin, a highly insoluble polymer of GlcNAc, is produced in massive and structural proteins. These include at least one, and probably quantities in the marine environment. Fortunately for survival of several, extracellular chitinases, chemotaxis systems specific for aquatic ecosystems, chitin is rapidly catabolized by marine bacte- chitin oligosaccharides (highly potent chemoattractants), a ‘‘nu- ria. Here we describe a bacterial two-component hybrid sensor͞ trient sensor’’ that allows the cells to bind to the chitin as long kinase (of the ArcB type) that rigorously controls expression of Ϸ50 as the extracellular environment contains all ingredients neces- genes, many involved in chitin degradation. The sensor gene, chiS, sary for protein synthesis, a specific chitoporin in the outer was identified in Vibrio furnissii and Vibrio cholerae (predicted membrane, at least two hydrolases specific for chitin oligosac- amino acid sequences, full-length: 84% identical, 93% similar). charides in the periplasmic space that yield the monosaccharide Mutants of chiS grew normally on GlcNAc but did not express (GlcNAc) and the disaccharide (GlcNAc)2, respectively, three extracellular chitinase, a specific chitoporin, or ␤-hexosaminidases, transport complexes in the inner membrane, and a minimum of nor did they exhibit chemotaxis, transport, or growth on chitin six cytoplasmic enzymes that convert the products of transport oligosaccharides such as (GlcNAc)2. Expression of these systems to fructose-6-P, NH3, and acetate. We have reported the mo- requires three components: wild-type chiS; a periplasmic high- lecular cloning of 10 of these genes and characterized the affinity chitin oligosaccharide, (GlcNAc)n (n > 1), binding protein corresponding proteins (5–13). (CBP); and the environmental signal, (GlcNAc)n. Our data are Expression of the chitinolytic genes is rigorously regulated, consistent with the following model. In the uninduced state, CBP and the proposed mechanism for this regulation is shown in Fig. binds to the periplasmic domain of ChiS and ‘‘locks’’ it into the 1. The model comprises three components: (i) the environmen- minus conformation. The environmental signal, (GlcNAc)n, disso- tal signal, (GlcNAc)2, and possibly higher chitin oligosaccha- ciates the complex by binding to CBP, releasing ChiS, yielding the rides; (ii) a periplasmic solute binding protein specific for plus phenotype (expression of chitinolytic genes). In V. cholerae,a (GlcNAc) (n Ͼ 1) designated here as CBP for chitin oligo- cluster of 10 contiguous genes (VC0620–VC0611) apparently com- n saccharide binding protein; and (iii) a hybrid sensor kinase of prise a (GlcNAc) catabolic operon. CBP is encoded by the first, 2 the Arc B type (14), designated ChiS. In sensors of this kind, the VC0620, whereas VC0619–VC0616 encode a (GlcNAc) ABC-type 2 protein consists of a short N-terminal peptide chain in permease. Regulation of chiS requires expression of CBP but not the cytoplasm, a membrane domain, a periplasmic domain, a (GlcNAc) transport. (GlcNAc) is suggested to be essential for 2 n second membrane domain, and a long polypeptide chain extend- signaling these cells that chitin is in the microenvironment. ing into the cytoplasm. The latter comprises three subdomains: HK or His kinase, RR or receiver (aspartate), and HPt. The wo-component signal transduction systems (also known as phosphoryl group is transferred sequentially from ATP to HK, THis–Asp phosphorelay systems) play essential roles in trans- to RR, to a His in HPt, and finally to Asp in a separate ferring information from the environment to the respective cytoplasmic cognate response regulator that interacts with the genomes of prokaryotes and some eukaryotes (1, 2). There are genome (not shown in Fig. 1). at least 29 His kinase signal transduction systems in Escherichia We propose that repression of expression of the chitinolytic coli. In this article we report that two marine Vibrios, Vibrio genes, the minus phenotype, is the result of binding of the furnissii and Vibrio cholerae express a unique two-component (GlcNAc)n periplasmic binding protein to the periplasmic do- signaling system that stringently regulates expression of many main of the sensor, ‘‘locking’’ it into an inactive conformation genes required for chitin catabolism. ␤ (Fig. 1A). The environmental signals are chitin oligosaccharides Chitin is a highly insoluble ,1-4-linked polymer composed derived from partial hydrolysis of chitin by chitinases. In Fig. 1B, primarily of GlcNAc and some glucosamine (GlcN) residues and (GlcNAc) is used as the example because it is the major product is one of the most abundant organic substances in nature. More 2 formed by virtually all bacterial and many eukaryotic chitinases. than 1011 tons are estimated to be produced annually in marine In addition, two enzymes in the periplasmic space hydrolyze waters alone, mostly from copepods. The consumption of these higher oligosaccharides to (GlcNAc) and some GlcNAc (9, 10). huge quantities of chitin is critical for maintaining the C and N 2 CBP does not bind the monosaccharide, GlcNAc. cycles in these waters. It is, in fact, rapidly consumed. Early in the The extracellular signals, (GlcNAc) or, more generally, last century it was shown that ocean sediments contained only 2 (GlcNAc)n, compete with the sensor ChiS for CBP. ChiS cannot traces of chitin despite the constant rain of the polysaccharide ͞ (‘‘marine snow’’) to the ocean floor. This apparent enigma was bind the CBP (GlcNAc)n complex and is thereby activated to the resolved in 1937 when Zobell and Rittenberg (3) reported that plus phenotype (Fig. 1B), whereon the chitinolytic genes are many marine bacteria were chitinivorous, i.e., they could use expressed. chitin as the sole source of C and N, and it has since been shown that marine snow rarely reaches the ocean bottom but is de- Abbreviations: CBP, chitin oligosaccharide binding protein; PNP-GlcNAc, p-nitrophenyl graded as it slowly settles in the ocean waters. ␤-D-N-acetylglucosaminide. The degradation process is exceedingly complex (partially *To whom correspondence should be addressed at: Department of Biology, Mudd Hall, reviewed in ref. 4). The cells must sense the chitin, bind to it, and Johns Hopkins University, 33rd and Charles Streets, Baltimore, MD 21218. E-mail: degrade it to fructose-6-P, acetate, and NH3. We estimated that [email protected]. MICROBIOLOGY the overall chitin catabolic cascade involved dozens of enzymes © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307645100 PNAS ͉ January 13, 2004 ͉ vol. 101 ͉ no. 2 ͉ 627–631 Downloaded by guest on September 28, 2021 induction of the ␤-hexosaminidases. The concentrations of antibiotics were 50 ␮g͞ml kanamycin and 75 ␮g͞ml ampicillin. Construction of Mutants. DNA preparation and analysis, restric- tion enzyme digests, ligation, and transformations were per- formed according to standard techniques (16). A Tn10 mutant library was constructed by transconjugation with V. furnissii strain ATCC 33813 as recipient and E. coli S17-1 harboring a suicide plasmid containing the Tn10 transposon with a chlor- amphenicol resistance gene derived from pNK2884 (17) as donor. The concentrations of antibiotics were 10 ␮g͞ml ampi- cillin and 30 ␮g͞ml chloramphenicol. V. cholerae deletion mutants were constructed by transconju- gation (18) with a temperature-sensitive suicide vector, pMAKSACA. V. cholera O1 E1 Tor N16961 strains with ⌬scrA⌬lacZ, KanR (VCXB21, wild type) or ⌬scrA⌬lacZ (VCXB36, wild type) were used as parental strains, and E. coli S17-1 cells harboring appropriate deletion vectors were used as donor strains. (i) CBP deletion strain: The EcoRI to SnaBI fragment of the cbp gene (VC0620) was deleted (373 of 556 aa; residues 43–416) and replaced with a kanamycin resistance cassette from plasmid pNK2859 (17). VCXB36 was used as the recipient strain. (ii) chiS deletion strain and chiS⌬cbp⌬ double- deletion strain: The chiS deletion strain contains fragments of the N and C termini of chiS, 48 and 50 bp, respectively, interrupted by a BglII restriction site. This construct was trans- ferred to VCXB21 as the recipient. The chiS⌬cbp⌬ double- deletion strain contains an additional AmpR (from pBR322) in ⌬ Fig. 1. Model for regulation of activity of the chitin catabolic sensor ChiS in the BglII site of the chiS deletion construct, using cbp as the V. furnissii and V. cholerae.(A) The minus phenotype. Three compartments, recipient. (iii) CBP-positive, permease deletion strain (VC0616– separated by two membranes are schematically illustrated: the extracellular VC0619⌬): The mutant contains 311 bp of the N terminus of space, the periplasmic space, and the cytoplasm. The outer membrane (OM) VC0619, a BglII site, followed by 329 bp at the C terminus contains the porins, including a chitin oligosaccharide specific porin (chito- of VC0616. A kanamycin resistance cassette from pNK2859 was porin). The periplasmic space contains the (GlcNAc)n high-affinity binding inserted into the BglII site. VCXB36 was used as the recipient protein (CBP) shown in pink. ChiS, the hybrid sensor, is green, and contains the strain. following domains: short amino terminal cytoplasmic domain, a periplasmic domain that separates two short membrane domains, and a large cytoplasmic ␤-Hexosaminidase Assay. domain comprising three subdomains: HK, the ATP-dependent, autophos- These enzymes were assayed with PNP- phorylatable His kinase͞phosphatase; RR, the Asp response regulator; and GlcNAc as described (10). In brief, V. cholerae cells were grown HPt, which contains a phosphorylatable His. The presumptive cognate recep- in the minimal lactate-ASW medium to mid-log phase, the cell 8 tor, a separate protein containing an active site Asp, is not shown. The inner density was adjusted to 5 ϫ 10 cells per ml, and the cells were ␮ ͞ membrane (IM) also contains the (GlcNAc)2 ABC type permease.