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The chitinolytic cascade in Vibrios is regulated by chitin and a two-component chitin catabolic sensor͞kinase

Xibing Li and Saul Roseman*

Department of , Johns Hopkins University, Baltimore, MD 21218

Contributed by Saul Roseman, November 19, 2003 Chitin, a highly insoluble of GlcNAc, is produced in massive and structural . These include at least one, and probably quantities in the marine environment. Fortunately for survival of several, extracellular , 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 synthesis, a specific chitoporin in the outer was identified in Vibrio furnissii and (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 Mutants of chiS grew normally on GlcNAc but did not express (GlcNAc) and the (GlcNAc)2, respectively, three extracellular , 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 -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 , (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 (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 (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 (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 ͞ (‘‘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 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 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. The periplas- treated with toluene at a ratio of 10 l ml of suspension. After mic binding protein binds to ChiS, locking it into an inactive conformation, vigorous shaking for 10 sec, the mixture was maintained at room resulting in repression of the chitinolytic genes. (B) The plus phenotype. temperature for 20 min, and 0.1-ml aliquots were mixed with 0.1 Extracellular chitinase(s) hydrolyze the polymer to oligosaccharides, the major ml of 1 mM PNP-GlcNAc in 20 mM Tris⅐HCl (pH 7.5) and product being (GlcNAc)2. The oligosaccharides enter the periplasmic space and incubated at 37°C. The reaction was stopped with 0.8 ml of 1 M bind to CBP, dissociating it from ChiS, which is now transformed into the active (ϩ) conformation. The chitinolytic genes are now expressed. Tris base (pH 11), cell debris was removed by centrifugation, and the absorbance of the supernatant was determined at 400 nm. Total hexosaminidase activity is expressed as p-nitrophenol Materials and Methods produced per minute per mg of protein. Materials. The following chemicals, reagents, and materials were Results purchased from the indicated sources. p-nitrophenyl ␤-D-N- Isolation of Sensor Mutants from V. furnissii. A transposon Tn10 acetylglucosaminide (PNP-GlcNAc) was from Sigma; Hepes mutant library was constructed in V. furnissii by transconjuga- buffer was from Fisher; oligonucleotide primers were from IDT tion, and the desired mutants were enriched by growing the cells (Coralville, IA); chitin oligosaccharides (GlcNAc)n, n ϭ 2–6, on a lethal analogue of (GlcNAc)2, i.e., (GlcNHCOCF3)2. They were prepared as described (15) or were from Seikagaku (Rock- were spread on chitin plates, and colonies that could not clear the ville, MD); DNA restriction and modifying enzymes were from chitin (negative chitinase expression) were selected for further New England Biolabs; and pGEM-T Vector System I was from study. Three of 6,000 transposon mutants showed the desired Promega. Other buffers and reagents were of the highest purity . First and most importantly, they fermented and commercially available. grew at normal rates on the monosaccharide, GlcNAc. Second, they could not be induced to express a number of proteins Growth and Maintenance of V. cholerae Strains. V. cholerae cells required for chitin degradation. DNA sequence analysis of the were grown either in LB containing 1% NaCl or minimal media three mutants showed that Tn10 had inserted into three different containing 50 mM Hepes (pH 7.5), 50% artificial sea water regions of the same gene, designated the chitin degradation (ASW), 0.1% NH4Cl, 0.001% K2HPO4, and 0.5% DL-lactate sensor (chiS) gene. (lactate-ASW). Cell cultures were grown at 37°C with aeration (30°C for V. furnissii) and growth measured by absorbance at 540 Relationship Between V. furnissii and V. cholerae. When the com- nm. (GlcNAc)2 was added at 0.6 mM in the minimal medium for plete sequence of the V. cholerae genome became available (19),

628 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307645100 Li and Roseman Downloaded by guest on September 28, 2021 Table 1. Comparison of predicted amino-acid sequences of proteins from V. furnissii and V. cholerae Identity, Similarity, V. furnissii proteins V. cholerae ORF (annotation) % %

(GlcNAc)2 phosphorylase VC0612 ( phosphorylase) 86 92 chiS (sensor) VC0622 (sensor) 84 93 Aryl ␤-N-acetylglucosaminidase VC0692 (␤-hexosaminidase) 81 90 Periplasmic ␤-N-acetylglucosaminidase VC0613 (␤-hexosaminidase) 76 86 Periplasmic chitodextrinase VCA0700 (chitodextrinase) 76 82 Glucosamine-specific kinase VC0614 (hypothetical protein) 75 82 Extracellular chitinase VCA0027 (chitinase) 75 83 Cellobiase (exoglucosidase) VC0615 (endoglucanase) 70 77 Chitoporin VC0972 (porin, putative) 49 60 NagE (IIGlcNAc) transporter VC0995 (IIGlcNAc) transporter 42 55

we found a surprising similarity between the predicted amino (GlcNAc)2 and possibly (GlcNAc)3. We have described the acid sequences of many V. cholerae gene products and those that kinetic behavior of this transporter (8). We therefore conclude we had cloned and characterized from V. furnissii. Table 1 that 8 of the 10 genes are required for (GlcNAc)2 uptake and summarizes these findings. The predicted amino acid sequences catabolism, 1 gene (VC0611) is annotated as a phosphohexose of 7 of 10 putative gene products are Ն70% identical, and Ն76% mutase, although we believe that it is likely to be a phospho- similar over the full length of the proteins. The subjects of this GlcNAc mutase, and the 10th gene, VC0615, has been shown to report, the sensor proteins, are 84% identical and 93% similar. encode a cellobiase (13) whose physiological function is un- Table 1 also gives the correct functional assignment to each of known. (V. cholerae neither grows on nor ferments cellobiose.) the gene products. Evidence shows that the 10 genes function as a (GlcNAc)2 catabolic operon (unpublished work). -A Potential N,N؅-diacetylchitobiose Operon in V. cholerae. An im Chitin Degradation Sensor. VC0620 is the first structural gene in portant relationship between the (GlcNAc)2 genes is also ap- Ј parent in Table 1, i.e., that four of the genes, VC0612–VC0615, the presumptive ‘‘chitobiose,’’ more correctly, the N,N - are clustered together in the V. cholerae genome. This region of diacetylchitobiose or (GlcNAc)2 catabolic operon. Upstream (917 bp) is VC0622, the structural gene for chiS. The sensor the genome is shown in Fig. 2, and the cluster of the four genes mutants isolated as described above showed the following phe- are part of 10 contiguous genes, from VC0611 to VC0620. notypic properties. They did not clear chitin plates (4); i.e., they VC0616–VC0620 encodes an ABC-type transporter specific for did not express the extracellular chitinase at normal levels. Additionally, they did not express the chitoporin (7), uptake of MeTCB, which is transported by the (GlcNAc)2 permease (8), ␤-N-acetylhexosaminidases (see below), or chemotaxis to chitin oligosaccharides (20). Finally, they neither fermented nor grew on (GlcNAc)2, whereas they grew normally on GlcNAc. When the V. cholerae and V. furnissii mutants were complemented with the respective intact genes, the defective functions were restored, although not necessarily to wild-type levels.

The Periplasmic Chitin Oligosaccharide Binding Protein (CBP). If the model shown in Fig. 1 is correct, the sensor is ‘‘locked’’ in the negative mode when it is held in this conformation by bound CBP. The periplasmic binding protein CBP has been purified to apparent homogeneity and is a CBP with a Kassoc for (GlcNAc)2 Ϸ ␮ Fig. 2. Putative V. cholerae (GlcNAc)2 catabolic operon. The two highlighted of 1 m. This constant is similar to the association constants genes, VC0620 and VC0622, are the subjects of this communication. The of periplasmic binding proteins (21, 22). CBP also binds ‘‘operon’’ consists of 10 genes, VC0620–VC0611. VC0622 (chiS) must be ex- higher chitin oligosaccharides (but not GlcNAc); these binding pressed for these genes to be derepressed. The annotations of five genes are constants remain to be determined. based on biochemical characterization of the proteins purified to apparent homogeneity, and of the phenotypes of mutants: VC0615 (13), VC0614 (12), Assay of Cells for Total ␤-N-Acetylglucosaminidase Activity. VC0613 (10), and VC0612 (11). The binding protein, VC0620, has been purified Al- though many processes are regulated by the sensor, for present to homogeneity and its binding properties have been established by equilib- ␤ rium and͞or flow dialysis (unpublished work). The four-gene cluster VC0619– purposes we chose to study the total exo- -N-acetylglucosamini- VC0616 is an ABC-type (GlcNAc)2 transporter based on the phenotypic behav- dase activity by using the generic substrate for these enzymes, ior of mutants in any or all of the genes [no transport of the analogue MeTCB PNP-GlcNAc. This compound is an excellent substrate for three (8)]. The assignment of VC0611 as a GlcNAc-1-P mutase is speculative. The well characterized exo-␤-hexosaminidases in the two Vibrios. following annotations were previously assigned to these genes in the V. These glycosidases are induced by (GlcNAc)2 and have been cholerae genomic sequence (19): VC0622, sensor; VC0620, periplasmic binding identified in the V. cholerae genome: VC2217, an outer mem- protein; VC0619–VC0616, polypeptide ABC-type transporter; VC0615, endo- ␤ glucanase; VC0614, hypothetical protein; VC0613, ␤-N-acetylglucosamini- brane bound -N-acetylglucosaminidase (23); VC0613, a periplasmic enzyme specific for chitin oligosaccharides (10); and dase; VC0612, cellobiose phosphorylase; VC0611, phosphohexose mutase. ␤ One short ORF (130 aa predicted) in the V. cholerae genome sequence, VC0692, an aryl -N-acetylglucosaminidase of unknown func-

VC0621, annotated as ‘‘hypothetical protein,’’ is not shown because it is not tion (6). Six other genes in the sequence are annotated as MICROBIOLOGY found in any other known Vibrio genomic sequence or in V. furnissii. putative chitinases or chitodextrinases; it seems likely that some

Li and Roseman PNAS ͉ January 13, 2004 ͉ vol. 101 ͉ no. 2 ͉ 629 Downloaded by guest on September 28, 2021 chitinase or the binding protein (25). Finally, two genes in Streptomyces thermoviolaceus, designated chiS and chiR, are located upstream of the chitinase gene, chi40 on the chromo- some. The putative regulatory genes, chiS and chiR, have an Ϸ2-fold effect on the induced level of chitinase activity (29).

Chitin Catabolic Sensor͞Kinase, chiS, and the Periplasmic Binding Protein CBP. ChiS has a global effect. It regulates expression of 50 genes, most of which are involved in chitin catabolism.† Fur- thermore, regulation by ChiS appears to be much tighter than by previously reported systems. The data presented here on the ␤-N-acetylglucosaminidases and determined elsewhere on other proteins and processes (unpublished work) all support the model shown in Fig. 1. There is no regulation without CBP. It serves to keep the sensor locked Fig. 3. The expression of total ␤-N-acetylglucosaminidase activity and its in its ‘‘default’’ state until the environmental signal, (GlcNAc)n, regulation [induction by (GlcNAc)2] requires both the sensor, ChiS, and the periplasmic binding protein, CBP. Preparation of mutants, growth conditions, removes CBP, activates the sensor, and derepresses the chitin induction, extraction, and the ␤-N-acetylglucosaminidase assay technique are catabolic genes. described in Materials and Methods. The error bars indicate the range of results obtained with a minimum of three different cell preparations. The CBP and Other Solute Specific Periplasmic Binding Proteins (PBP). At permease deletion is a deletion of the four genes required for (GlcNAc)2 least 30 PBP have been identified in E. coli (21, 22, 30), most or transport, VC0619–VC0616, but not of VC0620, the gene encoding CBP. all of which participate in the transport of their specific ligands. Essentially the same results were obtained when a single gene of the permease Five PBP bind and three of this group (, , group, VC0616, was deleted. and ) also signal the cells to respond via chemotaxis to the sugars as a result of binding of the sugar͞PBP complex to a ͞ of these may also be exo- rather than endohydrolases, in which methylating chemoreceptor protein. The maltose BP complex case they would split the generic substrate PNP-GlcNAc. (For has been the most extensively studied, especially by Manson and instance, VC0615 is annotated as an endoglucanase, such as his coworkers (31). The complex binds to the aspartate mem- cellulase, but it is an exo-␤-glucosidase (13).) The advantage of brane , Tar, which activates the phosphorylation cascade ͞ using the generic substrate is that the genes are scattered over the in the chemotaxis system. Similarly, the Gal Glc and Rib PBP two V. cholerae chromosomes, and quantitation of total enzy- bind to the Trg chemoreceptor. In an analogous system, Agrobac- matic activity is a measure of the global effect of the sensor on terium tumefaciens carries a set of virulence (vir) genes on Ti these genes. plasmids that cause the growth of crown gall tumors in infected Extracts of cells grown in the presence and absence of 0.6 mM plants. Two regulatory Ti genes encode a two-component His (GlcNAc)2 were therefore assayed as described in Materials and kinase sensor, VirA and VirG, which induce expression of the vir Methods, and the results are shown in Fig. 3. (i) Total hex- genes in the presence of certain plant phenols and sugars; the osaminidase activity is induced Ϸ17-fold in wild-type cells by sugars act via a chromosomally encoded PBP called ChvE. The ͞ (GlcNAc)2.(ii) The sensor deletion neither expresses the chiti- sugar ChvE complex is suggested to bind to a Trg-like site in the nolytic genes nor is induced by (GlcNAc)2. These are the periplasmic domain of VirA (32, 33), which transmits the signal expected results from the model in Fig. 1, i.e., ChiS must be to VirG. ChvE mutants do not respond to the sugars but activated for derepression of the chitinolytic genes. (iii) Accord- continue to respond to the phenols, although the plant host range ing to the model, one way to activate the sensor would be to becomes more limited. mutate or delete CBP, and such a mutant should express the The periplasmic binding protein reported here, CBP, is unique chitinolytic genes constitutively, i.e., without requiring induction because unlike all of the examples cited above, it apparently by (GlcNAc)2. This is precisely the result shown in Fig. 3. (iv)As interacts with the sensor ChiS in the absence of the sugar ligand. expected, a double mutant in both the sensor and binding protein Additionally, while CBP is induced 20- to 30-fold by (GlcNAc)2, genes could not be induced. (v) The (GlcNAc)2 transporter is the sensor is maintained in the negative phenotypic mode by the encoded by VC0616–VC0620; a mutation in any of the genes was constitutive, low levels of CBP. This result implies that CBP has unable to transport (GlcNAc)2 or the nonmetabolizable ana- a high affinity for the periplasmic domain of ChiS. logue, MeTCB (8). The results in Fig. 3 show that a deletion of Is CBP͞ChiS the sole system of this type, or is the CBP͞ChiS the permease, VC0616–VC0619, but not of VC0620, which system a paradigm for other binding proteins and sensors? It encodes the periplasmic binding protein, CBP, behaves like the would be surprising if the mechanism reported here was not a wild-type strain. It is fully inducible. Thus, cell signaling by general one because the PBP are ideally constituted for such (GlcNAc)2 does not require that it be transported but only that signaling. They are usually freely exposed to the low-molecular- the periplasmic binding protein CBP be expressed. weight putative signaling solutes in the environment, and they have high-affinity binding constants for these solutes. They Discussion should also bind with high affinity to the periplasmic domains of Chitin Degradation. Not only is chitin degradation exceedingly the relevant sensors because they must interact at constitutively complex, but it is stringently regulated. For instance, expression expressed concentrations, i.e., before induction of the PBP by the of extracellular chitinase is catabolite repressed (4) in the marine signaling molecules. Vibrios described here. has a similar effect on chitinase secretion by Streptomyces lividans (24). Expression of chitinase and a chitin binding protein by Serratia marcescens 2170 is †By using a DNA microarray assay, 41 genes were shown to be specifically induced and 9 genes were specifically repressed by (GlcNAc) or shells but not by GlcNAc. Most of the induced by chitin and by (GlcNAc)2 (25). In this organism, the n disaccharide is taken up by the bacterial phosphotransferase 41 induced genes were repressed in the sensor deletion; i.e., they do not respond to (GlcNAc)n. The nine repressed genes include the (GlcNAc)2-specific genes of the phos- system, first identified in E. coli (26–28). S. marcescens mutants phoenolpyruvate:glycose phosphotransferase system (PTS) (K. L. Meibom, X.L., A. T. defective in (GlcNAc)2 transport are unable to express either the Nielsen, C.-Y. Wu, S.R., and G. K. Schoolnik, unpublished work).

630 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307645100 Li and Roseman Downloaded by guest on September 28, 2021 Bacteria Sense the Presence of Chitin via the Specific Marker is astonishing. Our proposal was based on two observations: first, (GlcNAc)n. It appears logical that chitinolytic bacteria have de- that starving V. furnissii cells secrete large quantities of extra- veloped a system for regulating chitin catabolism based on the cellular chitinase, and second, that (GlcNAc)2 and higher oli- presence or absence of the environmental signal (GlcNAc)n, and gosaccharides are exceedingly potent bacterial chemoattractants not, for instance, on the monosaccharide GlcNAc. The rationale (20, 34). The inference is that the secreted chitinase from is as follows. starving cells§ comes into contact with chitin in the microenvi- ͞ The glycosidases that hydrolyze chitin give small quantities ronment and generates a disaccharide and or a (GlcNAc)n of products such as GlcNAc, glucosamine, oligosaccharides of gradient, and that the cells swim up this gradient to the both , and oligosaccharides that contain both or chitin. We can now extend this idea. (GlcNAc)n induces glucosamine and GlcNAc. However, the vast majority of bacte- expression of the chitin catabolic cascade genes under control of rial chitinases (as well as many eukaryotic chitinases) yield ChiS, including the extracellular chitinase, and the cells remain (GlcNAc)2 as the major end product. in this state as long as (GlcNAc)n is generated. When the chitin One pathway for the of (GlcNAc)2 is its further is completely depleted, the periplasmic binding protein again hydrolysis by a host of hexosaminidases to GlcNAc, a sugar that binds to ChiS and ‘‘locks’’ it into the minus configuration, and the is avidly used by the Vibrios. But GlcNAc is also derived from system is turned off. a wide variety of other sources including a large group of Further evidence must be obtained to establish the validity of called , and from glycopro- the model proposed in this article. For example, attempts will be teins and glycolipids as well. Thus, GlcNAc would be a poor, made to determine whether the purified periplasmic binding nonspecific signal for the cells to start expressing the chitin protein binds to the cloned periplasmic domain of the sensor, the catabolic machinery. (GlcNAc)n is derived specifically from Kassoc and stoichiometry of the binding, the effect of (GlcNAc)n, chitin and is therefore an excellent signal to these bacteria that etc. Additionally, we do not know the default state of the sensor chitin is in the vicinity.‡ (kinase or phosphatase), whether it is a or dimer, or The present results complement a previously suggested idea the identity of cognate response regulator(s). Further experi- (4) to answer the following question: How do these bacteria ments should answer some of these questions. sense and find chitin in the ocean waters? Their ability to do so

§ Both starvation and (GlcNAc)n independently signal the cells to secrete extracellular ‡The disaccharide is also found in blood glycoproteins where it links oligosaccharides to the chitinase(s). Whether these are the same or different chitinases remains to be determined. polypeptide chains of the proteins. These oligosaccharides contain a variety of sugars, and their hydrolysis requires many discrete enzymes. Thus, it is unlikely that these substances could yield significant quantities of (GlcNAc)2 in the marine environment. However, in a more limiting environment rich in glycoproteins and hydrolases, such as the intestine, We thank Dr. Ann Stock (Rutgers, The State University of New Jersey, conceivably sufficient (GlcNAc)2 could be generated to result in expression of the chiti- Piscataway) for valuable suggestions. This work was supported by nolytic genes. In this connection, it may be relevant to note that (GlcNAc)2 also signals V. National Institutes of Health Grant GM51215 and a grant from New cholerae to express special pili and other genes, possibly involved in the infective process. England Biolabs kindly made available by Dr. Donald Comb.

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