SummarySuccessful colonization of a eukaryotic host by a microbe involves complex microbe-microbe and microbe-host interactions. Previously, we identified in Vibrio fischeri a putative sensor kinase, RscS, required for initiating symbiotic colonization of its squid host Euprymna scolopes. Here, we analysed the role of rscS by isolating an allele, rscS1, with increased activity. Multicopy rscS1 activated transcription of genes within the recently identified symbiosis polysaccharide (syp) cluster. Wild-type cells carrying rscS1 induced aggregation phenotypes in culture, including the formation of pellicles and wrinkled colonies, in a syp-dependent manner. Colonies formed by rscS1-expressing cells produced a matrix not found in control colonies and largely lost in an rscS1-expressing sypN mutant. Finally, multicopy rscS1 provided a colonization advantage over control cells and substantially enhanced the ability of wildtype cells to aggregate on the surface of the symbiotic organ of E. scolopes; this latter phenotype similarly depended upon an intact syp locus. These results suggest that transcription induced by RscSmediated signal transduction plays a key role in colonization at the aggregation stage by modifying the cell surface and increasing the ability of the cells to adhere to one another and/or to squid-secreted mucus.

c Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae ("vibrios"), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone. Horizontally transmitted microbial symbioses entail specificity challenges for both partners during each host generation (17). The juvenile host often enters the world lacking its partner microbe(s) and recruits them from a complex environmental assemblage of bacteria; for their part, the bacterial symbionts find the correct host niche to the exclusion of nonsymbiotic and pathogenic bacteria. Colonization specificity has been well studied in nitrogen-fixing plant symbionts, revealing detailed chemical communication that takes place between host plants and colonizing rhizobia to direct the symbionts to the correct host (15, 32). For example, plant flavonoids induce the production of bacterial Nod factors, chitin derivatives decorated with oligosaccharides that are specific for their cognate host plant. In turn, these factors signal plant-specific receptor kinases that result in plant tissue development.In animal associations-especially those in marine environments, which often contain Ͼ10 6 bacterial cells per milliliter of seawater-the need for effective mechanisms to assure recruitment and host specificity is clear; however, the underlying molecular determinants are poorly understood. Colonization of the Hawaiian bobtail squid Euprymna scolopes by the bioluminescent bacterium Vibrio fischeri has proven to be an especially valuable platform for identifying and characterizing such determinants. A "winnowing" process has been described (30) during which first Gram-negative bacteria, then V. fischeri, and finally motile V. fischeri specifically are selected for their ability to colonize this model host. Within the mantle cavity of the squid, the ciliated surface epithelium of the nascent light-emitting organ plays an important role. In the presence of hos...

Newly hatched juveniles of the Hawaiian squid Euprymna scolopes rapidly become colonized by the bioluminescent marine bacterium Vibrio fischeri. Motility is required to establish the symbiotic colonization, but the role of chemotaxis is unknown. In this study we analyzed chemotaxis of V. fischeri to a number of potential attractants. The bacterium migrated toward serine and most sugars tested. V. fischeri also exhibited the unusual ability to migrate to nucleosides and nucleotides as well as to N-acetylneuraminic acid, a component of squid mucus.Upon hatching, the light organ of the juvenile Hawaiian squid, Euprymna scolopes, is specifically colonized by the luminous marine bacterium Vibrio fischeri (reviewed in references 7 and 13). V. fischeri cells present in the seawater aggregate in mucus secreted from the light organ and then appear to stream into the openings of the light organ (10), suggesting directed movement by the bacterium. The light-organ mucus, secreted upon bacterial exposure (10) and subsequently within the light organ in response to symbiotic colonization (9), contains two sugars, N-acetylgalactosamine (NAGal) and N-acetylneuraminic acid (NANA) (10). These sugars, as well as amino acids and peptides within the light organ (6), may serve as nutrient sources and/or chemoattractants to enhance entry by V. fischeri, which must be motile to colonize successfully (5). To begin investigating the potential role of chemotaxis in symbiotic initiation, we characterized the response of the bacterium to various nutrients.Motile cells inoculated onto a soft agar medium containing two attractants form an outermost ring in response to a spatial gradient that results from the consumption and subsequent diffusion of the preferred attractant (14). Similarly, an inner ring forms to the second attractant (11 and B. M. Pruss and A. J. Wolfe, unpublished data). When inoculated onto TB-SW soft agar plates (1% tryptone, 0.88% NaCl, 0.62% MgSO 4 , 0.072% CaCl 2 , 0.038% KCl, 0.25% agar), cells of V. fischeri strain ES114 (2) formed two concentric rings (Fig. 1A). Cells of Escherichia coli also form two rings on tryptone-based soft agar, with the outer and inner rings sensing serine and aspartate, respectively (1). We therefore tested whether V. fischeri also migrated to serine and aspartate. While the bacterium did not respond to aspartate (data not shown), the addition of increasing concentrations of serine to the soft agar slowed the migration of the inner ring of V. fischeri, indicating that the cells present in that ring consume, sense, and migrate to serine ( Fig. 1B and C). We used an excess of serine to disrupt the gradient and found that serine perturbed migration of the inner ring (arrows in Fig. 1 depict the location of the spot of serine). Closer inspection of the rings revealed that a doublet we occasionally observed (e.g., Fig. 1B) consisted of fastermigrating cells on the surface of the plate and slower-migrating cells deeper in the agar, both of which responded to serine. Because the doublet responded as ...

The bacterium Vibrio fischeri requires bacterial motility to initiate colonization of the Hawaiian squid Euprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences on V. fischeri motility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg 2؉ . At concentrations naturally present in seawater, Mg 2؉ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg 2؉ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.The symbiosis between the marine bacterium Vibrio fischeri and the Hawaiian squid Euprymna scolopes provides a model for exploring the communication that occurs between a bacterium and its host in a natural setting (30,39,45,55). Juveniles of E. scolopes hatch without V. fischeri cells present inside the symbiotic organ (the light organ), and rapidly acquire these bacteria from the surrounding seawater (31, 57). Colonization begins with aggregation of V. fischeri cells in mucus on the surface of the light organ (37, 38, 40), followed by movement of these bacteria through pores into ducts, apparently toxic passageways that limit nonspecific invaders (11, 40), and ultimately into crypts where the bacteria multiply (46).Only motile cells of V. fischeri initiate symbiotic colonization of E. scolopes. Nonmotile mutants fail to colonize (16,33,59), presumably because they fail to migrate out of the bacterial aggregates formed on the surface of the light organ (40). Apparently, normal initiation also requires optimal motility, because several hypermotile mutants colonize with delayed kinetics (32).Once V. fischeri cells initiate colonization, the majority of symbiotic bacteria within the E. scolopes light organ become nonflagellated (33, 46). However, within an hour of their release from the light organ into seawater, V. fischeri cells regrow their flagella (46). These observations suggest that environmental conditions inside the light organ inhibit flagellation, while those outside favor it (46).Environmental influences on the motility of the enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium have been well documented (for reviews, see references 3 and 29). These influences include nutrient availability, temperature, ionic composition, pH, and surface interactions (2,21,23,27,35,48,50). Most known environmental influences act at the level of transcription initiation or, to a lesser extent, message stability. These operate through at least one nucleoid protein (H-NS) and a host of transcription factors, including the cyclic AMP (cAMP) receptor protein, LrhA (CRP), and several two-component response regulators (1,7,14,15,21,22,26,41,(49)(50)(51)54). Control of message stability involves the small RNA-binding protein CsrA ...

c Flagellar motility and chemotaxis by Vibrio fischeri are important behaviors mediating the colonization of its mutualistic host, the Hawaiian bobtail squid. However, none of the 43 putative methyl-accepting chemotaxis proteins (MCPs) encoded in the V. fischeri genome has been previously characterized. Using both an available transposon mutant collection and directed mutagenesis, we isolated mutants for 19 of these genes, and screened them for altered chemotaxis to six previously identified chemoattractants. Only one mutant was defective in responding to any of the tested compounds; the disrupted gene was thus named vfcA (Vibrio fischeri chemoreceptor A; locus tag VF_0777). In soft-agar plates, mutants disrupted in vfcA did not exhibit the serinesensing chemotactic ring, and the pattern of migration in the mutant was not affected by the addition of exogenous serine. Using a capillary chemotaxis assay, we showed that, unlike wild-type V. fischeri, the vfcA mutant did not undergo chemotaxis toward serine and that expression of vfcA on a plasmid in the mutant was sufficient to restore the behavior. In addition to serine, we demonstrated that alanine, cysteine, and threonine are strong attractants for wild-type V. fischeri and that the attraction is also mediated by VfcA. This study thus provides the first insights into how V. fischeri integrates information from one of its 43 MCPs to respond to environmental stimuli. F lagellar motility is one of several behaviors used by bacteria to migrate through their surroundings (1). Migration to preferred environmental conditions is mediated by a behavior known as chemotaxis (2), which allows the bacterium to sense gradients of attractants and respond by controlling the directionality of the flagellar motor (3). Methyl-accepting chemotaxis proteins (MCPs) function as receptors that bind attractants or repellants, usually in the periplasm. Upon ligand binding, MCPs transduce a signal through the CheAY two-component system to affect a change in the tumbling frequency of the flagella (4). By altering the tumbling frequency, MCPs direct an average change in the direction of travel for the bacteria. In Escherichia coli K-12, a total of five MCPs enable sensing of numerous attractants, including amino acids, peptides, galactose, ribose, and oxygen (5-9). However, as more diverse bacterial species have been studied, we have learned that bacterial chemotaxis is frequently more complex than the E. coli paradigm (10, 11). Bioinformatics and increased genome sequence availability have revealed that both the number and the domain structure of predicted MCPs vary greatly between species (12-14). Work in Pseudomonas aeruginosa, a species that encodes 26 MCPs in strain PAO1, has characterized 9 MCPs that mediate chemotaxis toward ligands such as amino acids, trichloroethylene, and malate (15-18). However, even in this relatively well-studied organism, high-throughput attempts to identify MCP ligands have been largely inconclusive and, in one case, only successful when energy taxis, mediat...