Alterations in Vibrio fischeri motility correlate with a delay in symbiosis initiation and are associated with additional symbiotic colonization defects

Motility is required for Vibrio fischeri cells to interact with and specifically colonize the light-emitting organ of their host, the squid Euprymna scolopes. To investigate the influence of motility on the expression of the symbiotic phenotype, we isolated mutants of the squid symbiont V. fischeri ES114 that had altered migration abilities. Spontaneous hyperswimmer (HS) mutants, which migrated more rapidly in soft agar and were hyperflagellated relative to the wild type, were isolated and grouped into three phenotypic classes. All of the HS strains tested, regardless of class, were delayed in symbiosis initiation. This result suggested that the hypermotile phenotype alone contributes to an inability to colonize squid normally. Class III HS strains showed the greatest colonization defect: they colonized squid to a level that was only 0.1 to 10% that achieved by ES114. In addition, class III strains were defective in two capabilities, hemagglutination and luminescence, that have been previously described as colonization factors in V. fischeri. Class II and III mutants also share a mucoid colony morphology; however, class II mutants can colonize E. scolopes to a level that was 40% of that achieved by ES114. Thus, the mucoid phenotype alone does not contribute to the greater defect exhibited by class III strains. When squid were exposed to ES114 and any one of the HS mutant strains as a coinoculation, the parent strain dominated the resulting symbiotic light-organ population. To further investigate the colonization defects of the HS strains, we used confocal laser-scanning microscopy to visualize V. fischeri cells in their initial interaction with E. scolopes tissue. Compared to ES114, HS strains from all three classes were delayed in two behaviors involved in colonization: (i) aggregation on host-derived mucus structures and (ii) migration to the crypts. These results suggest that, while motility is required to initiate colonization, the presence of multiple flagella may actually interfere with normal aggregation and attachment behavior. Furthermore, the pleiotropic nature of class III HS strains provides evidence that motility is coregulated with other symbiotic determinants in V. fischeri.     

Role for cheR of Vibrio fischeri in the Vibrio-squid symbiosis

Upon hatching, the Hawaiian squid Euprymna scolopes is rapidly colonized by its symbiotic partner, the bioluminescent marine bacterium Vibrio fischeri . Vibrio fischeri cells present in the seawater enter the light organ of juvenile squid in a process that requires bacterial motility. In this study, we investigated the role chemotaxis may play in establishing this symbiotic colonization. Previously, we reported that V.?fischeri migrates toward numerous attractants, including N-acetylneuraminic acid (NANA), a component of squid mucus. However, whether or not migration toward an attractant such as squid-derived NANA helps the bacterium to localize toward the light organ is unknown. When tested for the ability to colonize juvenile squid, a V. fischeri chemotaxis mutant defective for the methyltransferase CheR was outcompeted by the wild-type strain in co-inoculation experiments, even when the mutant was present in fourfold excess. Our results suggest that the ability to perform chemotaxis is an advantage during colonization, but not essential.     

Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors

Vibrio fischeri possesses two quorum-sensing systems, ain and lux, using acyl homoserine lactones as signaling molecules. We have demonstrated previously that the ain system activates luminescence gene expression at lower cell densities than those required for lux system activation and that both systems are essential for persistent colonization of the squid host, Euprymna scolopes. Here, we asked whether the relative contributions of the two systems are also important at different colonization stages. Inactivation of ain, but not lux, quorum-sensing genes delayed initiation of the symbiotic relationship. In addition, our data suggest that lux quorum sensing is not fully active in the early stages of colonization, implying that this system is not required until later in the symbiosis. The V. fischeri luxI mutant does not express detectable light levels in symbiosis yet initiates colonization as well as the wild type, suggesting that ain quorum sensing regulates colonization factors other than luminescence. We used a recently developed V. fischeri microarray to identify genes that are controlled by ain quorum sensing and could be responsible for the initiation defect. We found 30 differentially regulated genes, including the repression of a number of motility genes. Consistent with these data, ain quorum-sensing mutants displayed an altered motility behavior in vitro. Taken together, these data suggest that the sequential activation of these two quorum-sensing systems with increasing cell density allows the specific regulation of early colonization factors (e.g., motility) by ain quorum sensing, whereas late colonization factors (e.g., luminescence) are preferentially regulated by lux quorum sensing.     

Evolutionary perspectives in a mutualism of sepiolid squid and bioluminescent bacteria: combined usage of microbial experimental evolution and temporal population genetics

The symbiosis between marine bioluminescent Vibrio bacteria and the sepiolid squid Euprymna is a model for studying animal-bacterial Interactions. Vibrio symbionts native to particular Euprymna species are competitively dominant, capable of outcompeting foreign Vibrio strains from other Euprymna host species. Despite competitive dominance, secondary colonization events by invading nonnative Vibrio fischeri have occurred. Competitive dominance can be offset through superior nonnative numbers and advantage of early start host colonization by nonnatives, granting nonnative vibrios an opportunity to establish beachheads in foreign Euprymna hosts. Here, we show that nonnative V. fischeri are capable of rapid adaptation to novel sepiolid squid hosts by serially passaging V. fischeri JRM200 (native to Hawaiian Euprymna scolopes) lines through the novel Australian squid host E. tasmanica for 500 generations. These experiments were complemented by a temporal population genetics survey of V. fischeri, collected from E. tasmanica over a decade, which provided a perspective from the natural history of V. fischeri evolution over 15,000-20,000 generations in E. tasmanica. No symbiont anagenic evolution within squids was observed, as competitive dominance does not purge V. fischeri genetic diversity through time. Instead, abiotic factors affecting abundance of V. fischeri variants in the planktonic phase sustain temporal symbiont diversity, a property itself of ecological constraints imposed by V. fischeri host adaptation.     

Contribution of pilA to competitive colonization of the squid Euprymna scolopes by Vibrio fischeri

Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.     

Population dynamics of Vibrio fischeri during infection of Euprymna scolopes

The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.     

O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: composition and analysis of their role in Euprymna scolopes light organ colonization

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.     

Chemoattraction of Vibrio fischeri to serine, nucleosides, and N-acetylneuraminic acid, a component of squid light-organ mucus

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.     

Role for phosphoglucomutase in Vibrio fischeri-Euprymna scolopes symbiosis

Vibrio fischeri, a luminescent marine bacterium, specifically colonizes the light organ of its symbiotic partner, the Hawaiian squid Euprymna scolopes. In a screen for V. fischeri colonization mutants, we identified a strain that exhibited on average a 10-fold decrease in colonization levels relative to that achieved by wild-type V. fischeri. Further characterization revealed that this defect did not result from reduced luminescence or motility, two processes required for normal colonization. We determined that the transposon in this mutant disrupted a gene with high sequence identity to the pgm (phosphoglucomutase) gene of Escherichia coli, which encodes an enzyme that functions in both galactose metabolism and the synthesis of UDP-glucose. The V. fischeri mutant grew poorly with galactose as a sole carbon source and was defective for phosphoglucomutase activity, suggesting functional identity between E. coli Pgm and the product of the V. fischeri gene, which was therefore designated pgm. In addition, lipopolysaccharide profiles of the mutant were distinct from that of the parent strain and the mutant exhibited increased sensitivity to various cationic agents and detergents. Chromosomal complementation with the wild-type pgm allele restored the colonization ability to the mutant and also complemented the other noted defects. Unlike the pgm mutant, a galactose-utilization mutant (galK) of V. fischeri colonized juvenile squid to wild-type levels, indicating that the symbiotic defect of the pgm mutant is not due to an inability to catabolize galactose. Thus, pgm represents a new gene required for promoting colonization of E. scolopes by V. fischeri.     

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.

Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.     

Characterization of two host-specific genes, mannose-sensitive hemagglutinin (mshA) and uridyl phosphate dehydrogenase (UDPDH) that are involved in the Vibrio fischeri–Euprymna tasmanica mutualism

While much has been known about the mutualistic associations between the sepiolid squid Euprymna tasmanica and the luminescent bacterium, Vibrio fischeri , less is known about the connectivity between the microscopic and molecular basis of initial attachment and persistence in the light organ. Here, we examine the possible effects of two symbiotic genes on specificity and biofilm formation of V. fischeri in squid light organs. Uridine diphosphate glucose-6-dehydrogenase (UDPDH) and mannose-sensitive hemagglutinin ( mshA ) mutants were generated in V. fischeri to determine whether each gene has an effect on host colonization, specificity, and biofilm formation. Both squid light organ colonization assays and transmission electron microscopy confirmed differences in host colonization between wild-type and mutant strains, and also demonstrated the importance of both UDPDH and mshA gene expression for successful light organ colonization. This furthers our understanding of the genetic factors playing important roles in this environmentally transmitted symbiosis.     

GacA regulates symbiotic colonization traits of Vibrio fischeri and facilitates a beneficial association with an animal host

The GacS/GacA two-component system regulates the expression of bacterial traits during host association. Although the importance of GacS/GacA as a regulator of virulence is well established, its role in benign associations is not clear, as mutations in either the gacS or gacA gene have little impact on the success of colonization in nonpathogenic associations studied thus far. Using as a model the symbiotic association of the bioluminescent marine bacterium Vibrio fischeri with its animal host, the Hawaiian bobtail squid, Euprymna scolopes, we investigated the role of GacA in this beneficial animal-microbe interaction. When grown in culture, gacA mutants were defective in several traits important for symbiosis, including luminescence, growth in defined media, growth yield, siderophore activity, and motility. However, gacA mutants were not deficient in production of acylated homoserine lactone signals or catalase activity. The ability of the gacA mutants to initiate squid colonization was impaired but not abolished, and they reached lower-than-wild-type population densities within the host light organ. In contrast to their dark phenotype in culture, gacA mutants that reached population densities above the luminescence detection limit had normal levels of luminescence per bacterial cell in squid light organs, indicating that GacA is not required for light production within the host. The gacA mutants were impaired at competitive colonization and could only successfully cocolonize squid light organs when present in the seawater at higher inoculum densities than wild-type bacteria. Although severely impaired during colonization initiation, gacA mutants were not displaced by the wild-type strain in light organs that were colonized with both strains. This study establishes the role of GacA as a regulator of a beneficial animal-microbe association and indicates that GacA regulates utilization of growth substrates as well as other colonization traits.     

Magnesium promotes flagellation of Vibrio fischeri

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, Mg2+. At concentrations naturally present in seawater, Mg2+ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.     

Draft genome sequence of Vibrio fischeri SR5, a strain isolated from the light organ of the Mediterranean squid Sepiola robusta

Here, we describe the draft genome sequence of Vibrio fischeri SR5, a squid symbiotic isolate from Sepiola robusta in the Mediterranean Sea. This 4.3-Mbp genome sequence represents the first V. fischeri genome from an S. robusta symbiont and the first from outside the Pacific Ocean.     

Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization

The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.