The twin arginine translocation system contributes to symbiotic colonization of Euprymna scolopes by Vibrio fischeri

In many bacteria, the twin arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane, and these proteins can play a role in symbiotic or pathogenic infections. A role for the Vibrio fischeri Tat system was identified during symbiotic colonization of its host Euprymna scolopes, demonstrating a function for the Tat system in host colonization by a member of the Vibrionaceae. Using bioinformatics, mutant analyses, and green fluorescent protein fusions, a set of Tat-targeted proteins in V. fischeri was identified.     

Vibrio fischeri and its host: it takes two to tango

The association of Vibrio fischeri and Euprymna scolopes provides insights into traits essential for symbiosis, and the signals and pathways of bacteria-induced host development. Recent studies have identified important bacterial colonization factors, including those involved in motility, bioluminescence and biofilm formation. Surprising links between symbiosis and pathogenesis have been revealed through discoveries that nitric oxide is a component of the host defense, and that V. fischeri uses a cytotoxin-like molecule to induce host development. Technological advances in this system include the genome sequence of V. fischeri, an expressed sequence tagged library for E. scolopes and the availability of dual-fluorescence markers and confocal microscopy to probe symbiotic structures and the dynamics of colonization.     

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.     

Colonization of Euprymna scolopes squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities, while the nutrient-rich host tissue provides V. fischeri with a protected niche. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment. Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.     

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.     

NO means 'yes' in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association

During colonization of the Euprymna scolopes light organ, symbiotic Vibrio fischeri cells aggregate in mucus secreted by a superficial ciliated host epithelium near the sites of eventual inoculation. Once aggregated, symbiont cells migrate through ducts into epithelium-lined crypts, where they form a persistent association with the host. In this study, we provide evidence that nitric oxide synthase (NOS) and its product nitric oxide (NO) are active during the colonization of host tissues by V. fischeri. NADPH-diaphorase staining and immunocytochemistry detected NOS, and the fluorochrome diaminofluorescein (DAF) detected its product NO in high concentrations in the epithelia of the superficial ciliated fields, ducts, and crypt antechambers. In addition, both NOS and NO were detected in vesicles within the secreted mucus where the symbionts aggregate. In the presence of NO scavengers, cells of a non-symbiotic Vibrio species formed unusually large aggregates outside of the light organ, but these bacteria did not colonize host tissues. In contrast, V. fischeri effectively colonized the crypts and irreversibly attenuated the NOS and NO signals in the ducts and crypt antechambers. These data provide evidence that NO production, a defense response of animal cells to bacterial pathogens, plays a role in the interactions between a host and its beneficial bacterial partner during the initiation of symbiotic colonization.     

Vibrio fischeri lux genes play an important role in colonization and development of the host light organ

The bioluminescent bacterium Vibrio fischeri and juveniles of the squid Euprymna scolopes specifically recognize and respond to one another during the formation of a persistent colonization within the host's nascent light-emitting organ. The resulting fully developed light organ contains brightly luminescing bacteria and has undergone a bacterium-induced program of tissue differentiation, one component of which is a swelling of the epithelial cells that line the symbiont-containing crypts. While the luminescence (lux) genes of symbiotic V. fischeri have been shown to be highly induced within the crypts, the role of these genes in the initiation and persistence of the symbiosis has not been rigorously examined. We have constructed and examined three mutants (luxA, luxI, and luxR), defective in either luciferase enzymatic or regulatory proteins. All three are unable to induce normal luminescence levels in the host and, 2 days after initiating the association, had a three- to fourfold defect in the extent of colonization. Surprisingly, these lux mutants also were unable to induce swelling in the crypt epithelial cells. Complementing, in trans, the defect in light emission restored both normal colonization capability and induction of swelling. We hypothesize that a diminished level of oxygen consumption by a luciferase-deficient symbiotic population is responsible for the reduced fitness of lux mutants in the light organ crypts. This study is the first to show that the capacity for bioluminescence is critical for normal cell-cell interactions between a bacterium and its animal host and presents the first examples of V. fischeri genes that affect normal host tissue development.     

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.