Microbial factor-mediated development in a host-bacterial mutualism. (1/147)

Tracheal cytotoxin (TCT), a fragment of the bacterial surface molecule peptidoglycan (PGN), is the factor responsible for the extensive tissue damage characteristic of whooping cough and gonorrhea infections. Here, we report that Vibrio fischeri also releases TCT, which acts in synergy with lipopolysaccharide (LPS) to trigger tissue development in its mutualistic symbiosis with the squid Euprymna scolopes. As components of PGN and LPS have commonly been linked with pathogenesis in animals, these findings demonstrate that host interpretation of these bacterial signal molecules is context dependent. Therefore, such differences in interpretation can lead to either inflammation and disease or to the establishment of a mutually beneficial animal-microbe association.  (+info)

Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners. (2/147)

Vibrio fischeri belongs to the Vibrionaceae, a large family of marine gamma-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio, including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes. Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.  (+info)

Magnesium promotes flagellation of Vibrio fischeri. (3/147)

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.  (+info)

Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors. (4/147)

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.  (+info)

Identifying components of the NF-kappaB pathway in the beneficial Euprymna scolopes-Vibrio fischeri light organ symbiosis. (5/147)

The Toll/NF-kappaB pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of Euprymna scolopes was used to identify members of the Toll/NF-kappaB pathway. Full-length transcripts were identified by using 5' and 3' RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-kappaB phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-kappaB pathway in the initiation of the beneficial symbiosis between E. scolopes and Vibrio fischeri.  (+info)

The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. (6/147)

The function of LuxR homologues as quorum sensors is mediated by the binding of N-acyl-L-homoserine lactone (AHL) signal molecules to the N-terminal receptor site of the proteins. In this study, site-directed mutagenesis was carried out of the amino acid residues comprising the receptor site of LuxR from Vibrio fischeri, and the ability of the L42A, L42S, Y62F, W66F, D79N, W94D, V109D, V109T and M135A LuxR mutant proteins to activate green fluorescent protein expression from a P(luxI) promoter was measured. X-ray crystallographic studies of the LuxR homologue TraR indicated that residues Y53 and W57 form hydrogen bonds to the 1-carbonyl group and the ring carbonyl group, respectively, of the cognate AHL signal. Based on the activity and signal specificity of the LuxR mutant proteins, and on molecular modelling, a model is suggested in which Y62 (corresponding to Y53 in TraR) forms a hydrogen bond with the ring carbonyl group rather than the 1-carbonyl group, while W66 (corresponding to W57 in TraR) forms a hydrogen bond to the 1-carbonyl group. This flips the position of the acyl side chain in the LuxR/signal molecule complex compared to the TraR/signal molecule complex. Halogenated furanones from the marine alga Delisea pulchra and the synthetic signal analogue N-(sulfanylacetyl)-L-homoserine lactone can block quorum sensing. The LuxR mutant proteins were insensitive to inhibition by N-(propylsulfanylacetyl)-L-homoserine lactone. In contrast, the mutations had only a minor effect on the sensitivity of the proteins to halogenated furanones, and the data strongly suggest that these compounds do not compete in a 'classic' way with N-3-oxohexanoyl-L-homoserine lactone for the binding site. Based on modelling and experimental data it is suggested that these compounds bind in a non-agonist fashion.  (+info)

New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. (7/147)

Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by approximately 99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.  (+info)

Cyclo(Phe-Pro) modulates the expression of ompU in Vibrio spp. (8/147)

Vibrio vulnificus was found to produce a chemical that induced the expression of Vibrio fischeri lux genes. Electron spray ionization-mass spectrometry and 1H nuclear magnetic resonance analyses indicated that the compound was cyclo(L-Phe-L-Pro) (cFP). The compound was produced at a maximal level when cell cultures reached the onset of stationary phase. Sodium dodecyl sulfate-polyacrylamide gel analysis of the total proteins of V. vulnificus indicated that expression of OmpU was enhanced by exogenously added synthetic or purified cFP. A toxR-null mutant failed to express ompU despite the addition of cFP. The related Vibrio spp. V. cholerae, V. parahaemolyticus, and V. harveyi also produced cFP, which induced the expression of their own ompU genes. cFP also enhanced the expression in V. cholerae of the ctx genes, which are known to be regulated by ToxR. Our results suggest that cFP is a signal molecule controlling the expression of genes important for the pathogenicity of Vibrio spp.  (+info)

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