The N-acetyl-D-glucosamine repressor NagC of Vibrio fischeri facilitates colonization of Euprymna scolopes.
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ABSTRACT:
SUBMITTER: Miyashiro T
PROVIDER: S-EPMC3212624 | biostudies-literature | 2011 Nov
REPOSITORIES: biostudies-literature
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The N-acetyl-D-glucosamine repressor NagC of Vibrio fischeri facilitates colonization of Euprymna scolopes.
Miyashiro Tim T Klein Will W Oehlert Dane D Cao Xiaodan X Schwartzman Julia J Ruby Edward G EG
Molecular microbiology 20111012 4
To successfully colonize and persist within a host niche, bacteria must properly regulate their gene expression profiles. The marine bacterium Vibrio fischeri establishes a mutualistic symbiosis within the light organ of the Hawaiian squid, Euprymna scolopes. Here, we show that the repressor NagC of V. fischeri directly regulates several chitin- and N-acetyl-D-glucosamine-utilization genes that are co-regulated during productive symbiosis. We also demonstrate that repression by NagC is relieved ...[more]
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Project description:The marine bacterium Vibrio fischeri is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes, and the establishment of this association involves a number of signaling pathways and transcriptional responses between both partners. We report here the first full RNA-Seq dataset representing host-associated V. fischeri cells from colonized juvenile E. scolopes, as well as comparative transcriptomes under both laboratory and simulated marine planktonic conditions. These data elucidate the broad transcriptional changes that these bacteria undergo during the early stages of symbiotic colonization. We report several previously undescribed and unexpected transcriptional responses within the early stages of this symbiosis, including gene expression patterns consistent with biochemical stresses inside the host, and metabolic patterns distinct from those reported in associations with adult animals. Integration of these transcriptional data with a recently developed metabolic model of V. fischeri provides us with a clearer picture of the metabolic state of symbionts within the juvenile host, including their possible carbon sources. Taken together, these results expand our understanding of the early stages of the squid-vibrio symbiosis, and more generally inform the transcriptional responses underlying the activities of marine microbes during host colonization.
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Project description: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.
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Project description:The light organ of the squid Euprymna scolopes is specifically colonized to a high density by the marine bacterium Vibrio fischeri. To date, only a few factors contributing to the specificity of this symbiosis have been identified. Using a genetic screen for random transposon mutants defective in initiating the symbiotic association or in colonizing the light organ to high density, we identified a mutant of V. fischeri that exhibited an apparent defect in symbiosis initiation. This mutant was not defective in motility, luminescence, or growth in minimal medium, suggesting that it lacks an essential, previously unidentified symbiotic function. By sequence analysis, we showed that the locus inactivated in this mutant encodes a predicted 927-amino-acid protein with a high degree of similarity to the sensor component of hybrid two-component regulatory systems. We have therefore designated this locus rscS, for regulator of symbiotic colonization-sensor. Sequence analysis revealed two hydrophobic regions which may result in the formation of a periplasmic loop involved in signal recognition; PhoA fusion data supported this proposed membrane topology. We have investigated the start site of rscS transcription by primer extension and identified a putative promoter region. We hypothesize that RscS recognizes a signal associated with the light organ environment and responds by stimulating a putative response regulator that controls protein function or gene expression to coordinate early colonization events. Further studies on RscS, its cognate response regulator, and the signaling conditions will provide important insight into the interaction between V. fischeri and E. scolopes.
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Project description:Sulfur is in cellular components of bacteria and is, therefore, an element necessary for growth. However, mechanisms by which bacteria satisfy their sulfur needs within a host are poorly understood. Vibrio fischeri is a bacterial symbiont that colonizes, grows, and produces bioluminescence within the light organ of the Hawaiian bobtail squid, which provides an experimental platform for investigating sulfur acquisition in vivo. Like other γ-proteobacteria, V. fischeri fuels sulfur-dependent anabolic processes with intracellular cysteine. Within the light organ, the abundance of a ΔcysK mutant, which cannot synthesize cysteine through sulfate assimilation, is attenuated, suggesting sulfate import is necessary for V. fischeri to establish symbiosis. Genes encoding sulfate-import systems of other bacteria that assimilate sulfate were not identified in the V. fischeri genome. A transposon mutagenesis screen implicated YfbS as a sulfate importer. YfbS is necessary for growth on sulfate and in the marine environment. During symbiosis, a ΔyfbS mutant is attenuated and strongly expresses sulfate-assimilation genes, which is a phenotype associated with sulfur-starved cells. Together, these results suggest V. fischeri imports sulfate via YfbS within the squid light organ, which provides insight into the molecular mechanisms by which bacteria harvest sulfur in vivo.
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Project description:BackgroundBiologists are becoming increasingly aware that the interaction of animals, including humans, with their coevolved bacterial partners is essential for health. This growing awareness has been a driving force for the development of models for the study of beneficial animal-bacterial interactions. In the squid-vibrio model, symbiotic Vibrio fischeri induce dramatic developmental changes in the light organ of host Euprymna scolopes over the first hours to days of their partnership. We report here the creation of a juvenile light-organ specific EST database.ResultsWe generated eleven cDNA libraries from the light organ of E. scolopes at developmentally significant time points with and without colonization by V. fischeri. Single pass 3' sequencing efforts generated 42,564 expressed sequence tags (ESTs) of which 35,421 passed our quality criteria and were then clustered via the UIcluster program into 13,962 nonredundant sequences. The cDNA clones representing these nonredundant sequences were sequenced from the 5' end of the vector and 58% of these resulting sequences overlapped significantly with the associated 3' sequence to generate 8,067 contigs with an average sequence length of 1,065 bp. All sequences were annotated with BLASTX (E-value < -03) and Gene Ontology (GO).ConclusionBoth the number of ESTs generated from each library and GO categorizations are reflective of the activity state of the light organ during these early stages of symbiosis. Future analyses of the sequences identified in these libraries promise to provide valuable information not only about pathways involved in colonization and early development of the squid light organ, but also about pathways conserved in response to bacterial colonization across the animal kingdom.
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Project description: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.
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Project description:Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg2+ to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide V. fischeri with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide Bradyrhizobium ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.
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