The haem-uptake gene cluster in Vibrio fischeri is regulated by Fur and contributes to symbiotic colonization.
ABSTRACT: Although it is accepted that bacteria-colonizing host tissues are commonly faced with iron-limiting conditions and that pathogenic bacteria often utilize iron from host-derived haem-based compounds, the mechanisms of iron acquisition by beneficial symbiotic bacteria are less clear. The bacterium Vibrio fischeri mutualistically colonizes the light organ of the squid Euprymna scolopes. Genome sequence analysis of V. fischeri revealed a putative haem-uptake gene cluster, and through mutant analysis we confirmed this cluster is important for haemin use by V. fischeri in culture. LacZ reporter assays demonstrated Fur-dependent transcriptional regulation of cluster promoter activity in culture. GFP-based reporter assays revealed that gene cluster promoter activity is induced in symbiotic V. fischeri as early as 14 h post inoculation, although colonization assays with the haem uptake mutant suggested an inability to uptake haem does not begin to limit colonization until later stages of the symbiosis. Our data indicate that the squid light organ is a low iron environment and that haem-based sources of iron are used by symbiotic V. fischeri cells. These findings provide important additional information on the availability of iron during symbiotic colonization of E. scolopes by V. fischeri, as well as the role of haem uptake in non-pathogenic host-microbe interactions.
Project description:Microbial symbioses are essential for the normal development and growth of animals. Often, symbionts must be acquired from the environment during each generation, and identification of the relevant symbiotic partner against a myriad of unwanted relationships is a formidable task. Although examples of this specificity are well-documented, the genetic mechanisms governing it are poorly characterized. Here we show that the two-component sensor kinase RscS is necessary and sufficient for conferring efficient colonization of Euprymna scolopes squid by bioluminescent Vibrio fischeri from the North Pacific Ocean. In the squid symbiont V. fischeri ES114, RscS controls light-organ colonization by inducing the Syp exopolysaccharide, a mediator of biofilm formation during initial infection. A genome-level comparison revealed that rscS, although present in squid symbionts, is absent from the fish symbiont V. fischeri MJ11. We found that heterologous expression of RscS in strain MJ11 conferred the ability to colonize E. scolopes in a manner comparable to that of natural squid isolates. Furthermore, phylogenetic analyses support an important role for rscS in the evolution of the squid symbiosis. Our results demonstrate that a regulatory gene can alter the host range of animal-associated bacteria. We show that, by encoding a regulator and not an effector that interacts directly with the host, a single gene can contribute to the evolution of host specificity by switching 'on' pre-existing capabilities for interaction with animal tissue.
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.
Project description:The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the Vibrio cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production.IMPORTANCE TcpP homologs are widespread throughout the Vibrio genus; however, the only protein in this family described thus far is a V. cholerae virulence regulator. Here, we show that HbtR, the TcpP homolog in V. fischeri, has both a biological role and regulatory pathway completely unlike those in V. cholerae Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of the E. scolopes light organ. While LitR becomes activated within the crypts and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion of V. fischeri cells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival of V. fischeri outside its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.
Project description:Flagella are essential and multifunctional nanomachines that not only move symbionts towards their tissue colonization site, but also play multiple roles in communicating with the host. Thus, untangling the activities of flagella in reaching, interacting, and signaling the host, as well as in biofilm formation and the establishment of a persistent colonization, is a complex problem. The squid-vibrio system offers a unique model to study the many ways that bacterial flagella can influence a beneficial association and, generally, other bacteria-host interactions. Vibrio fischeri is a bioluminescent bacterium that colonizes the Hawaiian bobtail squid, Euprymna scolopes. Over the last 15 years, the structure, assembly, and functions of V. fischeri flagella, including not only motility and chemotaxis, but also biofilm formation and symbiotic signaling, have been revealed. Here we discuss these discoveries in the perspective of other host-bacteria interactions.
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.
Project description: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.
Project description:Host immune and physical barriers protect against pathogens but also impede the establishment of essential symbiotic partnerships. To reveal mechanisms by which beneficial organisms adapt to circumvent host defenses, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri by colonization and growth within the light organs of the squid Euprymna scolopes. Serial squid passaging of bacteria produced eight distinct mutations in the binK sensor kinase gene, which conferred an exceptional selective advantage that could be demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion that were mediated by cell-associated matrices including symbiotic polysaccharide (Syp) and cellulose. binK variation also altered quorum sensing, raising the threshold for luminescence induction. Preexisting coordinated regulation of symbiosis traits by BinK presented an efficient solution where altered BinK function was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations.
Project description:Environmentally acquired beneficial associations are comprised of a wide variety of symbiotic species that vary both genetically and phenotypically, and therefore have differential colonization abilities, even when symbionts are of the same species. Strain variation is common among conspecific hosts, where subtle differences can lead to competitive exclusion between closely related strains. One example where symbiont specificity is observed is in the sepiolid squid-Vibrio mutualism, where competitive dominance exists among V. fischeri isolates due to subtle genetic differences between strains. Although key symbiotic loci are responsible for the establishment of this association, the genetic mechanisms that dictate strain specificity are not fully understood. We examined several symbiotic loci (lux-bioluminescence, pil?=?pili, and msh-mannose sensitive hemagglutinin) from mutualistic V. fischeri strains isolated from two geographically distinct squid host species (Euprymna tasmanica-Australia and E. scolopes-Hawaii) to determine whether slight genetic differences regulated host specificity. Through colonization studies performed in naïve squid hatchlings from both hosts, we found that all loci examined are important for specificity and host recognition. Complementation of null mutations in non-native V. fischeri with loci from the native V. fischeri caused a gain in fitness, resulting in competitive dominance in the non-native host. The competitive ability of these symbiotic loci depended upon the locus tested and the specific squid species in which colonization was measured. Our results demonstrate that multiple bacterial genetic elements can determine V. fischeri strain specificity between two closely related squid hosts, indicating how important genetic variation is for regulating conspecific beneficial interactions that are acquired from the environment.
Project description:Symbiosis between the bobtail squid Euprymna scolopes (Mollusca: Cephalopoda) and Vibrio fischeri bacteria has been a well-studied model for understanding the molecular mechanisms of colonization and adherence to host cells. For example, pilin expression has been observed to cause subtle variation in colonization for a number of Gram-negative bacteria with eukaryotic hosts. To investigate variation amongst pil genes of closely related strains of vibrios, we amplified pil genes A, B, C and D to determine orientation and sequence similarity to other symbiotic vibrios. The pilA gene was found to be upstream from all other pil genes, and not contiguous with the rest of the operon. The pilB, pilC and pilD loci were flanked at the 3' end by yacE, followed by a conserved hypothetical gene. DNA sequences of each pil gene were aligned and analysed phylogenetically using parsimony for both individual and combined gene trees. Results demonstrate that certain pil loci (pilB and pilD) are conserved among strains of V. fischeri, but pilC differs in sequence between symbiotic and free-living strains. Phylogenetic analysis of all pil genes gives better resolution of Indo-west Pacific V. fischeri symbionts compared with analysis of the 16S rRNA gene. Hawaiian and Australian symbiotic strains form one monophyletic tree, supporting the hypothesis that V. fischeri strain specificity is selected by the geographical location of their hosts and is not related to specific squid species.
Project description:The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization.