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Genetic and biochemical analyses reveal direct interactions between LitR and genes important for Vibrio fischeri physiology, including biofilm production

ABSTRACT: Bacteria can link gene expression to population density to promote group behaviors using quorum sensing. Quorum sensing controls a multitude of bacterial processes such as virulence, motility, and biofilm formation. In Vibrio fischeri, the quorum sensing-dependent transcription factor LitR inhibits biofilm formation. A previous study showed that LitR modestly inhibits transcription of the bcs locus, which comprises the genes responsible for producing the cellulose polysaccharide. However, beyond that, the mechanism of LitR’s inhibitory effect on biofilm formation was unknown. Here, we find that LitR transcriptionally activates pdeV, which encodes a c-di-GMP phosphodiesterase that indirectly promotes cleavage of the large adhesive protein LapV from the surface of V. fischeri, leading to biofilm dispersal. LitR also induces transcription of the sensor kinase VF_A1016, which we determined to be important for biofilm inhibition. Like loss of LitR, loss of VF_A1016 modestly increased bcs transcription. Through chromatin immunoprecipitation sequencing (ChIP-seq), we found that LitR directly binds to the VF_A1016 and pdeV regulatory regions. In total, we identified 147 LitR binding sites in the genome and confirmed LitR’s transcriptional control over a subset of these putative regulatory targets. Of note, we determined that LitR induces transcription of the genes encoding the diguanylate cyclase VF_1200 and the glyoxylate shunt protein AceB and inhibits expression of the putative transcription factor TfoY. LitR also differentially regulates the response regulator ArcA in static and shaking conditions. These data define the mechanism of LitR regulation of genes involved in biofilm formation and the physiology of V. fischeri.

ORGANISM(S): Aliivibrio fischeri

PROVIDER: GSE288643 | GEO | 2025/07/21

REPOSITORIES: GEO

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Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12M-BM-:C. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR. V. salmonicida wild type strain LFI1238 (control) and the isogenic DlitR mutant were grown as statical biofilm in SWT medium, at 4M-BM-0C and harvested after 72 hours. Biological replicates for each sample: 4 wild type, 4 DlitR mutant (including one dye swap), independently grown and harvested. One replicate per array.

Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12M-BM-:C. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR. V. salmonicida wild type strain LFI1238 (control) and the isogenic DlitR deletion mutant were grown in suspension in SWT medium at 8M-BM-0C with 200 rpm and harvested at OD=0.8. Biological replicates for each sample: 4 wild type, 4 DlitR mutant (including one dye swap), independently grown and harvested. One replicate per array.

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 V. 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 that 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 of its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.

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Genetic and biochemical analyses reveal direct interactions between LitR and genes important for Vibrio fischeri physiology, including biofilm production

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