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:Beneficial microbial symbionts are often horizontally acquired by their animal hosts from environmental sources, requiring the symbionts to complete a lifestyle transition from free-living in the environment to association with host tissues. In the model symbiosis between the Hawaiian bobtail squid and its microbial symbiont Vibrio fischeri, one mechanism used to make this transition during host colonization is the formation of biofilm-like aggregates on host mucosa. Extensive work has previously been conducted to isolate the critical factors controlling V. fischeri biofilm formation, yet much remains unknown regarding the full breadth of the biofilm-associated regulon. Here, we probed in vitro models of biofilm formation using transcriptomics, to identify novel regulatory pathways active within biofilms of the V. fischeri type strain ES114. Through comparing the gene-sets which became differentially regulated in multiple biofilm models, we discovered a shared set of 232 genes which demonstrated similar patterns in expression relative to uninduced controls. These genes contained representatives of multiple exopolysaccharide loci, genes involved in flagellar motility, and a diverse collection of other genes. Follow-up analysis suggested that these transcriptomic changes reflected true phenotypic effects, including changes in motility and cyclic-di-GMP production in biofilm-induced backgrounds. Beyond characterizing the shared biofilm response, we additionally profiled the regulatory activity of the sensor kinase RscS. This sensor kinase has previously been characterized to function as a phospho-donor within an established biofilm-inducing phospho-relay, yet our data suggests that RscS moonlights in at least one other phospho-relay that integrates downstream signaling from a homolog of the Vibrio cholerae response regulator VpsR, without a need for its established signaling partners. Overall, this study adds to our understanding of the genes involved in V. fischeri biofilm regulation, while revealing new regulatory pathways branching from previously characterized signaling networks.

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