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 Escherichia coli quorum-sensing regulator, SdiA, belongs to the LuxR family of transcriptional regulators and is responsible for detecting signals from other bacteria. Previously we found that SdiA is necessary for E. coli to control its biofilm formation with indole just as SdiA is necessary for E. coli to alter its biofilm formation in the presence of N-acylhomoserine lactones (AHLs). Here we engineered SdiA by random mutagenesis to further control biofilm formation in the presence of indole and AHLs. After screening of 477?? mutants with indole and two AHLs (N-butyryl-DL-homoserine lactone, and N-(3-oxooctanoyl)-L-homoserine lactone, C6HSL), five SdiA variants were obtained that altered biofilm with and without signals of indole and AHLs. Two truncation variants (1E11 and 14C3) lacking the C-terminal DNA-binding domain of SdiA showed the reduction of biofilm formation by 5-fold and 10-fold in LB and LB glu, respectively. DNA microarrays show that the evolved SdiA (1E11) compared to wild-type SdiA influences indole synthesis genes, AI-2 uptake genes, acid-resistance genes, motility related genes, cold-shock protein genes, and several regulatory protein genes. Corroborating DNA microarrays, SdiA variants produced various amounts of indole which led to different survivals in low pH and influenced swimming motility and final cell density. Also, an AHL sensitive variant (2D10) 2-fold increased biofilm formation in the presence of C6HSL, while another variant (6B12) lowered biofilm formation in the presence of C6HSL. Hence, the results clearly showed that mutation of SdiA itself directly controls biofilm formation and SdiA variants could be further recognized by the inter-species signal AHLs. This is the first random protein engineering to control biofilm formation. Experiment Overall Design: For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL LB ( 1 mM IPTG + Cm30) in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures diluted that were 1:100. The cells were shaken at 250 rpm and 30C for 12 hours to form biofilms on the glass wool, and RNA was isolated from the the biofilm cells.

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