Fig. 2: Wsp signaling system regulates biofilm formation in response to c-di-GMP.
a Effects of several selected Wsp signaling components on biofilm biomass through combinatorial mutations of the respective encoding genes. ΔwspFΔwspA, ΔwspFΔwspE or ΔwspFΔwspR represent double mutations. b Involvement of the key WspE autokinase residue (H56) or WspR phosphor-accepting residue (D72) in biofilm formation. ΔwspFΔwspE-wspE and ΔwspFΔwspE-wspEH56A represent native wspE or its variant (wspEH56A) that were inserted into the chromosome of ΔwspFΔwspE double mutant; ΔwspFΔwspR-wspR and ΔwspFΔwspR-wspRD72A represent the native wspR and its variant (wspRD72A) that were individually inserted into the chromosome of ΔwspFΔwspR. c The involvement of key WspF phosphor-receiving residue on biofilm biomass. ΔwspF-wspFD53A and ΔwspF-wspFD53E indicate the wspF variation (substitution of phosphor-receiving residue D53 to A53 or E53, respectively), which was individually inserted into the chromosome of wspF mutant. d The role of c-di-GMP concentration in biofilm biomass. ΔwspF (pdeH) denotes ΔwspF carrying plasmid-borne pdeH driven by a constitutive promoter of the vector, which produces active c-di-GMP PDE from E. coli. ΔwspF (pBBR) indicates ΔwspF carrying an empty vector. e The role of key GGEEF motif of WspR in biofilm biomass. ΔwspFΔwspR-wspRGGAAF represents a wspR variant by replacing of GGEEF motif with GGAAF, which was inserted into the chromosome of ΔwspFΔwspR. f WspR, but not the other two potential DGCs (Δ0901 and ΔlchD), mediates WspF signaling in biofilm biomass. Two delta symbols indicate gene double mutants. Statistical comparisons were performed using one-way ANOVA of GraphPad software (GraphPad, La Jolla, CA). In all assays, mean data ± SD from three experiments were shown, **P < 0.01. NS means not significant.
