Fig. 1: Intact Cpx-signalling is crucial for Yptb-YPIII biofilm formation on abiotic surface.

Dynamic biofilm formation was analysed in 96-well round-bottom microtiter plates. A serial dilution-based scheme (6-fold, 9 times) was applied to monitor development of sessile biofilm and growth of planktonic cells from the LB-cultured (24 h incubation at 26 °C, 125 rpm shaking) strains of Yptb-YPIII. Each dot in the graph represents a different dilution of the same culture. Parental, wild-type (WT) or mutants within the Cpx-signalling were seeded either at 0.1 OD₆₀₀ or both at 0.1 and 1.0 OD₆₀₀. (a) Loss of CpxA (ΔcpxA null-mutant) prevents biofilm formation. (b) Poor growth of the ΔcpxA null-mutant does not account for this biofilm formation defect. (c) Biofilm formation is restored to the ΔcpxA null-mutant via ectopic expression of wild-type copy of cpxA in ΔcpxA null-mutant of the Cpx-signalling. Error bars on the graphs represent standard error of mean from three biological and three technical replicates of each strain. Statistical differences between sessile biofilm formation of each strain uses a total biofilm biomass counts (represented by the area under each curve) in comparison with a respective reference control (WT for a, WT_01 OD for b and WT/Evct for c). Standard error of mean for each strain was calculated from three independent biological experiments containing three technical replicates. Extent of significance from the parent or negative control was determined using One-way ANOVA with Tukey’s multiple comparisons test, with a single pooled variance. The difference in variance with a p-value of <0.05 was considered significant. The p-values are indicated by <0.001 (***), <0.01 (**) and > 0.05 (ns).