Figure 2
From: Type I toxin-dependent generation of superoxide affects the persister life cycle of Escherichia coli
TisB expression induces the SoxRS regulon. An attenuated TisB variant was generated by amino acid exchange of lysine 12 to leucine. Ectopic expression of wild-type TisB and TisB-K12L was induced by 0.2% L-arabinose during mid-exponential growth. The empty vector (pBAD) was used as control. (a) Growth curves were recorded by monitoring OD600 in 30-min time intervals. At time point 120 min expression was induced by 0.2% L-arabinose (arrow). Data points represent the mean and error bars depict the standard deviation (n = 3). (b) Cellular depolarization was measured by staining with DiBAC4(3) at time points as indicated. Fluorescence signals were OD600-normalized. Data represents the mean and error bars depict the standard deviation (n = 3). For statistical analysis two-way ANOVA with post-hoc Tukey HSD was performed. Significance levels are indicated (ns: not significant, **P < 0.01). (c) Total RNA from toxin-overexpressing cultures was isolated at the indicated time points and analyzed on RNA quality gels. (d,e) Cultures in mid-exponential phase were either subjected to (d) TisB-K12L overexpression for 60 min or (e) CCCP treatment for 30 min. Total RNA was isolated from pre- and post-treatment cultures and analyzed by qRT-PCR to calculate log2 fold changes of relative transcript levels (RTL) for selected genes. Data represents the mean and error bars depict the standard deviation (n = 3). For statistical analysis two-way ANOVA with post-hoc Tukey HSD was performed. Significance levels for pre- versus post-treatment samples are indicated (ns: not significant, **P < 0.01). (f) ROS measurements in strains with impaired detoxification of superoxide or hydrogen peroxide. Toxins TisB and TisB-K12L were overexpressed for 60 min and H2DCFDA measurements performed with 0 min and 60 min samples. DCF fluorescence signals were measured in a microplate reader and OD600-normalized to calculate fold changes. Hpx− denotes a ΔkatG ΔkatE ΔahpF and SodAB− a ΔsodA ΔsodB deletion strain. Data represents the mean and error bars depict the standard deviation (n ≥ 3). For statistical analysis two-way ANOVA with post-hoc Tukey HSD was performed. Significance levels are indicated (ns: not significant, *P < 0.05, **P < 0.01).
