Figure 6. Cell survival under conditions of excessive backtracking depends on error-prone DSBs repair.
(A) SOS response activation by sub-lethal amounts of Cm and BCM in the presence or absence of GreB. Cm induced fluorescence in recA’::gfp (wt), and both Cm and BCM induced in recA’::gfp ΔgreB. SOS caused by Cm was suppressed by IPTG-induced overexpression of GreB (recA’::gfp ΔgreB(pGreB) cells). In each case, the basal level of fluorescence before induction was taken as 100%. Values are the mean ±SD from four experiments.
(B) GreB-deficient cells are more sensitive to Cm (top) and NA (bottom). Numbers indicate the antibiotic concentrations (μg/ml).
(C) Survival of RNAP backtracking-prone cells depends on the DSBs repair machinery. Exponentially grown wild type and mutant E. coli cells were challenged with Cm (100 μg/ml) for indicated time periods, washed, and then plated for overnight incubation to determine CFUs. The fraction of surviving cells (%) in relation to wild type is shown as the mean ±SD from three independent experiments.
(D) Mutation frequency increases in GreB deficient cells. Wild type and mutant strains grew to OD 0.6 and then spread over LB-agar surface containing 30 μg/ml of rifampicin (rif). Plates were incubated at 30°C for 24 hours to detect RifR colonies. CFU/ml was calculated by spreading serially diluted cultures over LB-agar plates. The estimated mutation frequency is shown as the mean ±SD from three independent experiments.