The SOS and RpoS Regulons Contribute to Bacterial Cell Robustness to Genotoxic Stress by Synergistically Regulating DNA Polymerase Pol II

Abstract

Mitomycin C (MMC) is a genotoxic agent that induces DNA cross-links, DNA alkylation, and the production of reactive oxygen species (ROS). MMC induces the SOS response and RpoS regulons in Escherichia coli SOS-encoded functions are required for DNA repair, whereas the RpoS regulon is typically induced by metabolic stresses that slow growth. Thus, induction of the RpoS regulon by MMC may be coincidental, because DNA damage slows growth; alternatively, the RpoS regulon may be an adaptive response contributing to cell survival. In this study, we show that the RpoS regulon is primarily induced by MMC-induced ROS production. We also show that RpoS regulon induction is required for the survival of MMC-treated growing cells. The major contributor to RpoS-dependent resistance to MMC treatment is DNA polymerase Pol II, which is encoded by the polB gene belonging to the SOS regulon. The observation that polB gene expression is controlled by the two major stress response regulons that are required to maximize survival and fitness further emphasizes the key role of this DNA polymerase as an important factor in genome stability.

Keywords: DNA polymerase II; Escherichia coli; RpoS; SOS; mitomycin C.

Figure 1

Induction of the RpoS regulon with MMC. The general stress response regulated by RpoS is induced during exponential growth of bacteria in response to the DNA-damaging agent MMC. (A) RS carrying RpoS-mCherry protein fusion was grown in LB (black line) or LB supplemented with MMC (red line). (B) Expression of the RpoS-regulated pbolA-GFP reporter in the RS. An increase in fluorescence between OD₆₀₀ 0.2 and 0.4 in MMC-treated and NT cells is shown. (C) Catalase activity of the RpoS-regulated monofunctional catalase HPII encoded by katE in different genetic backgrounds. All measured catalase activities were normalized with respect to that of the NT RS. For all measures, each point represents the mean ± SEM of at least three independent experiments. MMC, treated with 1 µg ml⁻¹ MMC. ** P < 0.01, *** P < 0.001, two-tailed Student’s t-test. MMC, mitomycin C; NT, nontreated; RS, reference strain.

Figure 2

Role of the two-component system BarA-UvrY in RpoS regulon activation. An increase in RpoS-mCherry protein fusion expression between OD₆₀₀ 0.2 and 0.4 in mitomycin C (MMC)-treated and nontreated (A) reference (RS) and ΔiraD strains, and (B) the ΔbarA and ΔuvrY strains. Catalase activity of the RpoS-regulated monofunctional catalase HPII encoded by katE in MMC-treated and NT (C) RS and ΔiraD strains, and (D) in the ΔbarA and ΔuvrY strains. All measured catalase activities were normalized to that of the NT RS. For all results, each point represents the mean ± SEM of at least three independent experiments. MMC, treated with 1 µg ml⁻¹ MMC. *P < 0.05, two-tailed Student’s t-test.

Figure 3

MMC generates oxidative stress in treated cells. (A) Growth of the ΔkatG ΔkatE ΔahpCF strain in LB in the presence or absence of MMC, and with or without the addition of the antioxidant AO. (B) Expression of the RpoS-mCherry protein fusion in the reference strain (RS) grown in LB in the presence or absence of MMC, and with or without the addition of the antioxidant thiourea. Each curve represents the mean ± SEM of at least three independent experiments. AO, addition of 50 mM thiourea. MMC, treated with 1 µg ml⁻¹ mitomycin C; NT, nontreated.

Figure 4

Effect of RpoS-regulon induction on the survival and mutagenesis of MMC-treated cells. (A) Survival of the MMC-treated RS, ΔrpoS, and ΔrssB strains. For a dose of 4 µg ml⁻¹ of MMC, the following differences were significant: RS vs. ΔrpoS, *** P; ΔrssB vs. ΔrpoS, **P. For a dose of 5 µg ml⁻¹ of MMC, the following differences were significant: RS vs. ΔrpoS, * P; ΔrssB vs. ΔrpoS, * P. * P < 0.05, ** P < 0.01, and *** P < 0.001, two-tailed Student’s t-test. (B) The effect of the RpoS regulon on MMC-induced mutagenesis. The frequency of Rif^R mutants arising in MMC-treated cells was measured. Each result represents the mean ± SEM of at least 18 independent cultures in at least three independent experiments. **P < 0.01, ***P < 0.001, nonparametric Mann-Whitney U test. MMC, treated with 1 µg ml⁻¹ mitomycin C; NT, nontreated; Rif^R, rifampicin-resistant; RS, reference strain.

Figure 5

Expression of the polB gene encoding SOS regulated Pol II is under RpoS regulation. polB, but not genes encoding the other two SOS-dependent polymerases, dinB and umuD, are under RpoS regulation in the exponentially growing phase during MMC treatment. An increase in green fluorescence levels during growth of the RS, ΔrpoS, and ΔrssB strains carrying reporter fusions. The bars represent the ratio of the fluorescence at OD₆₀₀ 0.2 and 0.4. (A) Expression of the polB promoter fusion to fluorescent protein. (B) Expression of the dinB promoter fusion to fluorescent protein. (C) Expression of the umuD promoter fusion to fluorescent protein. (D) Expression of the polB promoter fusion to fluorescent protein in a constitutively induced SOS background, lexA(def). (E) Expression of the polB promoter fusion to fluorescent protein in ΔbarA and ΔrpoS mutants. For all experiments, each point represents the mean ± SEM of at least three independent experiments. *** P < 0.001, two-tailed Student’s t-test. MMC, treated with 1 µg ml⁻¹ mitomycin C; NT, nontreated; RS, reference strain.

Figure 6

Pol II is responsible for the RpoS-adaptive response during the genotoxic stress caused by MMC. Survival of different MMC-treated strains. (A) For a dose of 4 µg ml⁻¹ of MMC, the following differences were significant: RS vs. ΔpolB, ** P; RS vs. ΔrssB ΔpolB, *** P; ΔrssB vs. ΔpolB, * P; ΔrssB vs. ΔrssB ΔpolB, ** P. For a dose of 5 µg ml⁻¹ of MMC, the following differences were significant: RS vs. ΔpolB, * P; RS vs. ΔrssB ΔpolB, * P. (B) Susceptibility of the Pol II-complemented strains to MMC. For a dose of 4 µg ml⁻¹ of MMC, the following differences were significant: ΔrpoS vs. ΔrpoS+ppolB, * P; ΔpolB vs. ΔpolB+ppolB, * P. For a dose of 5 µg ml⁻¹ of MMC, the following differences were significant: ΔrpoS vs. ΔrpoS+ppolB, * P. Relevant genotypes are indicated. Each value represents the mean ± SEM of at least three independent experiments. * P < 0.05, ** P < 0.01, and ***P < 0.001, two-tailed Student’s t-test. MMC, treated with 1 µg ml⁻¹ mitomycin C; NT, nontreated; RS, reference strain.