RecA and Specialized Error-Prone DNA Polymerases Are Not Required for Mutagenesis and Antibiotic Resistance Induced by Fluoroquinolones in Pseudomonas aeruginosa

Abstract

To cope with stressful conditions, including antibiotic exposure, bacteria activate the SOS response, a pathway that induces error-prone DNA repair and mutagenesis mechanisms. In most bacteria, the SOS response relies on the transcriptional repressor LexA and the co-protease RecA, the latter being also involved in homologous recombination. The role of the SOS response in stress- and antibiotic-induced mutagenesis has been characterized in detail in the model organism Escherichia coli. However, its effect on antibiotic resistance in the human pathogen Pseudomonas aeruginosa is less clear. Here, we analyzed a recA deletion mutant and confirmed, by conjugation and gene expression assays, that RecA is required for homologous recombination and SOS response induction in P. aeruginosa. MIC assays demonstrated that RecA affects P. aeruginosa resistance only towards fluoroquinolones and genotoxic agents. The comparison of antibiotic-resistant mutant frequency between treated and untreated cultures revealed that, among the antibiotics tested, only fluoroquinolones induced mutagenesis in P. aeruginosa. Notably, both RecA and error-prone DNA polymerases were found to be dispensable for this process. These data demonstrate that the SOS response is not required for antibiotic-induced mutagenesis in P. aeruginosa, suggesting that RecA inhibition is not a suitable strategy to target antibiotic-induced emergence of resistance in this pathogen.

Keywords: DinB; ImuBC; PolB; SOS response; homologous recombination; induced mutagenesis; resistance.

Figure 1

Confirmation of the essential role of RecA in homologous recombination and activation of the SOS response in P. aeruginosa. (a) Frequency of transconjugants obtained for P. aeruginosa PAO1, the ΔrecA mutant, and the complemented strain ΔrecA recA⁺ upon conjugation-mediated acquisition of the replicative plasmid pFLP2, the self-integrative suicide plasmid mini-CTX1 or the suicide plasmid pDM4ΔrsmA, that requires homologous recombination for chromosome integration. Frequencies are expressed as transconjugant CFUs/recipient CFUs ratio. (b) Relative mRNA levels of selected SOS response genes (recN, recX, lexA, and imuB) determined by qRT-PCR in P. aeruginosa PAO1 and the ΔrecA mutant cultured in MH in the absence or in the presence of 0.25 × MIC ciprofloxacin (CIP) (0.031 and 0.008 μg/mL for PAO1 and ΔrecA, respectively; Table 1). Values are the mean (± SD) of three independent experiments. Asterisks indicate a statistically significant increase in relative gene expression with respect to the untreated control (unpaired t test: ** p < 0.01; *** p < 0.001).

Figure 2

Fluoroquinolones induce mutagenesis in a RecA-independent manner in P. aeruginosa. Frequency of antibiotic-resistant mutants obtained on selective agar plates containing (a) gentamicin (GEN) at 20 × MIC, (b) colistin (COL) at 5 × MIC, (c) ofloxacin (OFX) at 5 × MIC, or (d) fosfomycin (FOS) at 4 × MIC, for P. aeruginosa PAO1 and the ΔrecA mutant. Strains were precultured in MH at 37 °C in the absence or in the presence of GEN, COL, OFX or ciprofloxacin (CIP) at 0.25 × MIC (as indicated below each graph). Values are the mean (± SD) of at least five independent experiments. Asterisks indicate a statistically significant difference in the frequency of mutants with respect to the corresponding untreated control (unpaired t test: * p < 0.05; ** p < 0.01; *** p < 0.001). No statistically significant differences were observed between PAO1 and ΔrecA.

Figure 3

Specialized DNA polymerases are not required for fluoroquinolone-induced mutagenesis in P. aeruginosa. (a) Relative mRNA levels of the DNA polymerase genes dinB, polB, polA, and polC, determined by qRT-PCR, in P. aeruginosa PAO1 and the ΔrecA mutant cultured in MH in the absence or in the presence of 0.25 × MIC ciprofloxacin (CIP) (0.031 and 0.008 μg/mL for PAO1 and ΔrecA, respectively; Table 1). Values are the mean (± SD) of three independent experiments. Asterisks indicate a statistically significant difference in the frequency of mutants with respect to the corresponding untreated control (unpaired t test: * p < 0.05; ** p < 0.01). (b) Frequency of gentamicin (GEN) resistant mutants obtained on agar plates containing GEN at 20 × MIC for P. aeruginosa PAO1, the ΔrecA mutant, and the cognate polB, dinB, and imuC mutants. Strains were precultured in MH at 37 °C in the absence or in the presence of CIP at 0.25 × MIC. Values are the mean (± SD) of four independent experiments. No statistically significant differences between strains were observed under the same experimental condition (absence or presence of CIP in the preinoculum).