Tolerance of Escherichia coli to fluoroquinolone antibiotics depends on specific components of the SOS response pathway

Abstract

Bacteria exposed to bactericidal fluoroquinolone (FQ) antibiotics can survive without becoming genetically resistant. Survival of these phenotypically resistant cells, commonly called "persisters," depends on the SOS gene network. We have examined mutants in all known SOS-regulated genes to identify functions essential for tolerance in Escherichia coli. The absence of DinG and UvrD helicases and the Holliday junction processing enzymes RuvA and RuvB leads to a decrease in survival. Analysis of the respective mutants indicates that, in addition to repair of double-strand breaks, tolerance depends on the repair of collapsed replication forks and stalled transcription complexes. Mutation in recF results in increased survival, which identifies RecAF recombination as a poisoning mechanism not previously linked to FQ lethality. DinG acts upstream of SOS promoting its induction, whereas RuvAB participates in repair only. UvrD directly promotes all repair processes initiated by FQ-induced damage and prevents RecAF-dependent misrepair, making it one of the crucial SOS functions required for tolerance.

Keywords: DNA repair; SOS response; fluoroquinolones; persisters; tolerance.

Figure 1

Survival of lon and sulA mutants after ciprofloxacin challenge. Strains were challenged with 0.1 μg/ml ciprofloxacin for 6 hr in exponential phase. CFU counts were determined by plating. The data are averages of three independent experiments, and error bars indicate standard error.

Figure 2

Survival of the wild-type and mutant strains in exponential and stationary phase. (A–C) Exponentially growing cells were treated with 0.1 μg/ml ciprofloxacin for 6 hr. Survival was determined by dilution and plating to count CFU. The graph represents at least 20 independent experiments, and error bars represent standard error. (D–F) Stationary-phase cultures were treated with 1 μg/ml ciprofloxacin. Viable counts were determined by plating. The graphs represent three independent experiments. Standard error is indicated by the error bars. wt, wild type.

Figure 3

Survival of dinG, uvrD, and ruvAB mutants (A, B, C, respectively) in a strain constitutively expressing SOS. Exponentially growing cells were treated with 0.1 μg/ml ciprofloxacin. Mutants were made in a strain constitutively expressing SOS [lexA(Def)]. Graphs represent 10 independent experiments, and error bars represent standard error.

Figure 4

SOS induction during ciprofloxacin treatment. Induction of SOS response was measured by assaying for β-galactosidase activity in strains carrying lacZ under control of the recA SOS-inducible promoter after 15 min of treatment with ciprofloxacin with the concentration indicated on the x-axis. (A) dinG mutant. (B) uvrD mutant. Graphs are an average of at least three experiments, and error bars represent standard error.

Figure 5

Single-cell analysis of SOS induction during ciprofloxacin treatment. Induction of SOS response was measured using a flow cytometry by following GFP expression in strains carrying gfp gene under control of the sulA SOS-inducible promoter. Exponential-phase cells were treated with 0.1 μg/ml ciprofloxacin. A total of 10,000 cells were analyzed before ciprofloxacin treatment and again at 120 and 240 min for (A) a wild type (wt), (B) a dinG, and (C) a uvrD mutant.

Figure 6

The role of HJ processing in persistence to fluoroquinolones. Exponentially growing cultures of cells were exposed to 0.1 μg/ml ciprofloxacin. CFU counts were determined by serial dilution and then plating. Error bars represent standard error. The data represent 10 independent experiments.

Figure 7

Role of DinG in survival under different growth conditions. Exponentially growing cultures of dinG mutant and wild type were treated with 0.1 μg/ml of ciprofloxacin for 6 hr, and then CFU counts were determined by serial dilution and plating. (A) Level of persistence in different minimal media; the medium type and generation time in minutes are indicated on the x-axis. (B) Different temperature regimes after 6 hr of treatment. Error bars represent standard error, and graphs represent four independent experiments.

Figure 8

The role of RecF in survival to ciprofloxacin treatment. Cells were diluted and grown to late exponential phase and then treated with 0.1 μg/ml of ciprofloxacin. Data are from at least 20 independent experiments, and error bars represent standard error.

Figure 9

UvrD overproduction suppresses SOS deficiency. Exponentially growing cultures were treated with 0.1 mM IPTG to induce UvrD vector expression 30 min prior to the addition of 0.1 μg/ml ciprofloxacin. The data are an average of at least five experiments, and error bars represent standard error.

Figure 10

Model of cellular events following exposure to fluoroquinolones. See Discussion for detailed explanation.