RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin

Abstract

We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation.IMPORTANCE Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli, the Gram-negative bacterial paradigm, and V. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage.

Keywords: DNA repair; R-loop; antibiotic resistance.

FIG 1

Growth of E. coli mutants in the presence of TOB at 50% of the MIC (0.25 μg/ml). Growth was measured with the Tecan Infinite plate reader. MH is rich medium without antibiotic. Each condition was tested 3 to 5 times. Standard deviations are represented. Statistical significance tests were performed on the slopes, and P values are represented in Table S2.

FIG 2

Quantification of DNA double-strand breaks in E. coli. TUNEL assays were performed (see Materials and Methods), and fluorescence was measured by flow cytometry (MACSQuant). Standard deviations are represented. Statistical significance tests (t tests) were performed, and P values are represented in Table S2. MH, no antibiotic; TOB, 0.2 μg/ml; p0, empty pTOPO vector; pRNH, pTOPO::rnhA_ec (plasmids pB352 and pI388 are shown in Table S1).

FIG 3

Effect of RNase H overexpression on growth of E. coli mutants in recB-deficient context. Growth was measured with the Tecan Infinite plate reader. MH is rich medium without antibiotic. An 0.2-μg/ml concentration of TOB was used in the recB-deficient context (instead of 0.25 μg/ml) because of decreased viability of recB mutants. Each condition was tested at least 3 times. Standard deviations are represented. Statistical significance tests were performed on the slopes, and P values are represented in Table S2. Plasmids are as in Fig. 2.

FIG 4

Effect of RadD in R-loop-dependent stable DNA replication in E. coli dnaA(Ts) mutant. Cultures were started at 30°C and were kept at 30°C (permissive) or shifted at 42°C (nonpermissive temperature) at time zero. (A, B, and D) Numbers of CFU are represented over time after time zero (hours). When a plasmid was present, carbenicillin (100 μg/ml) was added to the medium. (C) Overnight cultures were plated at 30°C and 42°C, and the ratios of CFU are shown. pempty, empty pTOPO vector; prnhA_eco, pradD_eco, pradD_vch, and pVC0498, plasmids expressing the corresponding genes (plasmids pB352, I388, I605, I468, and I391 are shown in Table S1).

FIG 5

RadD counteracts formation of DSBs arising from R-loops. We propose that sub-MIC TOB impedes translation, leading to transcription defects, thus enhancing R-loops/R-lesions at transcription sites, causing DSBs that are repaired by the RecBCD pathway. We hypothesize that RadD (possibly with RecQ) acts either at the level of translation-transcription coupling for the avoidance of R-loop formation or directly at the R-loop before DSBs arise. Shown in parentheses are genes that are mentioned in the text and steps where they could be involved.