Contribution of reactive oxygen species to thymineless death in Escherichia coli

Abstract

Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.

Figure 1. Association of ROS with TLD

a, ROS accumulation in a ΔthyA mutant (strain 3640). T-starvation extended for the indicated times; ROS accumulation was monitored by flow cytometry using carboxy-H2DCFDA-mediated fluorescence. b, Inhibition of TLD. Rapid death of a thymidine-starved ΔthyA culture (squares) was reduced by 2,2’-bipyridyl (Bip, triangles) or dimethyl sulfoxide (DMSO, circles), each at 0.5 × MIC. Bipyridyl at 0.5 × MIC slightly suppressed growth, but such growth inhibitory effects cannot account for the large protection from TLD (see Supplementary Fig. 14 for details). c, H₂O₂ accumulation during TLD. H₂O₂ was monitored at the indicated times after initiation of T-starvation by flow cytometry using Peroxy Orange-1-mediated fluorescence. d, Lipid peroxidation during TLD. Lipid peroxidation at the indicated times was detected by flow cytometry using C11-BODIPY-mediated fluorescence. e, Exacerbation of TLD by catalase/peroxidase deficiencies. Deletion of katG (strain 3641) or katE (strain 3645), when present with a ΔthyA mutation, decreased cell survival for samples taken at the indicated times during T-starvation. A nutrient-rich, defined medium (Hi-DEF Azure) was used for panels a-d; M9 minimal medium was used for panel e (see Supplementary Fig. 3d for KatG/E effects in Hi-DEF Azure medium). Panels a, c and d are representative for 3 independent experiments; data points are averages of three independent experiments in panels b and e; error bars represent standard deviation of the mean.

Figure 2. Suppression of ROS accumulation during T-starvation by rifampicin, chloramphenicol, or deficiencies in respiratory-chain genes

ROS was measured by flow cytometry as in Fig. 1a unless otherwise stated. Color coding for samples is shown above each panel. a, Effect of rifampicin. A ΔthyA E. coli strain (3640) was treated with 50 µg ml⁻¹ rifampicin when T-starvation was initiated; samples were removed for flow cytometry at the indicated times. b, Effect of chloramphenicol. Method was as in panel a except 40 µg ml⁻¹ chloramphenicol replaced rifampicin. c, Effect of mutations in respiratory-chain genes on ROS. Cells were starved for thymidine for 3 h before samples were taken for flow cytometry. Bacterial strains were ΔthyA-ΔubiG (3812), ΔthyA-ΔcydB (4012), and ΔthyA (3640). d, Effect of mutations in respiratory-chain genes and bipyridyl on TLD. Cultures of double-deletion mutants (ΔthyA-ΔubiG, ΔthyA ΔcydB) and ΔthyA-ΔubiG plus bipyridyl (0.5 × MIC) were starved for thymidine; percent survival was determined at indicated times. Panels a-c are representative of 3 independent experiments; data points in panel d are averages for three independent experiments, with error bars representing standard deviation of the mean. No growth defect occurred when deficiencies in cydB and thyA were combined (Supplementary Fig. 14); a small inhibitory effect was observed with a ΔthyA-ΔubiG double mutant and with addition of bipyridyl to a ΔthyA mutant culture (Supplementary Fig. 14); however, substantial TLD was observed after a greater slowing of growth by incubation of the ΔthyA single mutant at 30 °C (Supplementary Fig. 14), thereby arguing against the small growth inhibition by ΔubiG or bipyridyl being a major reason for protection from TLD.

Figure 3. Single-strand DNA regions are necessary but insufficient for TLD

a, Effect of deficiencies of RecF or RecR. Mutants deficient in thyA and recF (strain 3711), thyA and recR (strain 3955), or only ΔthyA (strain 3640) were starved for thymidine, and at the indicated times samples were taken for determination of percent survival. b, Effect of deficiencies in recQ and rnhA. Mutant strains were starved for thymidine, and at the indicated times percent survival was determined for ΔthyA (strain 3640), ΔthyA-ΔrecQ (strain 3952), and ΔthyA ΔrnhA (strain 3664). Data points in panels a and b are averages for three independent experiments; error bars represent standard deviation of the mean. c, Prevalence of ΔthyA cells (strain 4313) with single-strand DNA regions. Fluorescent foci due to cellular binding of pre-induced Ssb-YFP to single-strand DNA regions (upper panels) are indicated by arrows. Lower panels: bright-field views of corresponding upper panels. Scale bar (in panels c-g) represents 10 µm; numbers under the panels indicate the prevalence of cells exhibiting fluorescent foci. d, Prevalence of ΔthyA-ΔrecQ mutant cells with single-strand DNA regions. Methods were as in panel c but with strain 4335. e, Prevalence of ΔthyA-ΔrnhA mutant cells exhibiting single-strand DNA regions. Methods were as in panel c but with strain 4389. f, Prevalence of ΔthyA-ΔcydB mutant cells exhibiting single-strand DNA regions. Methods were as in panel c but with strain 4012. g, Effect of rifampicin on prevalence of cells having single-strand DNA. Rifampicin (50 µg ml⁻¹) was added to the ΔthyA culture (strain 4313) at the time T-starvation was initiated. Methods were as in panel c. Similar results were observed in 3 independent experiments for panels c-g; 200 cells for each group were randomly selected for analysis of generation of single-strand DNA regions.

Figure 4. Double-strand DNA breaks associated with TLD

a, Prevalence of ΔthyA cells with double-strand DNA breaks during T-starvation. Strain 4258 (recN-yfp) was pre-induced with 0.1% arabinose for 2.5 h and then starved for thymidine for the indicated times followed by examination by fluorescence (upper panels) or bright field microscopy (lower panels). Arrows indicate fluorescent foci. Scale bar (in all panels) represents 10 µm. Prevalence of cells with at least one fluorescent focus is shown as percentage below panels. b, Effect of rifampicin on presence of DNA breaks during T-starvation. Methods were as in panel a except rifampicin (50 µg ml⁻¹) was added immediately after thymidine removal. c, Effect of 2,2’-bipyridyl (Bip) on presence of DNA breaks during T-starvation. Methods were as in panel a except for addition of 0.5 × MIC Bip immediately after thymidine removal. d, Effect of recQ on formation of DNA breaks during T-starvation. Methods were as in panel a but with strain 4262 (ΔthyA-ΔrecQ). e, Co-localization of DNA breaks and single-strand DNA regions during T-starvation. Ssb-mCherry was constitutively expressed under the native ssb promoter in the chromosome of a ΔthyA recN-yfp mutant (strain 4315). Two typical thymidine-starved cells are shown for appearance of DNA breaks in the single-strand DNA region. Arrows indicate fluorescent foci. About 60% of DNA breaks (green RecN-YFP foci, n = 30) co-localized with regions of single-strand DNA. All images are typical from pictures obtained from 3 independent experiments. For panels a-d, 200 cells for each group were randomly selected for analysis of generation of DNA breaks. Detection of double-strand DNA breaks by E. coli RecN was verified by finding the same results with Bacillus subtilis RecN expressed in E. coli (see Supplementary Fig. 9).

Figure 5. Conversion of single-strand DNA regions into lethal breaks by exogenous ROS

a, Exogenous H₂O₂ reduced survival of rifampicin-treated cells starved for thymidine. Rifampicin was added at the time of thymidine removal. At the indicated times after thymidine removal, H₂O₂ was added to 3.5 mM (unless otherwise indicated) for 20 min before sampling for survival. Squares, ΔthyA (strain 3640) without rifampicin or H₂O₂ treatment; circles, ΔthyA (strain 3640) incubated with 200 µg ml⁻¹ thymidine plus rifampicin and H₂O₂ treatment; triangles, wild-type (strain 3001) plus rifampicin and H₂O₂ treatment; inverted triangles, ΔthyA (strain 3640) plus rifampicin and H₂O₂ treatment. b, Exogenous H₂O₂ increased the prevalence of cells exhibiting DNA breaks. Plasmid-borne RecN-YFP was pre-induced by 0.1% arabinose for 2.5 h during growth of strain 4282 before removal of thymidine. H₂O₂ (3.5 mM) was added to cultures after 30-min rifampicin treatment and T-starvation. Upper panels show fluorescence after 20 min H₂O₂ treatment; lower panels are corresponding bright-field views. Scale bar, 10 µm. Microscopy results are representative of three independent experiments. c, Prevalence of cells with double-strand breaks generated by exogenous H₂O₂ administered as in panel b. Abbreviations: DSB double-stranded DNA break; Rif, rifampicin; Thy, thymidine. Number of cells examined for the four groups (Rif, Rif + H₂O₂, Thy + Rif, and Thy + Rif + H₂O₂) were 1040, 640, 460, and 1190 cells, respectively. Data from three independent experiments were used for analysis. d, Effect of recQ and cydB deficiencies on survival of rifampicin-treated, thymidine-starved ΔthyA cells when exposed to exogenous H₂O₂. H₂O₂ treatment was as in panel a. Squares, ΔthyA (strain 3640); circles, ΔthyA-ΔcydB (strain 4012); triangles, ΔthyA-ΔrecQ (strain 3952). Error bars in panels a, c and d represent standard deviation of the mean determined from three independent experiments.

Figure 6. Scheme describing ROS-mediated conversion of single-strand DNA regions into lethal DSBs during TLD

(i) When growing cells are starved for thymine, DNA replication forks stall; they then advance slowly by recruiting thymine from degraded DNA. (ii) Single-strand DNA regions, located in the lagging strands behind replication forks and at sites of single-strand lesion repair, enlarge due to attempted repair in the absence of dTTP. The number and size of single-strand DNA regions increase due to DNA-damage-repair systems (RecQ/RecFOR) and due to degradation of RNA from RNA-DNA hybrids (RnhA). (iii) T-starvation triggers accumulation of intracellular ROS as byproducts of respiration. Disruption of the respiratory chain by deletion of ubiG or cydAB decreases ROS generation. Addition of RNA or protein synthesis-inhibiting agents (rifampicin or chloramphenicol) inhibits cell respiration, thereby suppressing the ROS surge. ROS accumulation is also inhibited by the presence of Fe²⁺ chelators, such as 2,2’-bipyridyl, or antioxidants, such as DMSO. These ROS-inhibiting agents have little effect on expansion of single-strand DNA regions (iv) DNA is attacked by ROS, especially hydroxyl radical. When ROS attack occurs in single-strand DNA regions, DSBs arise; ROS-mediated attack of double-strand DNA leads primarily to single-strand lesions, which could be expanded into large single-strand regions by abortive DNA repair in the absence of dTTP. (v) Extensive DNA breakage results in cell death, with deficiencies in DSB repair exacerbating TLD. Induction of SOS/sulA, which may occur at multiple steps in the scheme, can lower the ability of cells to resume growth after thymine becomes available.