(a) Thymine starvation by trimethoprim in E. coli induces mazEF-mediated cell death. Cells were grown at 37°C in M9 liquid minimal medium plus 0.1 μg of trimethoprim/ml as described in Materials and Methods. Viability (% survivors) is plotted against the time of exposure to trimethoprim of E. coli MC4100relA⁺ (WT), MC4100ΔmazEFrelA⁺ (ΔmazEF), and MC4100ΔmazEFrelA⁺/pmazEF (ΔmazEF/pmazEF). MC4100relA⁺ was also grown in thepresence of 0.1 μg of trimethoprim/ml plus 50 μg of thymine/ml (WT+thymine). We calculated cell survival by comparing the colony-forming ability of trimethoprim-treated cells to that of untreated cells at zero time. The numbers represent the results of one out of five similar experiments. (b to d) Effect of thymine starvation on the synthesis of DNA (b), RNA (c), and protein (d) in MC4100relA⁺ and its ΔmazEF derivative. Cells were grown at 37°C in M9 liquid medium as described in Materials and Methods. After overnight growth, the cultures were diluted to 10⁷ cells/ml and allowed to grow for 1 h in M9 medium plus inosine (50 μg/ml). Then, trimethroprim (2 μg/ml) was added; the cells were immediately labeled with either [6-³H]thymidine (b), [5,6-³H]uracil (c), or [³⁵S]methionine (d); and their incorporation into the acid-insoluble fraction was determined as described in Materials and Methods. As controls, we used MC4100relA⁺ (▴) and its ΔmazEF derivative (▪) without the addition of trimethoprim. ▵, trimethoprim-treated MC4100relA⁺; □, ΔmazEF derivative.

Thymine starvation by SMZ in E. coli induces mazEF-mediated cell death. Viability (% survivors) is plotted against time of exposure to SMZ (10 μg/ml) of E. coli MC4100relA⁺ (WT) and its ΔmazEF derivative (ΔmazEF) in M9 liquid medium at 37°C. MC4100relA⁺ was also grown in the presence of 10 μg of SMZ/ml plus 50 μg of thymine/ml (WT+Thymine). Cell survival was determined as described in the legend to Fig. 1a.

Thymine starvation in an E. coli thyA mutant induces mazEF-mediated cell death. Viability (% survivors) is plotted against times of growth of E. coli strains KL742thyA748::Tn10 (WT), KL742thyA748::Tn10ΔmazEF (ΔmazEF), and KL742thyA748::Tn10ΔclpP (clpP −) at 37°C in M9 liquid medium lacking thymine. Cell survival was determined as described in the legend to Fig. 1a.

(a) Thymine starvation by trimethoprim in E. coli MC4100relA⁺ reduces transcription from the mazEF promoter P_2. E. coli strain MC4100relA⁺ harboring plasmid pSK10Δ6-p_ef was treated with trimethoprim; RNA was extracted at the indicated times, primer