FIG. 2.

Effect of various stressful conditions on EDF production. (A) E. coli MC4100relA⁺ WT and (B) MC4100relA⁺ΔmazEF were grown in M9 medium (containing 0.5% glucose) with shaking (160 rpm) at 37°C for 12 h. Cells were then diluted 1:100 in M9 medium and were grown with shaking (160 rpm) at 37°C to mid-logarithmic phase (OD₆₀₀ of 0.6). The cells were incubated without shaking for 10 min and then one of the following stressful conditions was applied: (i) incubation at 37°C with chloramphenicol (45 μg/ml) for 20 min (Cam); rifampin (20 μg/ml) (Rif), nalidixic acid (1,000 μg/ml) (Nal), or mitomycin C (0.25 μg/ml) (Mit) for 10 min; or trimethoprim (2 μg/ml) (Tm) for 1 h; (ii) incubation for 10 min at 50°C; or (iii) overexpression of MazF. MC4100relA/pQEmazF was induced with IPTG (1 mM) at 37°C for 30 min. Supernatants were obtained as described in Materials and Methods. The supernatants from cultures that were induced by antibiotics were dialyzed in Tris buffer (1 mM) at 24°C for 8 h, followed by incubation at 4°C for 12 h. The EDF activities of the supernatants were quantified as described in Materials and Methods. The supernatants of an untreated culture (NT) served as a control.