FIG. 4.

The effects of toxin overexpression on persister formation. (A) Cells were cultured as described in the legend to Fig. 1. RelE was induced (▪) from pKD3035 (pBAD::relE) in MG1 (MC1000 ΔrelBE) at time zero by the addition of 0.2% arabinose, and MG1 with a blank vector (pBAD33) served as the control (▴). (B) Cells were cultured, and RelE was induced as described above. After 3 h of RelE induction, samples were removed and treated with either cefotaxime (100 μg/ml), mitomycin C (10 μg/ml), ofloxacin (5 μg ml), or tobramycin (25 μg/ml) for 3 h at 37°C with aeration. The control (MG1/pBAD) (black bars) was challenged at a cell density similar to that of the relE induced cells (white bars). (C) HM22 cells (K12 hipA7) were cultured as described above and at time zero were moved to 30°C (▪) or kept at 37°C (▴). (D) HM22 cells were challenged at 30°C (white bars) as described in panel B. Controls are HM22 (black bars) and HM21 (K-12 hipA wild type) (gray bars) cells were challenged at 37°C.