FIGURE 4.

Amp resistance induced by heterogeneous response to indole. (A) Growth was monitored by counting colony forming units (CFUs) in the presence of indole with 100 μg/ml Amp. (B) Growth rates in each condition at 0–4 h of incubation, as shown in Figure 4A. (C) Amp susceptibility test of cells taken from cultures in 100 μg/ml Amp with various concentrations of indole, as shown in Figure 4A. Cells were harvested at the indicated time points (8, 12, 16, 20, 24, and 30 h of incubation) and washed to remove residual chemicals. The same number of cells was spotted on plates containing Amp (0, 100, 150, and 200 μg/ml) and incubated for 24 h. The percentage of mutated cells (D,E) was determined as follows. Cells harvested from cultures grown in 100 μg/ml Amp with various concentration of indole, as shown in Figure 4A. The percentages of mutated cells showing Amp resistance (Amp^R) (D) and Rif resistance (Rif^R) (E) were determined from the relative percentages of CFU/ml [(CFU obtained from the antibiotic plate/total number of CFU obtained from the LB plate) × 100]. (F) Percentage of Amp-resistant (Amp^R) cells in the presence of an efflux pump inhibitor. Cells were inoculated into LB medium containing 100 μg/ml Amp and 1000 μM indole with or without 2 μg/ml PAβN and incubated for 30 h at 30°C. Appropriate dilutions of the cells were spread on LB plates containing 200 μg/ml Amp. The total number of CFUs was determined on LB agar plates. All data represent the average of three replicates, and the error bar indicates the standard deviation.