Fig. 4. Active efflux driven by PMF contributes partially to the formation of an antibiotic-tolerant subpopulation during starvation.

a–d Fluorescence intensity recorded by flow cytometry depicts the degree of antibiotic accumulation (BOCILLIN^TM FL Penicillin, 10 µg/mL) in wild type and the ΔpspA strain when subjected to 24 h starvation in the presence and absence of CCCP. The P2 and P3 gates indicate the population whose BOCILLIN fluorescent intensity is >10³RFU and 2 × 10⁴RFU, respectively. e The fluorescent efflux substrate Nile Red was used to stain the wild type and ΔpspA bacterial population, which had been subjected to 24 h starvation in the presence and absence of CCCP. n = 5 biologically independent experiments. P values depicting the degree of significance in differences between the wild type and ΔpspA, and the wild type and wild type + CCCP samples at 30 mins. Error bar represents standard deviation. f Variation in the population size of E. coli strain BW25113 and the ΔtolC gene knockout strain, which had been subjected to starvation for 24 h, followed by treatment with ampicillin for 144 h. A no ampicillin treatment control of each of the BW25113 and ΔtolC gene knockout strains was included. The effect of the efflux pump inhibitor PAβN on starvation-induced ampicillin tolerance of the BW25113 strain is also depicted. n = 3 biologically independent experiments. P values were tested between WT and PAβN + AMP, as well as ΔtolC and ΔtolC + AMP at 144 h. **indicate a P value of <0.01, ***indicate a P value of <0.001 by two-tailed Student’s test. Error bar represents standard deviation.