PMC

Efflux Gene Expression in Total and Persister Cells of Wild-Type E. coli Measured by RNA-seq and qPCR
(A) Heatmap showing relative transcript abundance of persistence related genes in total cells and persister cells. Scale below the heatmap indicates log2-normalized transcript abundance relative to the mean expression level (T, total cells; P, persister cells). See also .
(B) Scheme of the TolC related efflux system complexes, the number of cylinders indicating the copy number of the protein in each efflux complex.
(C) Scheme of the tolC operon.
(D) Relative gene expression level in total cells, persister cells, and regrown cells of wild-type E. coli measured by qPCR. The bars indicate mean of at least three independent experiments; error bar indicates SD.

Lower Intracellular Antibiotic Accumulation in Persister Cells of Wild-Type E. coli
(A) Experimental procedure for measuring antibiotic accumulation in bacterial cells.
(B and C) Histogram of antibiotic accumulation in total cells (n = 209) and persister cells (n = 86) of wild-type E. coli.
(D) Time-lapse microscopy showing reduced accumulation of antibiotic in persisters (from ). The first five images are merged bright field and fluorescence images, revealing that antibiotic accumulation accompanies cell death (separate images are shown in F). The different media added during experiment is indicated below (killing medium: 90% [v/v] M9 + 10% [v/v] LB + 150 μg/ml carbenicillin + 20 μg/ml BOCILLIN + 0.15% [w/v] methylcellulose; growth medium: 90% [v/v] M9 + 10% [v/v] LB + 5% [w/v] methylcellulose) (). The persister cell (arrow) shows low antibiotic accumulation and regrows after removal of antibiotic (Scale bar, 3 μm; t = time in min).
See also and .

High Efflux Activity in Persister Cells
(A and B) Histogram of antibiotic accumulation in total cells (n = 103) and persister cells (n = 144) of ΔompF strain.
(C and D) Histogram of antibiotic accumulation in total cells (n = 116) and persister cells (n = 109) of ΔompC strain.
(E and F) Histogram of antibiotic accumulation in total cells (n = 138) and persister cells (n = 84) of ΔtolC strain.
(G) Experimental procedure for measuring efflux rate of fluorescent antibiotic in bacterial cells.
(H) Intracellular fluorescent intensity decay after removing antibiotic in the medium is well fit by a single exponential function. Fluorescent intensity in a persister cell (red) decayed more rapidly than in a susceptible cell (green), while a cell of ΔtolC mutant (purple) showed little change in fluorescence intensity with time.
(I) Statistical analysis showing that the efflux rate of antibiotic in persister cells is significantly higher than that in total cells of wild-type E. coli (TC, total cells; PER, persisters) (p < 0.0001).
See also and .

System-wide Comparison of the Contribution of Persistence Genes to Bacterial Drug Tolerance
(A) Heatmap showing relative transcript abundance of persistence related genes in total cells and persister cells. Scale below the heatmap indicates log2-normalized transcript abundance relative to the mean expression level (T, total cells; P, persister cells). See also .
(B) Bar plot representing cell survival rate (percentage log scale) after 4 hr carbenicillin treatment of two groups sorted by flow cytometer from fluorescently labeled tolC, soxS, uvrD, hipA, plsB, pspA, relE, and phoU strains, respectively.
(C) Bar plot representing cell survival rate (in logarithm scale) after 4 hr carbenicillin treatment of knockout strains ΔtolC, ΔansA, Δcrp, ΔdinG, ΔdnaK, ΔdnaJ, ΔdksA, ΔhipA, ΔhupA, ΔhupB, Δlon, ΔplsB, ΔpspA, ΔruvA, ΔruvB, ΔuvrD, and ΔyigB. The bars indicate mean of at least three independent experiments; error bar indicates SD (^∗p value < 0.1; ^∗∗p value < 0.01; ^∗∗∗p value < 0.001; ^∗∗∗∗p value < 0.0001).

Persister Formation Frequency Positively Correlates with Efflux Gene Expression Level and Negatively Correlates with Intracellular Antibiotic Accumulation. Antibiotics Lethal Effects Were Enhanced by Addition of Efflux Inhibitors
(A) Relative tolC expression level of the four strains measured by qPCR. WT (wild-type strain BW25113), OVER (tolC overexpression strain, by pBAD::tolC in BW25113, induced by 10^-5% arabinose), ΔtolC (tolC knockout strain, Key::JW5503), and RESC (tolC rescued strain, by pBAD::tolC in ΔtolC strain, induced by 10^-6% arabinose).
(B) Relative antibiotic accumulation in the four strains determined by fluorescence microscopy.
(C) Persister formation frequency of the four strains determined by antibiotic susceptibility measurement. The correlation coefficiency between intracellular antibiotic accumulation level and tolC expression level is −0.776; between intracellular antibiotic accumulation level and probability of persistence is −0.989; between tolC expression level and probability of persistence is 0.848.
(D–G) (D) Persister formation frequency of the four strains under antibiotic treatment of different concentrations. Antibiotics lethal effects were enhanced by efflux inhibitors; carbenicillin (E), cloxacillin (F), and nalidixic acid (G). Abbreviations: Car, carbenicillin. NA, nalidixic acid. Clo, cloxacillin. PAβN, Phenylalanine arginyl β-naphthylamide. NMP, 1-(1-Naphthylmethyl) piperazine. The bars indicate mean of at least three independent experiments; error bar indicates SD (^∗p value < 0.1; ^∗∗p value < 0.01; ^∗∗∗p value < 0.001; ^∗∗∗∗p value < 0.0001).
See also .

The Dependence between Persister Formation Frequency and TolC Expression Level
(A) TolC expression level in total cells of TC tag-TolC strain measured by Tetracysteine-based protein detection (FlAsH imaging, upper panel); TolC expression level in persister cells that survived antibiotic treatment (lower panel).
(B) Time-lapse microscopy showing that persister cells express a high level of TolC (from ). The first and fifth images are the merged bright field and fluorescent images (separate bright field and fluorescent images are shown in D). The different media added during the experiment are indicated below (killing medium: 90% [v/v] M9 + 10% [v/v] LB + 150 μg/ml carbenicillin + 0.15% [w/v] methylcellulose; growth medium: 90% [v/v] M9 + 10% [v/v] LB + 5% [w/v] methylcellulose). The persister cell (arrow), shows a significantly higher expression of TolC, and regrows after removal of antibiotic (from ). (Scale bar, 3 μm; t = time in min).
(C) Experimental procedure for sorting cells by expression level of persistence related genes.
(D) Distribution of fluorescence intensity indicating TolC expression levels of 76RP strain. Stationary-phase cells were sorted into three groups: group A containing the total cells (100.0%), group B including the majority of total cells except for those with highest fluorescence intensity (97.8%), and group C containing a sub-population with highest fluorescence intensity (1.0%).
(E) Bar plot representing cell survival rate (percentage log scale) after 4 hr carbenicillin treatment of three groups sorted by flow cytometer, revealing that the survival rate significantly increases in group C. The bars indicate mean of at least three independent experiments; error bar indicates SD.
See also and .