Fig. 4. High-persistence nuo* mutants have no impaired antibiotic uptake and do not solely depend on a decreased ATP:ADP ratio.

a Single-cell fluorescence intensity distributions measured via cytometry where fluorescence corresponds to DiBAC₄(3) uptake and serves a proxy for the extend of electrical depolarization. The data were plotted as “1-cumulative distributions”, showing the percentage of cells with an intensity above the fluorescence value specified in the x-axis to aid comparison between the strains. nuo* mutants in stationary phase show a minor decrease in electrical potential, indicated by the higher fraction of cells (higher curves) that have high DiBAC₄(3) uptake (starting from a fluorescence value of ±10) (one representative of n = 2). Control samples treated with CCCP (in black, average across all repeats and strains), a protonophore and potent inducer of antibiotic tolerance⁷⁰, show the expected increase in dye uptake in all cells for a depolarized sample. Cyan lines, arrows and numbers were added to put the results in biological context: the wild-type persister level (0.1% of the population, see Fig. 2e) corresponds to fluorescent level above 650. Only <1% of the nuo* mutants have such a high level of DiBAC₄(3), whereas these strains carry on average 50% persister cells (Fig. 2d, e, Supplementary Fig. 2f–i). b Intracellular amikacin concentrations of stationary phase cultures of nuo* mutants measured in a bioassay are similar to the wild type or higher, confirming that antibiotic uptake is not decreased in the high-persistence mutants (means ±stdev, n = 3; *p < 0.01 for comparison to the wild type from a one-way ANOVA with Dunnett’s posttest). c In stationary phase (16–20 h after 1/100 dilution of an overnight culture), ATP:ADP ratios are significantly decreased as measured by the ratiometric GFP, Perceval (means ±stdev, n = 3; *p < 0.0001 for comparison to the wild type from a one-way ANOVA with Dunnett’s posttest). However, nuo* mutants show similar decreases in ATP:ADP ratios in exponential phase when they do not display increased persistence (Supplementary Figs. 4b, 2h). See also Supplementary Fig. 4. Source data are provided as a Source Data file.