Fig. 6. RpoS and decreased protein synthesis contribute to increased persistence in nuo* mutants.

a, b Increased tolerance of the nuo* mutants is partially RpoS-dependent in a stationary phase (amikacin for 5h with 400 µg  ml⁻¹) and b upon the glucose-to-fumarate shift (amikacin for 4h with 400 µg ml⁻¹) (means ±stdevs, n≥4; * p<0.05, ** p<0.01, *** p<0.001 and ns = non-significant for within-strain comparison from on a linear mixed model with Šidák’s posttest). c Proteome changes after a shift from growth on glucose to fumarate (red dots) indicate that the proteome of high-persistence nuo* mutants is in a locked state (means ±stdevs, n≥2). The wild type with functional RpoS (closed gray symbols) shows an activation of the RpoS regulon as it moves down on the stress dimension of the PCA plot over time (see Methods), while removing rpoS (open gray symbols) leads to an adaptation to steady-state growth on fumarate and a migration along the growth rate dimension. Starting from 30 min after the shift to fumarate, the proteome of nuo* mutants remains unchanged, regardless of the presence of RpoS (in blue; average of nuoM* and nuoN* mutants). The nuoL* mutant was omitted as, in this particular experiment, it did not show strong and persistent acidification and hence, displayed a deviating proteome change (Supplementary Fig. 5f, g), which further substantiates our finding. d Translation activity is lower in nuo* mutants in stationary phase as measured by the incorporation of radioactive H³-leucine (means ±stdevs, n ≥ 2; *p < 0.05 for a phenotype-level comparison from a linear mixed model). e Chloramphenicol (CAM) supplementation (25 µg ml⁻¹ at the time of the shift) induces tolerance towards amikacin (for 4h with 400 µg ml⁻¹; 30 min after the shift) upon shifting from glucose to fumarate in the WT with/without rpoS and the nuoL* and nuoN* mutant lacking rpoS (means ±stdevs, n = 4; *p < 0.05, ***p < 0.001 for within-strain comparison between presence/absence of CAM from a linear mixed model with Šidák’s posttest). See also Supplementary Figs. 5, 6. Source data are provided as a Source Data file.