Fig. 4.

Lysine 12 is essential for TisB-dependent antibiotic persistence. Wild type MG1655 (WT), a tisB deletion strain, and two chromosomal amino acid substitutions (D5N and K12L) were treated with CIP (10 µg/mL) during exponential phase for six hours. (a) TisB-dependent ATP depletion. Pre- and post-treatment samples were analyzed using a luciferase-based assay to measure cellular ATP levels (nM per OD₆₀₀). Bars represent the mean of three biological replicates. Error bars indicate the standard deviation. ANOVA with post-hoc Tukey HSD test was performed (*** p < 0.001; ** p < 0.01; n.s.: not significant). (b) Persister cell survival. Cells before and after treatment were plated on LB agar plates to determine relative persister levels. Bars represent the mean of at least four biological replicates (WT: n = 8; D5N: n = 4; K12L: n = 5; ΔtisB: n = 4). Error bars represent the standard deviation. ANOVA with post-hoc Tukey HSD test was performed, and a compact letter display was applied to present significant groups. (c) Persister cell recovery. The ScanLag method was applied to determine the colony appearance time after CIP treatment. Colony appearance times are illustrated as violin box plots. Colonies from at least two biological replicates were combined (WT: n = 741; D5N: n = 930; K12L: n = 165; ΔtisB: n = 375). The white dot indicates the mean. The respective median appearance time (white bar) is shown on top of each plot. Strains were compared using a pairwise Wilcoxon rank sum test (*** p < 0.0001; n.s.: not significant).