Fig. 3.

TisB-induced stress tolerance. (a) Northern blot analysis of tisB induction. Wild type MG1655 (WT) and a tisB deletion strain were treated with either 10 µg/mL CIP, 10 mM H₂O₂, or 200 µg/mL KAN during exponential phase for one hour. Total RNA was separated using urea-polyacrylamide gels and blotted onto nylon membranes. A radioactive probe was applied for specific tisB mRNA detection. Numbers refer to tisB primary (+ 1) and processed mRNAs (+ 42 and + 106). 5S rRNA was probed as loading control. For unedited northern blot images, see Supplementary Fig. S3. (b) Stress tolerance after TisB induction. Wild type MG1655, harboring either TisB-tisB (TisB) or an empty pBAD plasmid (CTR), were treated with L-ara (0.2%) during exponential phase for 30 min to induce tisB expression. Cells were subsequently treated with either 10 µg/mL CIP for four hours, 10 mM H₂O₂ for two hours, or 200 µg/mL KAN for four hours. Cells before (0 min), after L-ara (30 min), and after treatment with stress agents (120–240 min) were plated on LB agar plates to determine colony counts (CFU/mL). Bars represent the mean of at least two biological replicates (CIP: TisB: n = 3; CTR: n = 3 | H₂O₂: TisB: n = 3; CTR: n = 3 | KAN: TisB: n = 3; CTR: n = 2). Error bars indicate the standard deviation. ANOVA with post-hoc Tukey HSD test was performed independently for each treatment, and a compact letter display was applied to present significant groups. (c) Persistence induced by TisB. Wild type MG1655, harboring TisB-tisB (TisB) and variants with different amino acid substitutions, were treated with L-ara (0.2%) during exponential phase for 30 min to induce tisB expression. An empty pBAD plasmid (CTR) was used as control. Cells were subsequently treated with 10 µg/mL CIP for four hours. Cells before L-ara and after CIP were plated on LB agar plates to determine relative persister levels. Bars represent the mean of at least three biological replicates (TisB: n = 10; CTR: n = 9; D5L: n = 6; K12L: n = 3; Q19L: n = 3; D22L: n = 3; K26L: n = 3; K29L: n = 3). 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.