Fig 4. Accumulation of off-pathway pili on the cell membrane activate the CroRS system.
(A) qRT-PCR analysis of croRS expression in the WT (white bars), ΔsrtAΔhtrA (black bars), and croR::tnΔsrtAΔhtrA (light grey bars). qRT-PCR was performed in biological triplicates and analyzed by the ΔΔCT method, using gyrB as a housekeeping gene. Fold change indicates the change in croR and croS transcription compared to WT. croR::tnΔsrtAΔhtrA was used as the negative control for croRS transcription. Statistical analysis was performed by the 1-way ANOVA and Tukey’s comparison test using GraphPad. **** P ≤ 0.0001. (B) Statistical analysis of pili expressing cells labeled with α-EbpC immune serum and Alexa Fluor 568 secondary antibody. Results are represented as bar graphs with standard error of mean. Combined data from three independent experiments were shown. Statistical analysis was performed by the 2-way ANOVA and Tukey multiple comparison test using GraphPad. * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001; P > 0.05, differences not significant (ns). (C) qRT-PCR analysis of ebpA (black bars), ebpB (light gray bars) and ebpC (white bars) expression in the WT, ΔsrtAΔhtrA and croR::tnΔsrtAΔhtrA strains. qRT-PCR was performed in biological triplicates and analyzed by the ΔΔCT method using gyrB as a housekeeping gene. Fold change indicates the change in ebpABC gene transcription compared to WT. Statistical analysis was performed by the 2-way ANOVA and Tukey multiple comparisons test using GraphPad. **** P ≤ 0.0001. (D) List of CroR-dependent genes identified by Timmler et al in bacitracin-treated OG1 [39] that are differentially expressed in the ΔsrtAΔhtrA strain (compared to OG1RF WT), in the croR::tnΔsrtAΔhtrA strain (compared to ΔsrtAΔhtrA), or in both. Log2FC values are indicated in blue for upregulated genes and orange for downregulated genes. Genes highlighted in bold font and yellow background are restored, at least in part, to WT levels upon croR inactivation.