Fig. 1: Identifying transcriptional regulators of rifampicin tolerance.

A An actively growing TFI library was divided into two, and one part is induced with ATc for 20 hours, followed by steady state 10 X MIC (0.1 µg/mL) rifampicin exposure for 16 days in a 24-well transwell plate along with uninduced controls. Cultures from multiple wells were harvested at regular intervals and plated on hygromycin-containing agar media with and without rifampicin. Figure generated using BioRender. B Rifampicin time-kill kinetics, based on total CFU counts of a TFI library exposed to rifampicin as described in (A) above. Each dot represents the CFU from a separate well with at least 4 replicates from day 0 and day 4, 8 replicates from day 12, and 12 replicate cultures from day 16 were considered. C Competitive drug-free growth versus rifampicin time-kill kinetics of the TFI library based on deep sequencing. The relative abundance (ATc-induced versus uninduced) of each TFI strain in the library cultured in the absence of antibiotics (X-axis) is plotted against their relative abundance (ATc-induced versus uninduced) after four days in the presence of steady state rifampicin (Y-axis). The relative abundance of each strain was measured by deep sequencing TF specific regions of the TFI library using a pool of reverse primers specific to each TF contained in the library and one universal forward primer. Enlarged red dots show selected rifampicin tolerant TFI strains while green dots show rifampicin hypersusceptible TFI strains. Empty vector control (VC) is indicated by the black dot. Strains with no significant difference, low abundance or growth defects are shown in gray. D Volcano plot depicting selected TFI strains that developed significant rifampicin hypersusceptibility or tolerance upon ATc induction compared to their uninduced controls. X-axis denotes the fold difference in the relative abundance of the ATc induced versus uninduced TFI library strains and the Y-axis denotes significance. The TFI strain harboring an empty vector control is indicated by a black dot. Significance is estimated using multiple unpaired t-test without correction for multiple comparisons using GraphPad Prism 9.1.