Summary of the applied drug treatments and their phenotypic consequences

Cell length distribution of M. smegmatis cells treated with different drugs. Horizontal lines represent the mean of the plotted data points (n = 84–212). The inset shows the fold changes in cell length compared to the untreated control on a log2 axis, highlighting the phenotypic effect of each treatment. * indicates data significantly different from the control at p = 0.0001. Numerical values and additional statistical parameters are provided in Table S1.

Mutation accumulation (MA) experiment and the resulting genotypic and phenotypic changes in wild-type M. smegmatis mc2155 strains under antibiotic pressure. A) Experimental design. B) Mutation rates determined through genome sequencing of the drug treated cells as an output of the MA process. UV(+) serves as a control reference for DNA damage. Columns represent averages, and error bars indicate the standard deviations of three individually sequenced samples. Statistical significance is marked by an asterisk (*), with a p-value of 0.05. C) Phenotypic drug sensitivity in drug-treated strains. Three individual MIC determinations are presented, with the mean indicated by a horizontal line.

Changes in the expression of DNA repair genes upon stress treatments. Gene expression changes are normalized to the mock treated control using the SigA and Ffh reference genes. Upregulation is numerically interpreted as fold change; downregulation is interpreted as -1/ (fold change) in the heatmap. * p<0.1; ** p<0.05.

First line antituberculotic treatments and DNA damaging agents alter dNTP concentrations in the cell. A-F) Cellular dNTP concentrations in drug treated M. smegmatis. dNTP levels were measured in cellular extracts and normalized to the average cell volume for each treatment, yielding the concentrations shown. Each drug treatment and dNTP quantification included a corresponding control to account for potential fluctuations in growth and experimental conditions. Note the different scales on the Y-axis. Data bars represent the averages of three biological replicates each carried out in three technical replicates; error bars represent SE. The p-values from the t-tests calculated for the measured differences are provided in Table S6, with significance indicated in the figure by asterisks as follows (**) for p < 0.04 and (*) for p < 0.07. G) dNTP pool compositions of drug treated bacteria. The large error bars in the control data arise from the combination of individual controls measured for each treatment. H) Summed molar concentration of all four dNTPs compared to the control for each treatment. The Y-axis is on a log2 scale to equally represent both increases and decreases. I) Correlation of relative cell size (determined from cell lengths, compared to control cells) to relative total dNTP concentration for each treatment.

Phenotypic CIP tolerance assay. A) Scheme of the fluctuation test used in the study. B) Development of phenotypic resistance to a selecting CIP concentration following preincubation with a sublethal CIP concentration for various time periods. Data bars represent the averages of three biological replicates each carried out in three technical replicates; error bars represent SE.