Fig. 1.

Genetic strategy to define essential bacterial functions that alter drug efficacy in vitro. (A) Growth of H37Rv in minimal media with glycerol, acetate, and cholesterol as the sole carbon source and increasing concentrations of RIF. Results shown as mean from three biological replicates with SDs. (B) Normalized growth inhibition of WT Mtb across increasing concentrations of INH, RIF, and MOX in minimal media with glycerol (black), acetate (blue), and cholesterol (red) as sole carbon sources. Results shown as means from three biological replicates with SDs. (C) Barcoded hypomorph mutants were pooled and grown in 96-well plates containing minimal media with glycerol, acetate, or cholesterol as the sole carbon source for 14 d. Antibiotics were added to individual wells as well as untreated controls. Chromosomal barcodes were PCR amplified and pooled for Illumina NGS. Barcodes were analyzed to quantify changes in fitness of individual strains from different conditions. (D) Boxplot representing changes in relative abundances of rpoB, gyrA, and ndh mutants grown in glycerol during RIF, MOX, and INH treatment, respectively. The highest drug concentrations tested for RIF, MOX, and INH represent the concentrations at which most of the strains in the library were inhibited. Data represent five biological replicates. Significance was calculated using unpaired t test and compared to untreated conditions, *P < 0.05, **P < 0.01, ***P < 0.001. (E) Heatmap representing changes in fitness of individual mutants, shown as log2 fold change, during INH, RIF, and MOX treatment in glycerol growth conditions. Mutants were chosen from previous association with respective antibiotic. sspB numbers denote sspB expression level of mutant. Results shown as means from five biological replicates.