Fig. 5: Mutation in mlaD influences AMP susceptibilities through antagonistic mutational effects.

a Relative change in MICs of the mlaD overexpression and deletion strains (ΔmlaD) to a representative set of membrane-targeting and intracellular-targeting AMPs. MICs were compared to corresponding wild-type control strains (see Supplementary Figures 12, 13). Dashed lines represent previously defined cut-offs for resistance ( ≥ 1.2 x MIC of the control) and sensitivity ( ≤ 0.8 x MIC of the control)²⁵. b Decreased net negative surface charge of the mlaD overexpression and deletion strains. Significant differences: **P = 0.0021 and P = 0.0021 for WT + empty vector vs overexpression and WT vs deletion strain, respectively, from two-sided Mann–Whitney U test, n = 6 biological replicates for each genotype. Charge measurement was done using FITC-labeled poly-L-lysine (FITC-PLL) assay where the fluorescence signal is proportional to the binding of the FITC-PLL molecules. A lower binding of FITC-PLL indicates a less net negative surface charge of the outer bacterial membrane (see Methods). c Increased membrane potentials of the mlaD overexpression and deletion strains. Significant differences: **P = 0.007, P = 0.0079 and P = 0.0079 for WT + empty vector CCCP control vs WT + empty vector, WT + empty vector vs WT + mlaD overexpression and WT vs. ΔmlaD, respectively, two-sided Mann–Whitney U test, n = 5 biological replicates for each genotype. Relative membrane potential was measured by determining relative fluorescence (RFU) using a carbocyanine dye DiOC2(3) assay (see Methods). Red/green ratios were calculated using population mean fluorescence intensities. WT E. coli carrying the empty vector treated with cyanide-m-chlorophenylhydrazone (CCCP, a chemical inhibitor of proton motive force) was used as an experimental control for diminished membrane potential. Central horizontal lines represent mean values of biological replicates. Source data are provided as a Source Data file and in Supplementary Fig. 16.