Growth and permeability assays for lpt and lpx knockdowns.

(A) Box plot of significant negative CG scores for lpt or lpx genes. Asterisk denotes p < 0.05 (Student’s <i>t</i>-test). (B) Colistin and levofloxacin MIC test strip assays for <i>lptA</i> or <i>lpxC</i> knockdown, LOS^-, or nontargeting guide control strains. Plates supplemented with 1mM IPTG. Approximate MICs indicated below images. (C) Heatmap of CG scores of <i>lpx</i> knockdowns across conditions, showing muted phenotypes compared to <i>lpt</i> knockdowns. (D) 10 uL spots of ten-fold serial dilutions on plates with and without induction (N=3 biological replicates). The <i>lptA</i> knockdown, but not the <i>lpxC</i> knockdown, exhibits a minor growth defect upon induction. LOS^- strains show major growth defects. (E) Ethidium bromide permeability assay for <i>lptA</i> knockdown and nontargeting control in 19606 or LOS- backgrounds without induction; increased fluorescence over time indicates membrane permeability. Ribbons represent standard deviation (N=4 biological replicates). Without induction, the strain containing the <i>lptA</i> guide behaves similarly to a nontargeting guide control. (PDF)