Fig. 1.

MRL-494 impairs biogenesis of OMPs. (A) Chemical structure of MRL-494. (B) MRL-494 potentiates rifampicin. MIC measurements for rifampicin with or without a sublethal dose of MRL-494 were performed. (C and D) Midlog cultures of TJS101 carrying a PmicA-gfp reporter were treated with MRL-494 at 1×, 0.5×, and 0.25× MIC or a DMSO control (vehicle [Veh]) and grown for 1.5 h. (C) MRL-494 treatment decreases OMP abundance. After 1.5 h of treatment, cells were harvested, normalized to OD, and assayed for protein abundance. The heavy line indicates separate biological replicates. Data are representative of 2 independent experiments. (D) MRL-494 treatment results in a dose-dependent increase in σ^E activity. GFP fluorescence posttreatment was measured as a reporter of σ^E activity and normalized to OD600. Data are shown as the average fold value to the DMSO treatment group ± SD of triplicate samples. (E) OMP-biogenesis mutants demonstrate increased susceptibility to MRL-494. The resistance to MRL-494 of strains with the indicated deletion or mutation in a tolC⁺ background was assayed by MIC. Log₂ fold changes relative to the parent strain are shown. Data are representative of 2 independent experiments. (F) Batimastat treatment increases susceptibility to MRL-494. Cells were treated as in B and C and were additionally treated with 0.25× MIC batimastat where indicated. OD600 was assayed posttreatment. Data are shown as the average of triplicate samples ± SD. (G) OMP biogenesis is inhibited by MRL-494 and restored by bamA_E470K. Midlog cultures of strains expressing wild-type (WT) bamA or bamA_E470K in haploid were treated with DMSO (Veh), 0.25× MIC batimastat, or 0.25× MIC batimastat with 0.25× wild-type MIC MRL-494 for 1.5 h. The conformational state and total abundance of LamB were assayed. Data are representative of 2 independent experiments.