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The β‐barrel assembly machinery (Bam) complex facilitates the assembly of outer membrane proteins (OMPs) in gram‐negative bacteria. The Bam complex is conserved and essential for bacterial viability and consists of five subunits, BamA‐E. BamA is the transmembrane component, and its β‐barrel domain opens laterally to allow folding and insertion of incoming OMPs. The remaining components are regulatory, among which only BamD is essential. Previous studies suggested that BamB regulates BamA directly, while BamE and BamC serve as BamD regulators. However, specific molecular details of their functions remain unknown. Our previous research demonstrated that BamE plays a specialized role in assembling the complex between the lipoprotein RcsF and its OMP partners, required for the Regulator of Capsule Synthesis (Rcs) stress response. Here, we used RcsF/OmpA as a model substrate to investigate BamE function. Our results challenge the current view that BamE only serves as a BamD regulator. We show that BamE also directly interacts with BamA. BamE interaction with both BamA and BamD is important for function. Our genetic and biochemical analysis shows that BamE stabilizes the Bam complex and promotes bidirectional signaling interaction between BamA and BamD. This BamE function becomes essential when direct BamA/BamD communication is impeded. [ABSTRACT FROM AUTHOR]

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents. [ABSTRACT FROM AUTHOR]