The hallmark of Gram-negative bacteria and organelles such as mitochondria and chloroplasts is the presence of an outer membrane (OM). In bacteria such as Escherichia coli, the OM is a unique asymmetric lipid bilayer with lipopolysaccharide (LPS) in the outer leaflet. Integral, transmembrane proteins assume a β-barrel structure (OMPs) and their assembly is catalyzed by the heteropentomeric Bam complex containing the OMP BamA and four lipoproteins, BamB-E. How the Bam complex assembles a great diversity of OMPs into a membrane without an obvious energy source is a particularly challenging problem because folding intermediates are predicted to be unstable in either an aqueous or a hydrophobic environment. Two models have been put forward, the budding model based largely on structural data, and the BamA-assisted model based on genetic and biochemical studies. Here we offer a critical discussion of the pros and cons of each.

RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of β-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all β-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the β-barrel assembly machine.membrane biogenesis | protein folding | signal transduction | envelope stress response

Myxococcus xanthus has a lifecycle characterized by several social interactions. In the presence of prey, M. xanthus is a predator forming cooperatively feeding colonies, and in the absence of nutrients, M. xanthus cells interact to form multicellular, spore-filled fruiting bodies. Formation of both cellular patterns depends on extracellular functions including the extracellular matrix and intercellular signals. Interestingly, the formation of these patterns also depends on several activities that involve direct cell-cell contacts between M. xanthus cells or direct contacts between M. xanthus cells and the substratum, suggesting that M. xanthus cells have a marked ability to distinguish self from nonself. Genome-wide analyses of the M. xanthus genome reveal a large potential for protein secretion. Myxococcus xanthus harbours all protein secretion systems required for translocation of unfolded and folded proteins across the cytoplasmic membrane and an intact type II secretion system. Moreover, M. xanthus contains 60 ATP-binding cassette transporters, two degenerate type III secretion systems, both of which lack the parts in the outer membrane and the needle structure, and an intact type VI secretion system for one-step translocation of proteins across the cell envelope. Also, analyses of the M. xanthus proteome reveal a large protein secretion potential including many proteins of unknown function.

Lipoprotein RcsF is the OM component of the Rcs envelope stress response. RcsF exists in complexes with β-barrel proteins (OMPs) allowing it to adopt a transmembrane orientation with a lipidated N-terminal domain on the cell surface and a periplasmic C-terminal domain. Here we report that mutations that remove BamE or alter a residue in the RcsF trans-lumen domain specifically prevent assembly of the interlocked complexes without inactivating either RcsF or the OMP. Using these mutations we demonstrate that these RcsF/OMP complexes are required for sensing OM outer leaflet stress. Using mutations that alter the positively charged surface-exposed domain, we show that RcsF monitors lateral interactions between lipopolysaccharide (LPS) molecules. When these interactions are disrupted by cationic antimicrobial peptides, or by the loss of negatively charged phosphate groups on the LPS molecule, this information is transduced to the RcsF C-terminal signaling domain located in the periplasm to activate the stress response.DOI: http://dx.doi.org/10.7554/eLife.15276.001

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.