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The outer membrane (OM) of Gram-negative bacteria serves as an effective permeability barrier and confers intrinsic antibiotic resistance. This barrier function requires distinct distribution of lipids across the bilayer, yet how phospholipids, the most basic building block, get transported and assembled into the OM is not well understood. In this study, we describe the observation revealing that three putative phospholipid transporters are mostly present at the cell poles in Escherichia coli , highlighting possible polar localization of lipid transport to ultimately support OM biogenesis during growth and division. Our work sets the stage for studying how phospholipid transport impacts OM stability, lipid asymmetry, and/or function, thus informing future strategies for antibiotics development against these processes.

The outer membrane (OM) of bacteria like Escherichia coli and Shigella flexneri forms a barrier that protects cells against antibiotics and immune effectors. The surface-exposed leaflet is filled by lipopolysaccharides (LPS) decorated with long “O-antigen” (O-Ag) polysaccharides. The benefit of covering the surface with O-Ag is well appreciated; these long polysaccharides shield against host assaults. Our study reveals a hidden cost to these long O-Ag polysaccharides: transporting and assembling LPS modified with O-Ag compromises integrity of the OM antibiotic barrier, rendering bacteria vulnerable to antibiotics. Cells must balance O-Ag across two parameters—protection from the host and preserving OM integrity. Our findings also present an inherent benefit to not producing O-Ag, a common feature among diverse bacterial pathogens.

The complex envelope of gram-negative bacteria is a critical structure. It is assembled and maintained by multiple essential pathways, all of which, including the β-barrel assembly machinery (BAM) complex, have been the focus of novel antibiotic discovery efforts. Species in the genus Klebsiella encode a paralog to the BAM complex component BamA, called BamK, but the importance and role of this protein has remained a mystery. Leveraging mutants resistant to a recently discovered BamA inhibitor, we describe how activation of the BaeSR envelope stress response system can activate bamK expression to overcome the loss of BamA or its function both in vitro and in vivo . These findings provide important insights into BamK, Klebsiella biology, gram-negative stress responses, and targeting outer membrane protein folding as an antibacterial strategy.

Abstract Pseudomonas fluorescens strain PCL1210, a competitive tomato root tip colonization mutant of the efficient root colonizing wild type strain WCS365, is impaired in the two-component sensor-response regulator system ColR/ColS. Here we show that a putative methyltransferase/wapQ operon is located downstream of colR/colS and that this operon is regulated by ColR/ColS. Since wapQ encodes a putative lipopolysaccharide (LPS) phosphatase, the possibility was studied that the integrity of the outer membrane of PCL1210 was altered. Indeed, it was shown that mutant PCL1210 is more resistant to various chemically unrelated antibiotics which have to pass the outer membrane for their action. In contrast, the mutant is more sensitive to the LPS-binding antibiotic polymyxin B. Mutant PCL1210 loses growth in competition with its wild type when grown in tomato root exudate. Mutants in the methyltransferase/wapQ operon are also altered in their outer membrane permeability and are defective in competitive tomato root tip colonization. A model for the altered outer membrane of PCL1210 is discussed.

The multilayered cell envelope of Gram-negative bacteria provides natural resistance to antibiotics. Understanding cell envelope synthesis and regulation is crucial for the identification of new antimicrobial targets and improved drug design. LpxC inhibitors, a new and promising class of antibiotics, impede function of the committed enzyme in lipopolysaccharide synthesis. Here, we characterize a new mechanism of resistance to the LpxC inhibitor PF-5081090, where the accumulation of lysophospholipids signals a reduction in cellular glycerophospholipid levels to repair outer membrane balance. This work proposes a new pathway to restore outer membrane asymmetry, which is a critical aspect of cell envelope integrity, and describes a role for lysophospholipids in bacterial cell signaling when lipopolysaccharide synthesis is disrupted.

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.

The outer membrane structure is common in Gram-negative bacteria, mitochondria and chloroplasts, and contains outer membrane β-barrel proteins (OMPs) that are essential interchange portals of materials 1 – 3 . All known OMPs share the antiparallel β-strand topology 4 , implicating a common evolutionary origin and conserved folding mechanism. Models have been proposed for bacterial β-barrel assembly machinery (BAM) to initiate OMP folding 5 , 6 ; however, mechanisms by which BAM proceeds to complete OMP assembly remain unclear. Here we report intermediate structures of BAM assembling an OMP substrate, EspP, demonstrating sequential conformational dynamics of BAM during the late stages of OMP assembly, which is further supported by molecular dynamics simulations. Mutagenic in vitro and in vivo assembly assays reveal functional residues of BamA and EspP for barrel hybridization, closure and release. Our work provides novel insights into the common mechanism of OMP assembly. The structural basis of the late-stage intermediate assembly of outer membrane β-barrel proteins mediated by the bacterial β-barrel assembly machinery is determined.

The defining feature of the Gram-negative cell envelope is the presence of two cellular membranes, with the specialized glycolipid lipopolysaccharide (LPS) exclusively found on the surface of the outer membrane. The surface layer of LPS contributes to the stringent permeability properties of the outer membrane, which is particularly resistant to permeation of many toxic compounds, including antibiotics. As a common surface antigen, LPS is recognized by host immune cells, which mount defences to clear pathogenic bacteria. To alter properties of the outer membrane or evade the host immune response, Gram-negative bacteria chemically modify LPS in a wide variety of ways. Here, we review key features and physiological consequences of LPS biogenesis and modifications.Lipopolysaccharide is a key component of the Gram-negative cell envelope and functions, for example, as a permeability barrier or determinant of host immune responses. In this Review, Simpson and Trent guide us through lipopolysaccharide biogenesis and modifications and their functional and therapeutic implications.

It has long been appreciated that the Gram-negative outer membrane acts as a permeability barrier, but recent studies have uncovered a more expansive and versatile role for the outer membrane in cellular physiology and viability. Owing to recent developments in microfluidics and microscopy, the structural, rheological and mechanical properties of the outer membrane are becoming apparent across multiple scales. In this Review, we discuss experimental and computational studies that have revealed key molecular factors and interactions that give rise to the spatial organization, limited diffusivity and stress-bearing capacity of the outer membrane. These physical properties suggest broad connections between cellular structure and physiology, and we explore future prospects for further elucidation of the implications of outer membrane construction for cellular fitness and survival.In this Review, Huang and colleagues explore the emerging physical and mechanical properties of the Gram-negative outer membrane. They discuss recent studies that revealed key molecular factors and interactions that give rise to the spatial organization, limited diffusivity and stress-bearing capacity of the outer membrane.

In Pseudomonas aeruginosa , MexAB–OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB–OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available. In this study, we present the structure of wild-type MexAB–OprM in the presence or absence of drugs at near-atomic resolution. The structure reveals that OprM does not interact with MexB directly, and that it opens its periplasmic gate by forming a complex. Furthermore, we confirm the residues essential for complex formation and observed a movement of the drug entrance gate. Based on these results, we propose mechanisms for complex formation and drug efflux. In Pseudomonas aeruginosa , MexAB–OprM plays a central role in multidrug resistance by ejecting various drug compounds. Here the authors present the structure of wild-type MexAB–OprM in the presence or absence of drugs and propose mechanisms for complex formation and drug efflux.