Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

Colicins are Escherichia coli–specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A colicins typically parasitize the proton-motive force–linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A colicins.

The outer membrane (OM) of Gram-negative bacteria is a robust protective barrier that excludes major classes of antibiotics. The assembly, integrity and functioning of the OM is dependent on β-barrel outer membrane proteins (OMPs), the insertion of which is catalyzed by BamA, the core component of the β-barrel assembly machine (BAM) complex. Little is known about BamA in the context of its native OM environment. Here, using high-affinity fluorescently-labelled antibodies in combination with diffraction-limited and super-resolution fluorescence microscopy, we uncover the spatial and temporal organization of BamA in live Escherichia coli K-12 cells. BamA is clustered into ~150 nm diameter islands that contain an average of 10-11 BamA molecules, in addition to other OMPs, and are distributed throughout the OM and which migrate to the poles during growth. In stationary phase cells, BamA is largely confined to the poles. Emergence from stationary phase coincides with new BamA-containing islands appearing on the longitudinal axis of cells, suggesting they are not seeded by pre-existing BamAs but initiate spontaneously. Consistent with this interpretation, BamA-catalyzed OMP biogenesis is biased towards non-polar regions. Cells ensure the capacity for OMP biogenesis is uniformly distributed during exponential growth, even if the growth rate changes, by maintaining an invariant density of BamA-containing OMP islands (~9 islands/μm2) that only diminishes as cells enter stationary phase, the latter change governing what OMPs predominate as cells become quiescent. We conclude that OMP distribution in E. coli is driven by the spatiotemporal organisation of BamA which varies with the different phases of growth.SignificanceThe integrity and functioning of the outer membrane (OM) of Gram-negative bacteria depends on the β-barrel assembly machinery (BAM). Although the structure and the mechanism of the complex have been widely explored, little information exists about the organization of the BAM complex and how it dictates protein distribution in the OM. Here, we utilized highly specific monoclonal antibodies to study the spatiotemporal organization of BamA, the key component of this complex. We reveal that BAM organization is dynamic and tightly linked to the cell’s growth phase. We further discover that the rate of BAM facilitated OMP biogenesis is significantly reduced near the poles. In turn, these features govern the biogenesis patterns and the distribution of OMPs on the cell surface.