Phase separation in the outer membrane of Escherichia coli

Antimicrobial resistance is particularly prevalent in gram-negative bacteria, as antibiotics that act inside the cells must overcome their outer membrane. So far, technical limitations have prevented us from determining how outer-membrane proteins and lipids are organized to form this functional barrier. Here, we use nanoscale imaging of live bacteria to reveal that the most abundant outer-membrane proteins form a network that spans the entire bacterial surface, leaving only small gaps of phase-separated lipopolysaccharide. This tendency to phase separate is further emphasized by the formation of new domains when phospholipids are mislocated at the surface, rendering cells more susceptible to some antibiotics. Overall, the phase-separated nature of the outer membrane defines a perspective on its integrity and barrier function

Phase separation in the outer membrane of Escherichia coli

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