Phase separation in the outer membrane of .

TitlePhase separation in the outer membrane of .
Publication TypeJournal Article
Year of Publication2021
AuthorsBenn, G, Mikheyeva, IV, Inns, PGeorge, Forster, JC, Ojkic, N, Bortolini, C, Ryadnov, MG, Kleanthous, C, Silhavy, TJ, Hoogenboom, BW
JournalProc Natl Acad Sci U S A
Volume118
Issue44
Date Published2021/11/02
ISSN1091-6490
KeywordsBacterial Outer Membrane, Bacterial Outer Membrane Proteins, Biomolecular Condensates, Cell Membrane, Cell Wall, Escherichia coli, Escherichia coli Proteins, Lipid Bilayers, Lipopolysaccharides, Molecular Dynamics Simulation, Phospholipids, Porins
Abstract

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 strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the 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.

DOI10.1073/pnas.2112237118
Alternate JournalProc Natl Acad Sci U S A
PubMed ID34716276
PubMed Central IDPMC8612244
Grant ListF32 GM139232 / GM / NIGMS NIH HHS / United States
R35 GM118024 / GM / NIGMS NIH HHS / United States
BB/R000042/1 / BB_ / Biotechnology and Biological Sciences Research Council / United Kingdom
MR/R000328/1 / MRC_ / Medical Research Council / United Kingdom