BioAFM Journal Club

Phase Separation in the Outer Membrane of Escherichia coli

by Georgina Benn et al.

Key Points

  • The most abundant outer-membrane proteins in gram-negative bacteria form a network with only small gaps of phase-separated lipopolysaccharide across the entire bacterial surface ;
  • New domains form when phospholipids are mislocated at the surface, which renders cells more susceptible to some antibiotics;
  • Overall, the phase-separated nature of the outer membrane in gram-negative bacteria informs membrane integrity and barrier function.



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Proc. Nat. Acad. Sci. U.S.A. 2021, 118 (46), e2112237118
DOI: 10.1073/pnas.2112237118

In this article, high-resolution AFM was used to study the organization of the outer membrane of living bacterial E. coli strains at the nanometer scale. The outer membrane (OM) envelope of Gram-negative bacteria is composed of a functional barrier made up of phospholipids in its inner leaflet, lipopolysaccharides (LPS) in its outer leaflet, and a variety of outer membrane proteins (OMP) clustered across the surface. In order to address antimicrobial resistance, which is particularly prevalent in Gram-negative bacteria, it is necessary to achieve a deeper insight into the supramolecular organization of OMPs, which could lead to a structure-oriented rational design of antibiotics that would improve the efficacy of drug entry into pathogenic bacteria.

Using high-resolution AFM phase imaging on live, metabolically active bacteria complemented by specific OMP labeling, the authors identified large-scale, near-static, protein-rich networks interspersed with LPS-rich nanoscale domains on the surface of the bacteria. Structural analysis, assisted by single-particle tracking, identified pores and pore-free patches in the OM that can be attributed to hexagonal lattices of densely packed porin trimers (pores) superposed on to a smooth 2-5 nm high background (LPS patches). Using genetically engineered bacterial strains, the authors suggest that the predominant porin, OmpF, is recruited in the formation of the near-static network across the surface, whereas OmpA is embedded within the porin network and interacts non-covalently with the bacterial cell wall. Furthermore, in mutants, the spatial redistribution of phospholipids in the outer membrane, from the inner to the outer leaflet, correlates with the formation of new domains that trigger bacterial susceptibility to harsh environments.

      KEY TERMS:

  • High-resolution AFM, Amyloid, Gram-negative bacteria, E. coli, Outer membrane protein labeling, Antibiotic drug resistance