Immobile lipopolysaccharides and outer membrane proteins differentially segregate in growing Escherichia coli
Author(s)
Type
Journal Article
Abstract
The outer membrane (OM) of gram-negative bacteria is a robust, impermeable barrier that excludes many classes of antibiotics. Contrary to the classical model of an asymmetric lipid bilayer, recent evidence suggests the OM is predominantly an asymmetric proteolipid membrane (APLM). Outer leaflet lipopolysaccharides (LPS) that surround integral β-barrel outer membrane proteins (OMPs) are shared with other OMPs to form a supramolecular network in which the levels of OMPs approach those of LPS. Some of the most abundant OMPs in the Escherichia coli OM are trimeric porins. How porins and LPS are incorporated into the OM of growing bacteria is poorly understood. Here, we use live-cell imaging and microfluidics to investigate how LPS, labeled using click chemistry, and the porin OmpF, labeled using the bacteriocin colicin N, are incorporated into the E. coli OM. Diffraction-limited fluorescence microscopy shows OmpF and LPS to be uniformly distributed and immobile. However, clustering of both macromolecules becomes evident by superresolution microscopy, which is also the case for their biogenesis proteins, BamA and LptD, respectively. Notwithstanding these common organizational features, OmpF insertion into the OM is cell-cycle-dependent leading to binary partitioning and strong polar accumulation of old OmpF. Old LPS on the other hand is diluted ~50% at each division cycle by new LPS, resulting in only mild polar accumulation of preexisting LPS. We conclude that although LPS and OMPs are destined to form the APLM their insertion dynamics are fundamentally different, which has major implications for understanding how the OM is assembled.
Date Issued
2025-03-11
Date Acceptance
2025-01-07
Citation
Proceedings of the National Academy of Sciences of USA, 2025, 122 (10)
ISSN
0027-8424
Publisher
National Academy of Sciences
Journal / Book Title
Proceedings of the National Academy of Sciences of USA
Volume
122
Issue
10
Copyright Statement
© 2025 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).
License URL
Identifier
10.1073/pnas.2414725122
Publication Status
Published
Article Number
e2414725122
Date Publish Online
2025-03-03