Lipopolysaccharide insertion and vesicle mediated turnover drive growth independent outer membrane adaptation in Escherichia coli.

The Gram-negative outer membrane is a load-bearing permeability barrier dependent on ordered lipopolysaccharide (LPS) packing in its outer leaflet. How this organisation is maintained after LPS insertion, and whether bacteria can remodel LPS independently of growth, remain unclear. Existing models attribute LPS turnover to passive dilution during elongation and division, limiting adaptation as growth slows. Here we show that, as Escherichia coli enters stationary phase and elongation slows, new LPS insertion continues while pre-existing LPS is preferentially removed through outer membrane vesicles, enabling growth-independent surface remodelling. Pulse-chase metabolic labelling and super-resolution microscopy reveal that newly inserted LPS localises to discrete sites and remains segregated from pre-existing LPS. Spatiotemporal analysis supports an insertion-trapping model in which localised insertion and restricted lateral diffusion maintain LPS-rich patches without coarsening into larger domains. Time-lapse imaging, biochemical fractionation and nanoparticle tracking identify vesicle release as a route that uncouples LPS turnover from cell growth.