Persistent LPS insertion, spatial segregation and vesicle biogenesis drive growth-independent adaptation of the Escherichia coli outer membrane

The barrier and load-bearing functions of the Gram-negative bacterial outer membrane (OM) depend on ordered, dense packing of lipopolysaccharide (LPS), the major constituent of its outer leaflet. Despite its importance, LPS spatiotemporal dynamics and turnover mechanisms remain poorly understood. Existing models posit that LPS turnover only occurs via passive dilution during growth-dependent OM expansion, restricting adaptive LPS turnover, especially in nutrient-limited conditions. Here, using innovative pulse-chase LPS metabolic labelling techniques in combination with super-resolution microscopy, we demonstrate that Escherichia coli maintains OM homeostasis through continuous removal of pre-existing LPS and insertion of new LPS, even during stationary phase. We show that newly inserted LPS localises at discrete sites across the OM, remaining spatially segregated from pre-existing, background LPS. These observations challenge established OM organisational principles, suggesting an insertion-trapping mechanism can maintain LPS-rich clusters independent of a thermodynamically-driven phase separation. Time-lapse super-resolution imaging, biochemical assays, and nanoparticle tracking collectively reveal that OM vesicle (OMV) release mediates background LPS clearance. Our findings reveal bacteria have the capacity to remodel their surface architecture through OMV-mediated LPS turnover in a growth- independent manner. These insights redefine OM homeostasis and establish OMV biogenesis as fundamental to OM adaptation in Gram-negative bacteria.