Olea-Ozuna, R. J. J.; Furlan, B.; Gong, H.; Massidda, O.; Boll, J. M.

Gram-negative bacteria rely on an asymmetric outer membrane (OM) for barrier integrity, with phospholipids confined to the inner leaflet and glycolipids such as lipopolysaccharide (LPS) or lipooligosaccharide (LOS) forming the outer leaflet. Although LPS/LOS was long considered essential, recent findings challenge this view, leaving the mechanistic basis and evolutionary flexibility unclear. Here, we identify lipid asymmetry as a structural checkpoint that governs access to LOS-independent survival. Using Acinetobacter baumannii as a model, we show that disrupting retrograde phospholipid transport and surface phospholipid degradation destabilizes OM lipid balance, creating a permissive state that enables emergence of LOS-deficient, colistin-resistant variants. Integrated lipidomic and transcriptomic analyses reveal a staged remodeling program that reinforces lipoprotein scaffolds, rewires peptidoglycan synthesis, and expands trafficking pathways to stabilize a glycolipid-free envelope. Critically, loss of LOS coincides with sharp repression of PBP1A, and maintaining its activity blocks adaptation, demonstrating interdependence between OM and peptidoglycan homeostasis. We propose a three-state model--basal, permissive, adapted--that explains how envelope architecture gates evolutionary trajectories to antibiotic resistance.