Insertion of phospholipids into the outer leaflet fluidizes the bacterial outer membrane

The Gram-negative outer membrane (OM) is an asymmetric bilayer composed of porins, lipopolysaccharides (LPS), and phospholipids (PLs). Acting as both a permeability barrier and a mechanical structure, the OM provides essential protection against harmful chemicals and mechanical stresses. Investigating the fundamental physical properties of this membrane offers key insights into its structure and assembly. Using fluorescence recovery after photobleaching (FRAP), we quantified LPS fluidity in live cells under perturbations to the OM composition and demonstrated, in a mutant with reduced OM protein synthesis due to bamD deletion, that PL insertion into the outer leaflet drastically fluidizes the OM by disrupting LPS packing. In contrast, reduction in LPS or weakening of LPS crosslinks—achieved by inhibiting LPS synthesis or transport, or chelating divalent cations with EDTA—resulted in only a modest increase in fluidity. Combined with OM stiffness measurements, these assays revealed that reductions in porins versus LPS cause distinct changes in OM fluidity and stiffness, suggesting that porins and LPS contribute differently to the OM's physical properties.