Independent tuning of outer membrane fluidity and mechanics in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria forms a protective barrier that combines selective permeability with mechanical load-bearing capacity, properties linked to its asymmetric bilayer structure with lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet. Unlike most PL bilayers, the OM typically exhibits limited lateral diffusion, resulting in a gel-like surface with spatially organized proteins and LPS. The molecular basis of this physical state and its relationship with envelope mechanics remain unclear. Here, we show that increasing PL levels in the outer leaflet or truncating LPS core oligosaccharides increases OM fluidity by disrupting LPS packing. In contrast, reduced LPS abundance or disruption of divalent cation-mediated crosslinking primarily reduces OM stiffness with little effect on fluidity. Thus, OM fluidity and mechanical stiffness can be tuned independently through distinct molecular interactions. This separation of physical control mechanisms provides a framework for understanding how Gram-negative bacteria modulate OM properties during environmental adaptation and envelope homeostasis.