Phospholipid flipping into the outer leaflet of the bacterial outer membrane compensates for severe reduction in protein content

The Gram-negative bacterial outer membrane (OM) is a critical and versatile surface layer that provides selective permeability to nutrients and antimicrobial agents and protection against environmental mechanical stress. Studying the biophysical properties of the OM, e.g., mechanical stiffness and permeability, and their underlying molecular interactions offers structural insights into the essentiality of the OM and mechanisms to destabilize or bypass this barrier for conquering antimicrobial resistance. Here, we characterized an E. coli mutant with a global reduction in OM proteins due to the deletion of bamD, which causes reduction in OM stiffness, increased membrane permeability, and OM rupture in spent medium. The suppression of OM rupture required the additional deletion of pldA, which encodes a phospholipase that removes mislocated phospholipids on the outer leaflet of the OM, suggesting that phospholipids flipping into the outer leaflet is required to fill the space vacated by the loss of major OM proteins, which otherwise causes imbalanced material content between leaflets and an interleaflet stress that weakens the OM. Despite rescuing OM rupture, these suppressor mutants with abundant phospholipids on the OM outer leaflet did not restore the mechanical stiffness of the OM and showed rapid cell lysis upon oscillatory hyperosmotic loading, suggesting that beta-barrel proteins are required for OM mechanical strength.