UNLABELLED: The outer membrane (OM) of Gram-negative bacteria such as is an asymmetric bilayer with the glycolipid lipopolysaccharide (LPS) in the outer leaflet and glycerophospholipids in the inner. Nearly all integral OM proteins (OMPs) have a characteristic β-barrel fold and are assembled in the OM by the BAM complex, which contains one essential β-barrel protein (BamA), one essential lipoprotein (BamD), and three non-essential lipoproteins (BamBCE). A gain-of-function mutation in enables survival in the absence of BamD, showing that the essential function of this protein is regulatory. We demonstrate that the global reduction in OMPs caused by BamD loss weakens the OM, altering cell shape and causing OM rupture in spent medium. To fill the void created by OMP loss, PLs flip into the outer leaflet. Under these conditions, mechanisms that remove PLs from the outer leaflet create tension between the OM leaflets, which contributes to membrane rupture. Rupture is prevented by suppressor mutations that release the tension by halting PL removal from the outer leaflet. However, these suppressors do not restore OM stiffness or normal cell shape, revealing a possible connection between OM stiffness and cell shape.

SIGNIFICANCE STATEMENT: The outer membrane (OM) is a selective permeability barrier that contributes to the intrinsic antibiotic resistance of Gram-negative bacteria. Biophysical characterization of the roles of the component proteins, lipopolysaccharides, and phospholipids is limited by both the essentiality of the OM and its asymmetrical organization. In this study, we dramatically change OM physiology by limiting the protein content, which requires phospholipid localization to the outer leaflet and thus disrupts OM asymmetry. By characterizing the perturbed OM of various mutants, we provide novel insight into the links among OM composition, OM stiffness, and cell shape regulation. These findings deepen our understanding of bacterial cell envelope biology and provide a platform for further interrogation of OM properties.