β-Barrel proteins dictate the effect of core oligosaccharide composition on outer membrane mechanics.

The outer membrane is the defining structure of Gram-negative bacteria. We previously demonstrated that it is a major load-bearing component of the cell envelope and is therefore critical to the mechanical robustness of the bacterial cell. Here, to determine the key molecules and moieties within the outer membrane that underlie its contribution to cell envelope mechanics, we measured cell-envelope stiffness across several sets of mutants with altered outer-membrane sugar content, protein content, and electric charge. To decouple outer membrane stiffness from total cell envelope stiffness, we developed a novel microfluidics-based "osmotic force-extension" assay. In tandem, we developed a method to increase throughput of microfluidics experiments by performing them on color-coded pools of mutants. We found that truncating the core oligosaccharide, deleting the β-barrel protein OmpA, or deleting lipoprotein outer membrane-cell wall linkers all had the same modest, convergent effect on total cell-envelope stiffness in Escherichia coli. However, these mutations had large, variable effects on the ability of the cell wall to transfer tension to the outer membrane during large hyperosmotic shocks. Surprisingly, altering the electric charge of lipid A had little effect on the mechanical properties of the envelope. Finally, the presence or absence of OmpA determined whether truncating the core oligosaccharide decreased or increased envelope stiffness (respectively), revealing sign epistasis between these components. Based on these data we propose a putative structural model in which the spatial interactions between lipopolysaccharides, β-barrel proteins, and phospholipids coordinately determine cell envelope stiffness.