Primary role of the Tol-Pal complex in bacterial outer membrane lipid homeostasis

Gram-negative bacteria are defined by an outer membrane (OM) that contributes to envelope integrity and barrier function. Building this bilayer require proper assembly of lipopolysaccharides, proteins, and phospholipids, yet how the balance of these components is achieved is unclear. One system long known for ensuring OM stability is the Tol-Pal complex, which has been implicated in maintaining OM lipid homeostasis. However, assignment of Tol-Pal function has been challenging, owing to its septal localization and associated role(s) during division. Here, we uncouple the function of Tol-Pal in OM lipid homeostasis from its impact on cell division in Escherichia coli, by engineering a chimeric complex that loses septal enrichment. We demonstrate that this peripherally-localized Tol-Pal complex is fully capable of maintaining lipid balance in the OM, thus restoring OM integrity and barrier. Our work establishes the primary function of the Tol-Pal complex in OM lipid homeostasis, independent of its role during division.