Tan, W. B.; Chng, S.-S.

Gram-negative bacteria are defined by an outer membrane (OM) that contributes to cell envelope integrity, and functions as an effective permeability barrier. The OM is thus critical for fitness and antibiotic resistance. Building this bilayer require proper assembly of lipopolysaccharides, proteins, and phospholipids, yet how the intricate balance of these components is achieved to ensure a stable OM is not well understood. One system long known to be important for OM stability is the Tol-Pal complex, which has recently been implicated in the maintenance of OM lipid homeostasis, possibly via retrograde phospholipid transport. However, assignment of its primary function has been challenging, owing to septal localization of Tol-Pal and its associated role(s) during cell division. Here, we uncouple the function of the Tol-Pal complex in OM lipid homeostasis from its impact on cell division in Escherichia coli, by preventing recruitment to mid-cell. The Tol-Pal complex comprises the inner membrane TolQRA and OM TolB-Pal subcomplexes. We engineer a TolA variant that forms a chimeric complex with ExbBD, which are homologous to TolQR, resulting in loss of septal enrichment of Tol-Pal. We demonstrate that this peripherally-localized complex cannot rescue division defects in strains lacking TolQRA, but restores OM integrity and barrier. Importantly, this chimeric complex is fully capable of maintaining lipid balance in the OM, independent of the role of Tol-Pal in cell division. Our work establishes the primary function of the Tol-Pal complex in OM lipid homeostasis, and provides novel insights on how this conserved system also contributes to cell division.