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

Abstract

AbstractGram-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 inEscherichia 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.Significance StatementThe Gram-negative bacterial outer membrane is a strong barrier against many antibiotics, limiting available options for treatment of infections. The stability of this protective barrier depends on the balance between different components in the membrane. ∼60 years ago, a conserved protein complex was identified to be important for outer membrane stability, but its true physiological function has remained elusive. Here, we establish that the primary role of this complex is to maintain outer membrane lipid homeostasis, and hence stability. Our work reshapes the existing paradigms of how this complex contributes to the expansion and division of the cell envelope, and will inform future strategies for the development of antibiotics.