Biogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well-studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans-envelope Tol-Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol-Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess PLs in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol-Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol-Pal complex may promote OM invagination during cell division.

Summary 15Biogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for

23The outer membrane (OM) is an essential component of the Gram-negative bacterial cell 24 envelope that protects cells against external threats such as antibiotics. To maintain a stable and 25 functional OM barrier, cells require distinct mechanisms to ensure a balance of proteins and 26 lipids in the membrane. Crucial to this is the proper transport and assembly of various OM 27 components, of which the process of phospholipid (PL) transport is least understood. How OM 28 assembly pathways are coordinated to achieve homeostasis is also unclear. In this study, we set 29 out to identify potential mechanism(s) that can alleviate OM lipid dyshomeostasis in Escherichia 30 coli. Cells lacking the Tol-Pal complex accumulate excess PLs in the OM due to defective 31 retrograde PL transport. Here, we isolated mutations in enterobacterial common antigen (ECA) 32 biosynthesis that restore OM barrier function in these strains; build-up of biosynthetic 33 intermediates along the ECA pathway is key to this rescue. Interestingly, these ECA mutations 34 re-establish OM lipid homeostasis in cells lacking the Tol-Pal complex yet do not act by 35 restoring retrograde PL transport. Furthermore, a novel diacylglycerol pyrophosphoryl-linked 36 ECA species structurally similar to PLs can be detected in the inner membrane of ECA mutants. 37 We therefore propose a model where these unique species may modulate anterograde PL 38 transport to overcome OM lipid dyshomeostasis. Our work provides insights into bacterial lipid 39 transport across the cell envelope and highlights previously unappreciated effects of ECA 40 intermediates in OM biology. 41 42 3 Author Summary 43 44Biological membranes define cellular boundaries, allow compartmentalization, and represent a 45 prerequisite for life; yet, our understanding of membrane biogenesis and stability remain 46 rudimentary. In Gram-negative bacteria, the outer membrane prevents entry of toxic substances, 47 conferring intrinsic resistance against many antibiotics. How the outer membrane is assembled, 48 specifically lipid trafficking processes are not well understood. How this membrane is stably 49 maintained is also unclear. In this study, we discovered that intermediates along the biosynthetic 50 pathway of an exopolysaccharide exhibit stabilizing effects on outer membranes with lipid 51 imbalance in Escherichia coli. Our work suggests that these intermediates modulate phospholipid 52 trafficking within the double-membrane cell envelope to achieve outer membrane lipid 53 homeostasis. Furthermore, it provides a starting point to begin identifying hitherto unknown 54 phospholipid transport systems in Gram-negative bacteria, which are potential targets for the 55 development of future antibiotics. 56 57 58 Gram-negative bacteria are surrounded by a multilayered cell envelope consisting of the 59 inner membrane (IM), the peptidoglycan layer, and the outer membrane (OM). This envelope 60 structure, in particular the OM, plays an essential role in preventing toxic molecules from 61 en...

Gram-negative bacteria are surrounded by a multilayered cell envelope consisting of the inner membrane (IM), the peptidoglycan layer, and the outer membrane (OM). This envelope structure, in particular the OM, plays an essential role in preventing toxic molecules from entering the cell, contributing to intrinsic resistance of Gram-negative bacteria against many antibiotics and detergents (Nikaido, 2003). The OM bilayer is asymmetric and has a unique lipid composition, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet. In the presence of divalent cations, LPS molecules in the outer leaflet pack together to form an impervious monolayer (Raetz and Whitfield, 2002); OM structure and lipid asymmetry are thus key determinants for its