Biosynthetic intermediates of the enterobacterial common antigen overcome outer membrane lipid dyshomeostasis in Escherichia coli

The outer membrane (OM) is an essential component of the Gram-negative bacterial cell envelope that protects cells against external threats such as antibiotics. To maintain a stable and functional OM barrier, cells require distinct mechanisms to ensure a balance of proteins and lipids in the membrane. Crucial to this is the proper transport and assembly of various OM components, of which the process of phospholipid (PL) transport is least understood. How OM assembly pathways are coordinated to achieve homeostasis is also unclear. In this study, we set out to identify potential mechanism(s) that can alleviate OM lipid dyshomeostasis in Escherichia coli . Cells lacking the Tol-Pal complex accumulate excess PLs in the OM due to defective retrograde PL transport. Here, we isolated mutations in enterobacterial common antigen (ECA) biosynthesis that restore OM barrier function in these strains; build-up of biosynthetic intermediates along the ECA pathway is key to this rescue. Interestingly, these ECA mutations re-establish OM lipid homeostasis in cells lacking the Tol-Pal complex yet do not act by restoring retrograde PL transport. Furthermore, a novel diacylglycerol pyrophosphoryl-linked ECA species structurally similar to PLs can be detected in the inner membrane of ECA mutants. We therefore propose a model where these unique species may modulate anterograde PL transport to overcome OM lipid dyshomeostasis. Our work provides insights into bacterial lipid transport across the cell envelope and highlights previously unappreciated effects of ECA intermediates in OM biology.