Phosphatidylglycerol Is the Lipid Donor for Synthesis of Phospholipid-Linked Enterobacterial Common Antigen

The Gram-negative outer membrane is a permeability barrier preventing cellular entry of antibiotics. However, outer membrane biogenesis pathways are targets for small molecule development. The Gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. In Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella, and Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECA(PG)). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECA(PG) contributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, in Escherichia coli K-12, the reaction synthesizing ECA(PG) from ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECA(PG). Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased the synthesis of ECA(PG), whereas alterations expected to decrease levels of PG decreased the synthesis of ECA(PG). We discovered depletion of PG levels in strains that could synthesize ECA(PG), but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECA(PG) can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECA(PG) and provide a key insight into the reaction producing ECA(PG). In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor.IMPORTANCE The Gram-negative outer membrane is a permeability barrier preventing cellular entry of antibiotics. However, outer membrane biogenesis pathways are targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECA(PG), found in the outer membrane of Enterobacterales (e.g., Escherichia, Salmonella, and Klebsiella). ECA(PG) consists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate-ECA is a phospholipid headgroup. The details of the reaction forming this molecule from polymerized ECA precursor are unknown. We determined the lipid donor providing the phospholipid moiety is phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECA(PG) provides the opportunity for development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat Gram-negative infections.