Phosphate-responsive ornithine lipid biosynthesis remodels outer membrane composition and contributes to polymyxin tolerance

Bacteria frequently encounter environments in which essential nutrients are limiting. Phosphate limitation constrains phospholipid synthesis and challenges maintenance of the cell envelope. While many Gram-negative bacteria respond by producing phosphorous-free membrane lipids, the mechanisms and consequences of this remodeling remain poorly understood in Enterobacterales. Here, we characterize the genetic and physiological basis of ornithine lipid (OL) biosynthesis in the opportunistic pathogens Enterobacter cloacae and Klebsiella pneumoniae . We show that OL biosynthesis depends on OlsF, a bifunctional acyltransferase that catalyzes both acylation steps required for OL synthesis. Phosphate limitation promotes OlsF-dependent OL accumulation through the PhoBR two-component system. Membrane fractionation revealed that OlsF-derived OLs are present in both membrane compartments but are enriched in the outer membrane. OlsF-dependent OL production enhances polymyxin tolerance during phosphate limitation, linking nutrient availability to changes in antimicrobial susceptibility. Together, these findings identify OlsF-dependent OL synthesis as a phosphate-responsive membrane remodeling pathway that reshapes outer membrane composition and influences the physiological properties of the Gram-negative cell envelope.