Abstract
Biological membranes, which comprise proteins, lipids, and glycans, serve as essential gatekeepers protecting cells from the external environment. In bacteria, phospholipids are a major class of membrane lipids, whose biology has extensively been studied in the Gram-negative organism Escherichia coli. As an adaptive mechanism, E. coli dynamically remodels its phospholipids in response to its environment, which may involve alterations of the structures and/or levels of existing lipids or the incorporation of exogenous substrates to form new phospholipid classes. Intriguingly, an unknown lipid was detected in E. coli and other Enterobacteriaceae. Detection of this lipid in E. coli grown in minimal media suggested its production using an endogenous metabolite. By coupling liquid chromatography mass spectrometry and metabolic incorporation, the lipid was identified as phosphatidylhomoserine (PHS). In E. coli, PHS was produced endogenously by phosphatidylserine synthase A (PssA), confirmed by the absence of PHS in an E. coli ΔpssA mutant, and its inability to incorporate exogenously supplied (L)-homoserine into its phospholipids. Furthermore, purified E. coli PssA (EcPssA) exhibited activity to utilize (L)-homoserine as an alternative substrate to make PHS in vitro. Interestingly, E. coli and other Enterobacteriaceae can decarboxylate PHS to form phosphatidylpropanolamine endogenously. When treated with (L)-homoserine, accumulation of PHS in E. coli was accompanied by a reduction in phosphatidylglycerol and phosphatidylethanolamine, due to competition for common metabolic intermediates. Overall, our findings on the endogenous production of PHS and phosphatidylpropanolamine re-established the baseline phospholipidome of E. coli and provided biochemical and cellular evidence on the substrate promiscuity of EcPssA.