Ca²⁺-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli. A novel membrane enzyme dependent upon phosphatidylethanolamine

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl₂ in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mM CaCl₂, is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl₂ is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mM CaCl₂ contain a novel pEtN transferase that uses the precursor Kdo₂-[4′-³²P]lipid IV_A as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mM MgCl₂, BaCl₂, or ZnCl₂. Hydrolysis of the in vitro reaction product, pEtN-Kdo₂-[4′-³²P]lipid IV_A, at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mM CaCl₂, which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mM CaCl₂ suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS.