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

Certain strains of Escherichia coil and Salmonella contain lipopolysacchari de (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, coil mutant WBB06 (Brabetz, W,, Muller-Loennies, S., Hoist, O , and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate t hat the critical parameter determining the presence or absence of pEtN is t he concentration of CaCl2 in the medium. As judged by mass spectrometry, ha lf the LPS in WBB06, grown on nutrient broth with 5 mM CaCl2, is derivatize d with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl 2 is not. Membranes from E, coil WBB06 or wild-type W3110 grown on 5-50 mM CaCl2 contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-P -32]lipid IVA as an acceptor. Transferase is not present in membranes of E. coil grown with 5 mM MgCl2, BaCl2, or ZnCl2, Hydrolysis of the in vitro re action product, pEtN-Kdo(2)-[4'-P-32]lipid IVA, at pH 4.5 shows that the pE tN substituent is located on the outer Kdo moiety, Membranes from an E, coi l pss knockout mutant grown on 50 mM CaCl2, which lack phosphatidylethanola mine, do not contain measurable transferase activity unless exogenous phosp hatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mM CaCl2 suggests possible role(s) in establishin g transformation competence or resisting environmental stress, and represen ts the first example of a regulated covalent modification of the inner core of E. coil LPS.