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

Margaret I. Kanipes, Shanhua Lin, Robert J. Cotter, Christian R.H. Raetz

Research output: Contribution to journalArticle

Abstract

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 IVA 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 IVA, 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.

Original languageEnglish (US)
Pages (from-to)1156-1163
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number2
DOIs
StatePublished - Jan 12 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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