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 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 CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(32)P]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 CaCl(2), 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 CaCl(2) 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.
Kanipes
M I
MI
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 , USA.
Lin
S
S
Cotter
R J
RJ
Raetz
C R
CR
D002118:Calcium / Q000494:pharmacology*; D002122:Calcium Chloride / Q000494:pharmacology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D004982:Ethanolaminephosphotransferase / Q000378:metabolism*; D004983:Ethanolamines / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000138:chemical synthesis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013400:Sugar Acids / Q000378:metabolism*
D004983:Ethanolamines; D008070:Lipopolysaccharides; D009844:Oligosaccharides; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; C005448:phosphorylethanolamine; D004982:Ethanolaminephosphotransferase; D002122:Calcium Chloride; D002118:Calcium