A phosphoethanolamine transferase specific for the outer 3-deoxy-D-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant.
Addition of a phosphoethanolamine (pEtN) moiety to the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue of lipopolysaccharide (LPS) in WBB06, a heptose-deficient Escherichia coli mutant, occurs when cells are grown in 5-50 mM CaCl2 (Kanipes, M. I., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 1156-1163). A Ca2+-induced, membrane-bound enzyme was responsible for the transfer of the pEtN unit to the Kdo domain. We now report the identification of the gene encoding the pEtN transferase. E. coli yhjW was cloned and overexpressed, because it is homologous to a putative pEtN transferase implicated in the modification of the beta-chain heptose residue of Neisseria meningitidis lipo-oligosaccharide (Mackinnon, F. G., Cox, A. D., Plested, J. S., Tang, C. M., Makepeace, K., Coull, P. A., Wright, J. C., Chalmers, R., Hood, D. W., Richards, J. C., and Moxon, E. R. (2002) Mol. Microbiol. 43, 931-943). In vitro assays with Kdo2-4'-[32P]lipid A as the acceptor showed that YhjW (renamed EptB) utilizes phosphatidylethanolamine in the presence of Ca2+ to transfer the pEtN group. Stoichiometric amounts of diacylglycerol were generated during the EptB-catalyzed transfer of pEtN to Kdo2-lipid A. EptB is an inner membrane protein of 574 amino acid residues with five predicted trans-membrane segments within its N-terminal region. An in-frame replacement of eptB with a kanamycin resistance cassette rendered E. coli WBB06 (but not wild-type W3110) hypersensitive to CaCl2 at 5 mM or higher. Ca2+ hypersensitivity was suppressed by excess Mg2+ in the medium or by restoring the LPS core of WBB06. The latter was achieved by reintroducing the waaC and waaF genes, which encode LPS heptosyl transferases I and II, respectively. Our data demonstrate that pEtN modification of the outer Kdo protected cells containing heptose-deficient LPS from damage by high concentrations of Ca2+. Based on its sequence similarity to EptA(PmrC), we propose that the active site of EptB faces the periplasmic surface of the inner membrane.
Reynolds
C Michael
CM
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kalb
Suzanne R
SR
Cotter
Robert J
RJ
Raetz
Christian R H
CR
D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002240:Carbohydrate Sequence; D002384:Catalysis; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004075:Diglycerides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D004982:Ethanolaminephosphotransferase / Q000737:chemistry*; D017353:Gene Deletion; D005822:Genetic Vectors; D006539:Heptoses / Q000737:chemistry; D006868:Hydrolysis; D007477:Ions; D007612:Kanamycin / Q000494:pharmacology; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013400:Sugar Acids / Q000737:chemistry*; D013997:Time Factors
D002412:Cations; D017931:DNA Primers; D004075:Diglycerides; D029968:Escherichia coli Proteins; D006539:Heptoses; D007477:Ions; D008055:Lipids; D008070:Lipopolysaccharides; D013400:Sugar Acids; C023023:lipid-linked oligosaccharides; C002532:2-keto-3-deoxyoctonate; D007612:Kanamycin; C501859:EptB protein, E coli; D017853:Phosphotransferases (Alcohol Group Acceptor); D004982:Ethanolaminephosphotransferase; D008274:Magnesium; D002118:Calcium