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J. Biol. Chem., Vol. 280, Issue 22, 21202-21211, June 3, 2005

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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^*

C. Michael Reynolds, Suzanne R. Kalb, Robert J. Cotter, and Christian R. H. Raetz¶

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

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 -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.

Received for publication, January 26, 2005 , and in revised form, March 28, 2005.

^* This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H. R.) and GM-64402 (to R. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Duke University Medical Center, P. O. Box 3711, Durham, NC. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.

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