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J. Biol. Chem., Vol. 279, Issue 47, 49470-49478, November 19, 2004
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MsbA Transporter-dependent Lipid A 1-Dephosphorylation on the Periplasmic Surface of the Inner Membrane
TOPOGRAPHY OF FRANCISELLA NOVICIDA LpxE EXPRESSED IN ESCHERICHIA COLI*
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From the
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the ¶Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
The lipid A anchor of Francisella tularensis lipopolysaccharide (LPS) lacks both phosphate groups present in Escherichia coli lipid A. Membranes of Francisella novicida (an environmental strain related to F. tularensis) contain enzymes that dephosphorylate lipid A and its precursors at the 1- and 4'-positions. We now report the cloning and characterization of a membrane-bound phosphatase of F. novicida that selectively dephosphorylates the 1-position. By transferring an F. novicida genomic DNA library into E. coli and selecting for low level polymyxin resistance, we isolated FnlpxE as the structural gene for the 1-phosphatase, an inner membrane enzyme of 239 amino acid residues. Expression of FnlpxE in a heptose-deficient mutant of E. coli caused massive accumulation of a previously uncharacterized LPS molecule, identified by mass spectrometry as 1-dephospho-Kdo2-lipid A. The predicted periplasmic orientation of the FnLpxE active site suggested that LPS export might be required for 1-dephosphorylation of lipid A. LPS and phospholipid export depend on the activity of MsbA, an essential inner membrane ABC transporter. Expression of FnlpxE in the msbA temperature-sensitive E. coli mutant WD2 resulted in 90% 1-dephosphorylation of lipid A at the permissive temperature (30 °C). However, the 1-phosphate group of newly synthesized lipid A was not cleaved at the nonpermissive temperature (44 °C). Our findings provide the first direct evidence that lipid A 1-dephosphorylation catalyzed by LpxE occurs on the periplasmic surface of the inner membrane.
Received for publication, August 9, 2004 , and in revised form, August 31, 2004.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY713119
* This work was supported by National Institutes of Health (NIH) Grants R37-GM-51796 (to C. R. H. Raetz) and GM-54882 (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.
Supported by NIH Grant GM-08558 in Biological Chemistry (to Duke University).
|| To whom correspondence should be addressed: Dept. of Biochemistry, Duke University Medical Center, Box 3711, Durham, NC 27710. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.
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