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J. Biol. Chem., Vol. 282, Issue 49, 36077-36089, December 7, 2007

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An Undecaprenyl Phosphate-Aminoarabinose Flippase Required for Polymyxin Resistance in Escherichia coli^*

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the PmrA transcription factor and is also present in E. coli, is necessary for the maintenance of polymyxin resistance. We previously elucidated the roles of pmrHFIJK in the biosynthesis and attachment of L-Ara4N to lipid A and renamed these genes arn-BCADT, respectively. We now propose functions for the last two genes of the operon, pmrL and pmrM. Chromosomal inactivation of each of these genes in an E. coli pmrA^c parent switched its phenotype from polymyxin-resistant to polymyxin-sensitive. Lipid A was no longer modified with L-Ara4N, even though the levels of the lipid-linked donor of the L-Ara4N moiety, undecaprenyl phosphate--L-Ara4N, were not reduced in the mutants. However, the undecaprenyl phosphate--L-Ara4N present in the mutants was less concentrated on the periplasmic surface of the inner membrane, as judged by 4-5-fold reduced labeling with the inner membrane-impermeable amine reagent N-hydroxysulfosuccin-imidobiotin. In an arnT mutant of the same pmrA^c parent, which lacks the enzyme that transfers the L-Ara4N unit to lipid A but retains the same high levels of undecaprenyl phosphate--L-Ara4N as the parent, N-hydroxysulfosuccinimidobiotin labeling was not reduced. These results implicate pmrL and pmrM, but not arnT, in transporting undecaprenyl phosphate--L-Ara4N across the inner membrane. PmrM and PmrL, now renamed ArnE and ArnF because of their involvement in L-Ara4N modification of lipid A, may be subunits of an undecaprenyl phosphate--L-Ara4N flippase.

Received for publication, July 26, 2007 , and in revised form, September 17, 2007.

^* This work was supported in part by National Institutes of Health Grant GM-51310 (to C. R. H. R.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Present address: Dept. of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706.

² Supported by the LIPID MAPS Large Scale Collaborative Grant GM-069338 from the National Institutes of Health.

³ To whom correspondence should be addressed: Dept. of Biochemistry, Duke University Medical Center, P. O. Box 3711, Durham, NC 27710. Tel.: 919-684-3384; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.

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