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J. Biol. Chem., Vol. 283, Issue 7, 4332-4343, February 15, 2008

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PagP Activation in the Outer Membrane Triggers R3 Core Oligosaccharide Truncation in the Cytoplasm of Escherichia coli O157:H7^*

Abigail E. Smith¹, Sang-Hyun Kim¹², Feng Liu, Wenyi Jia³, Evgeny Vinogradov^¶, Carlton L. Gyles^||, and Russell E. Bishop⁴

From the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada, the Departments of Laboratory Medicine and Pathobiology, and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8 Canada, the ^¶Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A OR6, Canada, and the ^||Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1 Canada

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagP5-14 allele, which mostly eliminates a periplasmic amphipathic -helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier.

Received for publication, October 2, 2007 , and in revised form, December 6, 2007.

^* 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.

¹ Both authors contributed equally to this work.

² Supported by the Canadian Institutes of Health Research (CIHR) training program (postdoctoral) on the structure and function of membrane proteins linked to disease (University of Toronto). Present address: The National Primate Research Center, KRIBB, Daejeon 305-806, South Korea.

³ Present address: Shanghai Asia United Antibody Medical Co. Ltd., Zhang Jiang Hi-tech Park, Pudong, Shanghai 201203, China.

⁴ Work in the laboratory of this author was supported by CIHR Operating Grant MOP-84329. To whom correspondence should be addressed: Dept. of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 28810); Fax: 905-522-9033; E-mail: bishopr{at}mcmaster.ca.

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