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J. Biol. Chem., Vol. 283, Issue 6, 3507-3518, February 8, 2008

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Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO^*

Wayne L. Miller¹², Mauricia J. Matewish¹³, David J. McNally⁴, Noboru Ishiyama^¶5, Erin M. Anderson, Dyanne Brewer, Jean-Robert Brisson, Albert M. Berghuis^¶6, and Joseph S. Lam⁷

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, the ^¶Departments of Biochemistry and Microbiology and Immunology, McGill University, Montreal, Quebec H3A 1A4, and the Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

Received for publication, October 29, 2007

^* This work was supported in part by operating grants from the Canadian Cystic Fibrosis Foundation and Canadian Institute of Health Research Grants MOP-14687 (to J. S. L.) and MT-13107 (to A. M. B.). 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 Tables S1-S4, Figs. S1-S4, and additional references.

¹ Both authors contributed equally to this work and should be considered first co-authors.

² Recipient of a doctoral research award from the Canadian Institute of Health Research. Present address: Dept. of Biochemistry, McGill University, Montreal, Quebec H3A 1A4, Canada.

³ Supported by a doctoral studentship and currently holds a postdoctoral fellowship from the Canadian Cystic Fibrosis Foundation. Present address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.

⁴ Present address: Dept. of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.

⁵ Recipient of a Studentship from the Canadian Cystic Fibrosis Foundation. Present address: Division of Signaling Biology, Ontario Cancer Institute, Toronto, Ontario M5G 1L7, Canada.

⁶ Holds a Canada Research Chair in Structural Biology.

⁷ Holds a Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology. To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Tel.: 519-824-4120 (Ext. 53832); Fax: 519-837-1802; E-mail: jlam{at}uoguelph.ca.

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