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J. Biol. Chem., Vol. 282, Issue 19, 14463-14475, May 11, 2007

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Targeted Metabolomics Analysis of Campylobacter coli VC167 Reveals Legionaminic Acid Derivatives as Novel Flagellar Glycans^*

David J. McNally, Annie J. Aubry, Joseph P. M. Hui, Nam H. Khieu, Dennis Whitfield, Cheryl P. Ewing^¶, Patricia Guerry^¶, Jean-Robert Brisson, Susan M. Logan¹, and Evelyn C. Soo²

From the National Research Council, Institute for Biological Sciences, Ottawa, Ontario K1A 0R6, Canada, the National Research Council, Institute for Marine Biosciences, Halifax, Nova Scotia B3H 3Z1, Canada, and the ^¶Naval Medical Research Center, Silver Spring, Maryland 20910

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

Received for publication, November 30, 2006 , and in revised form, March 19, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank^TM/EBI Data Bank with accession number(s) EF141522 [GenBank] .

^* The work was supported by the National Research Council of Canada, NIAID, National Institutes of Health Grant RO1 AI43559, and the Military Infectious Disease Work Unit 6000.RAD1.DA3.A0308 (to P. G.). 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 Figs. S1-S6 and Table S1.

¹ To whom correspondence may be addressed: Institute for Biological Sciences, National Research Council, Rm. 3037, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada. Tel.: 613-990-0839; Fax: 613-952-9092; E-mail: susan.logan{at}nrc-cnrc.gc.ca.

² To whom correspondence may be addressed: Institute for Marine Biosciences, National Research Council, 1411 Oxford St., Halifax, Nova Scotia B3H 3Z1, Canada. Tel.: 902-426-0780; Fax: 902-426-9413; E-mail: evelyn.soo{at}nrc-cnrc.gc.ca.

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