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J. Biol. Chem., Vol. 279, Issue 30, 31237-31250, July 23, 2004

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Chromosomal and Plasmid-encoded Enzymes Are Required for Assembly of the R3-type Core Oligosaccharide in the Lipopolysaccharide of Escherichia coli O157:H7^*

Natalia A. Kaniuk, Evgeny Vinogradov¶, Jianjun Li¶, Mario A. Monteiro¶||, and Chris Whitfield**

From the Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1 and ^¶Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada

The type R3 core oligosaccharide predominates in the lipopolysaccharides from enterohemorrhagic Escherichia coli isolates including O157:H7. The R3 core biosynthesis (waa) genetic locus contains two genes, waaD and waaJ, that are predicted to encode glycosyltransferases involved in completion of the outer core. Through determination of the structures of the lipopolysaccharide core in precise mutants and biochemical analyses of enzyme activities, WaaJ was shown to be a UDP-glucose:(galactosyl) lipopolysaccharide -1,2-glucosyltransferase, and WaaD was shown to be a UDP-glucose:(glucosyl)lipopolysaccharide -1,2-glucosyltransferase. The residue added by WaaJ was identified as the ligation site for O polysaccharide, and this was confirmed by determination of the structure of the linkage region in serotype O157 lipopolysaccharide. The initial O157 repeat unit begins with an N-acetylgalactosamine residue in a -anomeric configuration, whereas the biological repeat unit for O157 contains -linked N-acetylgalactosamine residues. With the characterization of WaaJ and WaaD, the activities of all of the enzymes encoded by the R3 waa locus are either known or predicted from homology data with a high level of confidence. However, when core oligosaccharide structure is considered, the origin of an additional -1,3-linked N-acetylglucosamine residue in the outer core is unknown. The gene responsible for a nonstoichiometric -1,7-linked N-acetylglucosamine substituent in the heptose (inner core) region was identified on the large virulence plasmids of E. coli O157 and Shigella flexneri serotype 2a. This is the first plasmid-encoded core oligosaccharide biosynthesis enzyme reported in E. coli.

Received for publication, February 20, 2004 , and in revised form, May 19, 2004.

^* This work was supported in part by grants (to C. W.) from National Sciences and Engineering Research Council and the Canadian Bacterial Disease Network. 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.

Recipient of a National Sciences and Engineering Research Council PGS-B graduate scholarship.

^|| Present address: Dept. of Chemistry and Biochemistry, University of Guelph, Ontario N1G 2W1, Canada.

^** To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 53361); Fax: 519-837-1802; E-mail: cwhitfie{at}uoguelph.ca.

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