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Journal of Bacteriology, April 2007, p. 2590-2598, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01592-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

Kerstin Steiner,¹ René Novotny,^1, Kinnari Patel,² Evgenij Vinogradov,³ Chris Whitfield,⁴ Miguel A. Valvano,² Paul Messner,¹ and Christina Schäffer^1*

Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria,¹ Infectious Diseases Research Group, Siebens-Drake Medical Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada,² Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada,³ Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada⁴

Received 13 October 2006/ Accepted 9 January 2007

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [2)--L-Rhap-(13)-ß-L-Rhap-(12)--L-Rhap-(1], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three -L-rhamnose residues, and a ß-D-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.

Published ahead of print on 19 January 2007.

Present address: Institut für Angewandte Genetik und Zellbiologie, Universität für Bodenkultur Wien, A-1190 Wien, Austria.

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