Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

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

^* To whom correspondence should be addressed. Email: christina.schaeffer{at}boku.ac.at.

	Abstract

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of [ 2)--L-Rhap-(13)--L-Rhap-(12)--L-Rhap-(1 ] repeating units, with a 2-O-methyl modification of the terminal trisaccharide at the non-reducing end of the glycan chain, a core saccharide composed of two to three -L-rhamnoses, and a -D-galactose residue as linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as 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.