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

Author:

Steiner Kerstin1,Novotny René1,Patel Kinnari2,Vinogradov Evgenij3,Whitfield Chris4,Valvano Miguel A.2,Messner Paul1,Schäffer Christina1

Affiliation:

1. Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria

2. Infectious Diseases Research Group, Siebens-Drake Medical Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada

3. Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada

4. Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Abstract

ABSTRACT The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [→2)-α- l -Rha p- (1→3)-β- l -Rha p -(1→2)-α- l -Rha p -(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 α- l -rhamnose residues, and a β- 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.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

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