. "The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)-adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3-(WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis." . . "J. Biol. Chem." . "Messner P." . "Schaffer C." . "Steiner K." . "Kosma P." . "Novotny R." . "Seeberger P.H." . "Hofinger A." . "Zarschler K." . "Werz D.B." . "2008"^^ . "21120-21133" . "Molecular basis of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus." . "283" . "doi:10.1074/jbc.M801833200" .