Membrane trafficking of activated intermediates in the glycosylation pathway of S-layer proteins

The goal of this research project is a comprehensive description of the biosynthetic pathway for glycan formation of the surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Due to striking structural similarities between S-layer glycans and O-antigens of lipopolysaccharides (LPS) of Gram-negative bacteria rather similar biosynthesis pathways had been assumed in the past. Very recently the sequences of sgsE, the structural gene coding for the S-layer protein of G. stearothermophilus NRS 2004/3a, and of the entire S-layer glycan biosyntheis cluster have been determined. Preliminary data base sequence homology comparisons have shown that in the cluster genes are localized which code for an ABC transporter-dependent pathway including an ATP binding protein for S-layer glycan biosynthesis. From molecular biological investigations of different bacterial polysaccharides it is known that involvement of an ABC transporter-dependent pathway for oligosaccharide biosynthesis implies a route different to the classical LPS O-antigen biosynthesis pathway. A typical feature of this pathway is that the insertion of monosaccharides takes place at the non-reducing end of the glycan chain. In addition, for chain growth no polymerase per se is required. In the proposed project it will be determined by high resolution mass spectrometric analyses which activated intermediate products are formed in G. stearothermophilus NRS 2004/3a, and how membrane trafficking of this material across the cytoplasmic membrane and the peptidoglycan layer is facilitated. In addition, the ligation process of the pre-assembled glycan chains to the S-layer polypeptide will be investigated. The collected data will be correlated with results of the genetic characterization of S-layer glycan biosynthesis (FWF proposal P15612- MOB) to develop a detailed model for glycan biosynthesis of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a in particular, and for S-layer glycoproteins in general. These investigations are intended to be seen in the general context of planned future applications of recombinant bacterial S-layer neoglycoproteins in biotechnology, medicine and nanotechnology.