Paenibacillus alvaei S-layer glycosylation pathway

Functional Biochemical Analysis and Crystallization of Selected Components from the S Layer Glycosylation Pathway of Paenibacillus alvei Paenibacillus alvei CCM 2051T is a Gram-positive, mesophilic bacterium that is completely covered with a glycosylated surface (S-) layer protein. Up to date, it is the only bacterium in which specific genetic manipulations of the inherent S-layer protein glycosylation pattern can be performed. Thus, it is an interesting candidate for the production of tailor-made, glycosylated S-layer proteins for nanobiotechnological applications. The genes involved in the glycosylation process are clustered in an slg (S-layer glycosylation) gene cluster of approximately 24 kB, which has been sequenced recently. By database sequence comparisons, glycosyltransferases, which are key in the assembly of the S-layer glycan chains, and genes for the biosynthesis of nucleotide-activated sugars as well as for transport and secretion of the cytoplasmatically assembled S-layer glycans have been identified. The function of comparable enzymes is well investigated in eukaryotes, but for those prokaryotes that synthesize glycoproteins, only scarce data is available. In the proposed research project three topics will be approached. 1. One work package is dealing with the expression and functional analysis of the so far not characterized glycosyltransferase WsfC. This presumably multifunctional enzyme possesses three different transferase domains and seems to be involved in the synthesis of the glycan side chain that is linked to the adaptor saccharide of the S layer glycan. Enzymes that are involved in branching of glycans are generally less investigated. Besides establishment of a functional assay for this enzyme, X-ray crystallographic studies will be performed in collaboration with Prof. H.M. Holden, University of Wisconsin, to learn about the functional mechanism of WsfC. 2. Since the S-layer glycoprotein (SGP) / secondary cell wall polymer (SCWP) complex of this bacterium, obtained after purification of the S-layer protein from native host cells, but also recombinant S-layer protein SpaA after expression in E. coli are completely water soluble, X-ray crystallography will be applied for characterization of the type of interaction between SGP and SCWP and, thus, the attachment of the SGP to the cell wall. 3. Tyrosine-linked O-glycans presumably are more stably linked than other O-glycans. In a systematic survey, the chemical stability of this type of linkage will be determined. Such stably linked glycans will definitely improve the application potential of tailor-made glycosylated S-layer proteins in nanobiotechnology.