Paenibacillus alvei S-layer glycosylation pathway

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.

The goals of the recently finished research project P22791-B11 comprised the structural and functional characterization of selected cell surface compounds which play a crucial role in the interaction between bacteria and the surrounding environment. In Gram-positive bacteria, non-classical secondary cell wall polymers (SCWPs) as a distinct class of glycopolymers attached to the multilayered peptidoglycan sacculus (PG), have been shown to mediate the attachment of bacterial cell surface layer proteins (S-layers) to PG. Pyruvate as an integral component of some SCWPs is of special interest because of its predicted involvement in the binding interaction to S-layer proteins which possess at their N-terminus surface layer homology (SLH) domains, usually present in triplicate. In Paenibacillus alvei CCM 2051T, the SCWP consists of [-3)-?-D-ManpNAc-(1-4)-?-D-GlcpNAc-(1-] disaccharide repeats with the ß-ManpNAc residue of each repeat modified with 4,6-linked pyruvate ketal. While the pyruvate ketal linked to ?-ManpNAc of the SCWP backbone is regarded as an ancestral and indispensable epitope for the binding interaction, its molecular basis is currently unknown. In close vicinity upstream of the S-layer structural gene spaA an ortholog of the SCWP biosynthesis key enzyme pyruvyltransferase CsaB is located on the bacterial chromosome. Initial modeling of the SLH region of the S-layer protein Sap of Bacillus anthracis revealed a pseudotrimer with critical amino acid motifs lining the grooves so that they would be accessible for SCWP binding. Our recent, to some extent delayed, research project on the 3D structure of the SpaA/SCWP complex of Paenibacillus alvei CCM 2051T aimed at getting insight into the structure and molecular interactions between pyruvylated SCWP and the SLH domain-containing S-layer protein SpaA. We hypothesized that distinct structural features of the pyruvate ketals and the amino sugar residues of the SCWP backbone are crucial for the binding interaction with critical amino acids of the SLH domains. Although the crystallization experiments are not yet finished we can expect that the 3D structure of the SpaA/SCWP complex of Paenibacillus alvei CCM 2051T will be solved in the near future. So far, no systematic study on the exact structural requirements of pyruvylated SCWP for interacting with SLH-domains of proteins has been performed. The occurrence of a pyruvyltransferase CsaB ortholog responsible for pyruvylation of SCWP in respective bacteria indicates a wide-spread utilization of this protein cell surface display mechanism. To extend our general knowledge on bacterial protein glycosylation for glycan engineering purposes we have in side projects also investigated specific glycosylation aspects of S-layer glycoprotein-carrying organisms like Sulfolobus acidocaldarius and Tannerella forsythia.