Functional analysis of surface layer glycoprotein assembly

This project focusses on the complete and detailed description of the genes located in the S-layer glycan biosynthesis cluster of the thermophilic, Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a. The work to be accomplished includes the characterization of all glycosyltransferases and other enzymes involved in the assembly of the S-layer glycoprotein glycan. In addition, for the first time, the interaction between the biosynthesis pathways of the S-layer protein of G. stearothermophilus NRS 2004/3a, which is encoded by the recently identified sgsE gene, and the S-layer glycan, which is mainly encoded by the S-layer biosynthesis cluster, will be investigated with respect to protein expression. These studies are required for potential applications of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a in the field of biomedicine and biotechnology. For this purpose, the S-layer protein glycosylation will be modified by the generation of specific gene knock-out mutants; furthermore, glycan chain microheterogeneity will be controlled at the molecular level. For the introduction of novel glycose residues into a mutant of this organism, bearing a truncated S-layer glycan, the heptose operon from Aneurinibacillus thermoaerophilus DSM 10155 has been chosen. The construction of an S-layer neoglycoprotein with defined, foreign glycan structures would point to the principal suitability of the bacterial system for the generation of recombinant, potentially medically relevant glycoproteins. In the future, the expression of several other important sugar receptor structures as S-layer neogycoproteins will be very likely. The outlined investigations constitute the foundations for the principal application potential of S-layer neoglycoproteins in biomedicine and nanotechnology. As bacteria have rather simple growth requirements and potential problems occurring with protein glycosylation can be excluded in a bacterial system, recombinant S-layer neoglycoproteins provide an attractive alternative to glycoproteins produced in eukaryotic cell lines.

The goals of the recently finished two-year project entitled "Functional Analysis of Surface Layer Glycoprotein Assembly" were the following: i. complete characterization of S-layer glycosylation by ahe detailed genetic analysis of the S-layer structural gene sgsE and the slg gene cluster of the Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a and attempts to modify of the naturally occurring S-layer glycosylation pattern of that organism, and ii. development of methods for a successful transformation of thermophilic bacilli. These studies are required for potential applications of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a in the field of biomedicine and biotechnology. For this purpose it was planned to modify S-layer protein glycosylation by appropriate genetic tools. Within the short duration of the project all attempts to transform the thermophilic bacilli, which included protoplast transformation and different protocols for electroporation, did not yield positive results. Even a collaboration with Prof. Neil E. Welker, Northwestern University, Evanston, IL, U.S.A., who is a pioneer in the field of genetic manipulation of thermophilic bacilli, proved to be unsuccessful. Besides the transformation attempts for specific G. stearothermophilus strains the entire slg gene cluster of G. stearothermophilus NRS 2004/3a was cloned and sequenced (GenBank accession no. AF328862). By Northern analysis and RT PCR experiments it could be demonstrated that the structural gene sgsE of that organism is transcribed monocistronically, whereas the slg gene cluster is transcribed polycistronically. In addition to these experiments the putative functions of all genes belonging to the slg gene cluster were predicted by data base sequence comparisons. This information is a prerequisite for the expression and functional characterization of the genes contained in the slg gene cluster. Due to the current inability to transform thermophilic bacilli successfully other expression systems will be used instead in future work. Only recently the heterologous expression of SgsE in Lactococcus lactis was possible and we are currently working on the functional expression of all genes of that gene cluster in different heterologous systems. Eventually, these approaches will lead to the creation of tailored S-layer neoglycoproteins for applications in biomedicine and nanotechnology. As bacteria have rather simple growth requirements and potential problems occurring with protein glycosylation might be excluded in a bacterial system, recombinant S-layer neoglycoproteins provide an attractive alternative to glycoproteins produced in eukaryotic cell lines.