Molecular biological characterization of surface layer glycan sythesis in gram-positive bacteria

The project has been granted by the Austrian Science Fund (FWF) in May 1998, started on October 1, 1998 and terminated on September 30, 2001. Project targets: 1. Genetic studies on the characterization of genes involved in the biosynthesis of the S-layer glycoprotein glycan of Aneurinibacillus thermoaerophilus DSM 10155. 2. The obtained data should be complemented by biochemical analyses on activated intermediate products of A. thermoaerophilus. 3. Electron microscopical analyses of antibody-labelled ultra-thin sections should demonstrate the localization of enzymes participating in glycan synthesis of S-layer glycoproteins. At the beginning of the genetic studies there was work dealing with the biosynthesis of dTDP-L-rhamnose in Salmonella, which was part of a co-operation with Prof. C. Whitfield, University of Guelph, Canada. After the functional characterization of the four enzymes involved in this reaction, crystallization studies performed by Prof. J.H. Naismith, University of St. Andrews, Scotland, UK, lead to an exact understanding of the examined enzymatic reactions. Within the framework of the experiments on the genetic characterization of dTDP-L-rhamnose biosynthesis in the Gram-positive bacterium A. thermoaerophilus DSM 10155 we were successful to isolate a DNA fragment carrying the genes for the biosynthesis of activated rhamnose as well as activated heptose. Previously, heptose was known only as a sugar component of Gram-negative bacteria. To sequence neighbouring regions of already analyzed DNA fragments effectively, a simple method of "chromosome walking" was developed. In the course of biochemical examinations, the structural analysis of the core-oligosaccharides from the S-layer glycoproteins of A. thermoaerophilus DSM 10155 was performed. For the first time the heterogenity of core- oligosaccharides had been examined in detail describing the occurrence of novel glycosidic linkages in these bacterial glycoproteins. Comparative studies with other S-layer glycoproteins led to a better understanding of the general assembly of these glycoconjugates. Besides the structural analysis of the glycans of A. thermoaerophilus DSM 10155 their biosynthesis was partially characterized for the first time. Presently, mass spectrometrical analyses are in progress in co-operation with Prof. Dr. J. Peter-Katalinic, University of Muenster, Germany. Electron microscopical studies of the pathway of nucleotide-activated L-rhamnose in A. thermoaerophilus DSM 10155 confirmed the results of the genetic and biochemical examinations. Summary With this project (P12966-MOB) a gradual molecular biological characterization of S-layer glycoprotein glycan biosynthesis of Gram-positive bacteria occurred successfully for the very first time. In part the results of the studies point to absolutely new biosynthesis pathways. The elucidation of glycan chain structures confirmed in one respect common knowledge. On the other hand substantially new insights into the general architecture of bacterial glycoproteins have been achieved. The results of this project can be considered basis for future applications of S- layer glycoproteins in biotechnology, biomedicine and nanotechnology. In such a way, for example, the distinct higher thermal stability of the glycosylation enzymes from the thermophilic organism Aneurinibacillus thermoaerophilus, in comparison to the same enzymes from the mesophilic strain Salmonella enterica, could lead to interesting biotechnological applications of these enzymes, e.g. their application on a "glyco chip" for the high- throughput screening.