The Surface Layer (S-layer) Glycoprotein of Geobacillus stearothermophilus NRS 2004/3a

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→] n= 13–18, with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core →3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→ and the linkage glycose β-d-Galp inO-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.