Glycosylation is a frequent and heterogeneous posttranslational protein modification occurring in all domains of life. While protein N-glycosylation at asparagine and Oglycosylation at serine, threonine or hydroxyproline residues have been studied in great detail, only few data are available on O-glycosidic attachment of glycans to the amino acid tyrosine. In this study, we describe the identification and characterization of a bacterial protein tyrosine O-glycosylation system. In the Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051^T, a polysaccharide consisting of [→3)-β-D-Galp-(1[α-DGlcp-(1→6)] →4)-β-D-ManpNAc-(1→] repeating units is O-glycosidically linked via an adaptor with the structure -[GroA-2→OPO₂→4-β-D-ManpNAc-(1→4)] →3)-α-LRhap-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-DGalp-(1→ to specific tyrosine residues of the S-layer protein SpaA. A ~24.3-kb S-layer glycosylation (slg) gene cluster encodes the information necessary for the biosynthesis of this glycan chain within 18 open reading frames (ORF). The corresponding translation products are involved in the biosynthesis of nucleotide-activated monosaccharides, assembly and export as well as in the transfer of the completed polysaccharide chain to the S-layer target protein. All ORFs of the cluster, except those encoding the nucleotide sugar biosynthesis enzymes and the ATP binding cassette (ABC) transporter integral transmembrane proteins, were disrupted by the insertion of the mobile group II intron Ll. LtrB, and S-layer glycoproteins produced in mutant backgrounds were analyzed by mass spectrometry. There is evidence that the glycan chain is synthesized in a process comparable to the ABC-transporter-dependent pathway of the lipopolysaccharide O-polysaccharide biosynthesis.Furthermore, with the protein WsfB, we have identified an O-oligosaccharyl:protein transferase required for the formation of the covalent β-D-Gal→Tyr linkage between the glycan chain and the S-layer protein.