Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

SummaryRecently, the Surface (S)-layer glycoprotein of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius was found to be N-glycosylated with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose. However, genetic analyses of genes involved in the N-glycosylation process in Crenarchaeota were missing so far. In this study we identify a gene cluster involved in the biosynthesis of sulfoquinovose and important for the assembly of the S-layer N-glycans. A successful markerless in-frame deletion of agl3 resulted in a decreased molecular mass of the S-layer glycoprotein SlaA and the flagellin FlaB, indicating a change in the Nglycan composition. Analyses with nanoLC ES-MS/MS confirmed the presence of only a reduced trisaccharide structure composed of Man 1GlcNAc2, missing the sulfoquinovose, a mannose and glucose. Biochemical studies of the recombinant Agl3 confirmed the proposed function as a UDP-sulfoquinovose synthase. Furthermore, S. acidocaldarius cells lacking agl3 had a significantly lower growth rate at elevated salt concentrations compared with the background strain, underlining the importance of the N-glycosylation to maintain an intact and stable cell envelope, to enable the survival of S. acidocaldarius in its extreme environment.

c Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man 2 Glc 1 GlcNAc 2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agl16 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the ⌬agl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the ⌬agl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N-glycan size.