Agl22 and Agl23 are involved in the synthesis and utilization of the lipid‐linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica.

Like both eukaryotes and bacteria, archaea can decorate proteins with N‐ and O‐linked glycans. Whereas pathways and roles of N‐glycosylation have been studied in several model archaeal organisms, little is known of O‐glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S‐layer glycoproteins are modified by an N‐linked glucose‐α‐(1, 2)‐[sulfoquinovosamine‐β‐(1, 6)‐]galactose trisaccharide and an O‐linked glucose‐α‐(1, 4)‐galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose‐DolP onto galactose‐DolP to form the glucose‐α‐(1, 4)‐galactose‐DolP precursor of the N‐glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase‐associated protein responsible for flipping of hexose‐DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N‐ and O‐linked glycans onto target protein occurs on the outer surface of the cell using hexose‐DolPs as sugar donors. Deletion mutant showed that N‐ and O‐glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica. [ABSTRACT FROM AUTHOR]