Abstract

Recent advances in the field of prokaryotic N-glycosylation have established a foundation for the pathways and proteins involved in this important posttranslational protein modification process. To continue the study of the Methanococcus voltae N-glycosylation pathway, characteristics of known eukaryotic, bacterial, and archaeal proteins involved in the N-glycosylation process were examined and used to select candidateM. voltae genes for investigation as potential glycosyl transferase and flippase components. The targeted genes were knocked out via linear gene replacement, and the resulting effects on N-glycan assembly were identified through flagellin and surface (S) layer protein glycosylation defects. This study reports the finding that deletion of two putative M. voltae glycosyl transferase genes, designated aglC (for archaeal glycosylation) and aglK, interfered with proper N-glycosylation. This resulted in flagellin and S-layer proteins with significantly reduced apparent molecular masses, loss of flagellar assembly, and absence of glycan attachment. Given previous knowledge of both the N-glycosylation pathway in M. voltae and the general characteristics of N-glycosylation components, it appears that AglC and AglK are involved in the biosynthesis or transfer of diacetylated glucuronic acid within the glycan structure. In addition, a knockout of the putative flippase candidate gene (Mv891) had no effect on N-glycosylation but did result in the production of giant cells with diameters three to four times that of wild-type cells.