title: Identification of a gene involved in the biosynthesis pathway of the terminal sugar of the archaellin N-linked tetrasaccharide in Methanococcus maripaludis.
abstract: In Methanococcus maripaludis, the three archaellins which comprise the archaellum are modified at multiple sites with an N-linked tetrasaccharide with the structure of Sug-4-β-ManNAc3NAmA6Thr-4-β-GlcNAc3NAcA-3-β-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-L-erythro-hexos-5-ulo-1,5-pyranose, so far found exclusively in this species. In this study, a six-gene cluster mmp1089-1094, neighboring one of the genomic regions already known to contain genes involved with the archaellin N-glycosylation pathway, was examined for its potential involvement in the archaellin N-glycosylation or sugar biosynthesis pathway. The co-transcription of these six genes was demonstrated by RT-PCR. Mutants carrying an in-frame deletion in mmp1090, mmp1091 or mmp1092 were successfully generated. The Δmmp1090 deletion mutant was archaellated when examined by electron microscopy and mass spectrometry analysis of purified archaella showed that the archaellins were modified with a truncated N-glycan in which the terminal sugar residue and the threonine linked to the third sugar residue were missing. Both gene annotation and bioinformatic analyses indicate that MMP1090 is a UDP-glucose 4-epimerase, suggesting that the unique terminal sugar of the archaellin N-glycan might be synthesised from UDP-glucose or UDP-N-acetylglucosamine with an essential early step in synthesis catalysed by MMP1090. In contrast, no detectable phenotype related to archaellin glycosylation was observed in mutants deleted for either mmp1091 or mmp1092 while attempts to delete mmp1089, mmp1093 and mmp1094 were unsuccessful. Based on its demonstrated involvement in the archaellin N-glycosylation pathway, we designated mmp1090 as aglW.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, K7L 3N6, Canada.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, K7L 3N6, Canada.
authors.forename: Gareth M
authors.initials: GM
authors.lastname: Jones
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, K7L 3N6, Canada.
authors.forename: Cedric
authors.initials: C
authors.lastname: Brimacombe
authors.affiliation: Department of Life Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima, 727-0023, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Uchida
authors.affiliation: Department of Life Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima, 727-0023, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Aizawa
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council, Ottawa, K1A 0R6, Canada.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Logan
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council, Ottawa, K1A 0R6, Canada. john.kelly@nrc-cnrc.gc.ca.
authors.forename: John F
authors.initials: JF
authors.lastname: Kelly
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, K7L 3N6, Canada. jarrellk@queensu.ca.
authors.forename: Ken F
authors.identifier: http://orcid.org/0000-0001-5254-8818
authors.initials: KF
authors.lastname: Jarrell
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D002240:Carbohydrate Sequence; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D019848:Gene Expression Regulation, Archaeal; D019766:Genes, Archaeal; D006031:Glycosylation; D017017:Methanococcus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D005810:Multigene Family; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis; D014534:UDPglucose 4-Epimerase / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D014534:UDPglucose 4-Epimerase
keywords: Archaea; Archaella; In-frame deletion; Mass spectrometry; Methanogens; N-linked glycosylation