Identification of genes involved in the acetamidino group modification of the flagellin N-Linked glycan of Methanococcus maripaludis

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DOIResolve DOI: http://doi.org/10.1128/JB.06686-11
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TypeArticle
Journal titleJournal of Bacteriology
ISSN0021-9193
Volume194
Issue10
Pages26932702; # of pages: 10
Subjectammonia; flagellin; glycan; glycosyltransferase; threonine; article; bacterial gene; bacterial growth; bacterium mutant; carbohydrate synthesis; controlled study; flagellum; gene deletion; gene identification; gene targeting; mass spectrometry; Methanococcus maripaludis; mmp1081 gene; mmp1082 gene; mmp1083 gene; nonhuman; operon; priority journal; Western blotting; Amino Acid Sequence; Blotting, Western; Carbohydrate Conformation; Flagellin; Gene Deletion; Gene Expression Regulation, Archaeal; Methanococcus; Mutation; Polysaccharides; Reverse Transcriptase Polymerase Chain Reaction; Archaea; Methanococcus maripaludis
AbstractN-linked glycosylation of protein is a posttranslational modification found in all three domains of life. The flagellin proteins of the archaeon Methanococcus maripaludis are known to be modified with an N-linked tetrasaccharide consisting of N-acetylgalactosamine (GalNAc), a diacetylated glucuronic acid (GlcNAc3NAc), an acetylated and acetamidino-modified mannuronic acid with a substituted threonine group (ManNAc3NAmA6Thr), and a novel terminal sugar residue [(5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in biosynthesis of the component sugars of this glycan, three genes, mmp1081, mmp1082, and mmp1083, were targeted for in-frame deletion, based on their annotation and proximity to glycosyltransferase genes known to be involved in assembly of the glycan. Mutants carrying a deletion in any of these three genes remained flagellated and motile. A strain with a deletion of mmp1081 had lower-molecular-mass flagellins in Western blots. Mass spectrometry of purified flagella revealed a truncated glycan with the terminal sugar absent and the threonine residue and the acetamidino group missing from the third sugar. No glycan modification was seen in either the Δmmp1082 or Δmmp1083 mutant grown in complex Balch III medium. However, a glycan identical to the Δmmp1081 glycan was observed when the Δmmp1082 or Δmmp1083 mutant was grown under ammonia-limited conditions. We hypothesize that MMP1082 generates ammonia and tunnels it through MMP1083 to MMP1081, which acts as the amidotransferase, modifying the third sugar residue of the M. maripaludis glycan with the acetamidino group. © 2012, American Society for Microbiology.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Institute for Biological Sciences (IBS-ISB)
Peer reviewedYes
NPARC number21269230
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Record identifier0761ecf8-9b64-4876-8c00-15af48aee1dc
Record created2013-12-12
Record modified2016-05-09
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