Identification of genes involved in the assembly and attachment of a novel flagellin N-linked tetrasaccharide important for motility in the archaeon Methanococcus maripaludis

David J. Vandyke, John Wu, Susan M. Logan, John F. Kelly, Shino Mizuno, Shin Ichi Aizawa, Ken F. Jarrell

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

Recently, the flagellin proteins of Methanococcus maripaludis were found to harbour an N-linked tetrasaccharide composed of N-acetylgalactosamine, di-acetylated glucuronic acid, an acetylated and acetamidino-modified mannuronic acid linked to threonine, and a novel terminal sugar [(5S)-2-acetamido-2,4- dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in the assembly and attachment of this glycan, in-frame deletions were constructed in putative glycan assembly genes. Successful deletion of genes encoding three glycosyltransferases and an oligosaccharyltransferase (Stt3p homologue) resulted in flagellins of decreased molecular masses as evidenced by immunoblotting, indicating partial or completely absent glycan structures. Deletion of the oligosaccharyltransferase or the glycosyltransferase responsible for the transfer of the second sugar in the chain resulted in flagellins that were not assembled into flagella filaments, as evidenced by electron microscopy. Deletions of the glycosyltransferases responsible for the addition of the third and terminal sugars in the glycan were confirmed by mass spectrometry analysis of purified flagellins from these mutants. Although flagellated, these mutants had decreased motility as evidenced by semi-swarm plate analysis with the presence of each additional sugar improving movement capabilities.

Original languageEnglish
Pages (from-to)633-644
Number of pages12
JournalMolecular Microbiology
Volume72
Issue number3
DOIs
Publication statusPublished - May 1 2009
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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