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

研究成果: 雜誌貢獻文章

69 引文 (Scopus)

摘要

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.
原文英語
頁(從 - 到)633-644
頁數12
期刊Molecular Microbiology
72
發行號3
DOIs
出版狀態已發佈 - 五月 1 2009
對外發佈Yes

指紋

Methanococcus
Flagellin
Archaea
Glycosyltransferases
Polysaccharides
Genes
Acetylgalactosamine
Glucuronic Acid
Flagella
Gene Deletion
Threonine
Immunoblotting
Mass Spectrometry
Electron Microscopy
Proteins

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

引用此文

Identification of genes involved in the assembly and attachment of a novel flagellin N-linked tetrasaccharide important for motility in the archaeon Methanococcus maripaludis. / Vandyke, David J.; Wu, John; Logan, Susan M.; Kelly, John F.; Mizuno, Shino; Aizawa, Shin Ichi; Jarrell, Ken F.

於: Molecular Microbiology, 卷 72, 編號 3, 01.05.2009, p. 633-644.

研究成果: 雜誌貢獻文章

Vandyke, David J. ; Wu, John ; Logan, Susan M. ; Kelly, John F. ; Mizuno, Shino ; Aizawa, Shin Ichi ; Jarrell, Ken F. / Identification of genes involved in the assembly and attachment of a novel flagellin N-linked tetrasaccharide important for motility in the archaeon Methanococcus maripaludis. 於: Molecular Microbiology. 2009 ; 卷 72, 編號 3. 頁 633-644.
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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.",
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