Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci

Linh Chi Nguyen, Masanobu Yamamoto, Mayumi Ohnishi-Kameyama, Salamah Andi, Fumiko Taguchi, Masako Iwaki, Mitsuru Yoshida, Tadashi Ishii, Tomoyuki Konishi, Kazuhiko Tsunemi, Yuki Ichinose

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Glycosylation of flagellin contributes to swimming and swarming motilities, adhesion ability, and consequently virulence in Pseudomonas syringae pv. tabaci 6605. Glycans attached to six serine residues are located in the central region of the flagellin polypeptide. The glycan structure at position Ser 201 was recently revealed to consist of two l-rhamnoses and one modified 4-amino-4,6-dideoxyglucose (viosamine). To clarify the mechanisms for glycosylation of modified viosamine, genes encoding dTDP-viosamine aminotransferase (vioA), dTDP-viosamine acetyltransferase (vioB), and viosamine-derivative transferase (vioT) were isolated and defective mutants were generated. MALDI-TOF-MS analysis of a lysyl endopeptidase-digested peptide including all six glycosylation sites from each flagellin indicated that the molecular masses of the three flagellin mutants were reduced with highly heterogeneous patterns at regular intervals of 146 Da in the mass range from m/z 13,819 to 15,732. The data indicated that the glycopeptides obtained from mutants had glycans consisting only of deoxyhexose instead of the flagellin glycans including the viosamine derivatives determined previously. The motility and virulence on host tobacco leaves were strongly impaired in the ΔvioA mutant and were weakly reduced in the ΔvioB and ΔvioT mutant strains. These results suggest that the genes vioA, vioB, and vioT are essential for glycosylation of flagellin, and accordingly are required for bacterial virulence.

Original languageEnglish
Pages (from-to)595-605
Number of pages11
JournalMolecular Genetics and Genomics
Volume282
Issue number6
DOIs
Publication statusPublished - Dec 2009

Fingerprint

Pseudomonas syringae
Flagellin
Glycosylation
Polysaccharides
Transferases
Transaminases
Genes
Virulence
lysyl endopeptidase
Peptides
Acetyltransferases
Glycopeptides
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Serine
Tobacco
4-amino-4,6-dideoxyglucose

Keywords

  • Flagellin glycosylation
  • Lipopolysaccharide
  • Motility
  • O-antigen
  • Viosamine

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci. / Nguyen, Linh Chi; Yamamoto, Masanobu; Ohnishi-Kameyama, Mayumi; Andi, Salamah; Taguchi, Fumiko; Iwaki, Masako; Yoshida, Mitsuru; Ishii, Tadashi; Konishi, Tomoyuki; Tsunemi, Kazuhiko; Ichinose, Yuki.

In: Molecular Genetics and Genomics, Vol. 282, No. 6, 12.2009, p. 595-605.

Research output: Contribution to journalArticle

Nguyen, LC, Yamamoto, M, Ohnishi-Kameyama, M, Andi, S, Taguchi, F, Iwaki, M, Yoshida, M, Ishii, T, Konishi, T, Tsunemi, K & Ichinose, Y 2009, 'Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci', Molecular Genetics and Genomics, vol. 282, no. 6, pp. 595-605. https://doi.org/10.1007/s00438-009-0489-8
Nguyen, Linh Chi ; Yamamoto, Masanobu ; Ohnishi-Kameyama, Mayumi ; Andi, Salamah ; Taguchi, Fumiko ; Iwaki, Masako ; Yoshida, Mitsuru ; Ishii, Tadashi ; Konishi, Tomoyuki ; Tsunemi, Kazuhiko ; Ichinose, Yuki. / Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci. In: Molecular Genetics and Genomics. 2009 ; Vol. 282, No. 6. pp. 595-605.
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AU - Ohnishi-Kameyama, Mayumi

AU - Andi, Salamah

AU - Taguchi, Fumiko

AU - Iwaki, Masako

AU - Yoshida, Mitsuru

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AB - Glycosylation of flagellin contributes to swimming and swarming motilities, adhesion ability, and consequently virulence in Pseudomonas syringae pv. tabaci 6605. Glycans attached to six serine residues are located in the central region of the flagellin polypeptide. The glycan structure at position Ser 201 was recently revealed to consist of two l-rhamnoses and one modified 4-amino-4,6-dideoxyglucose (viosamine). To clarify the mechanisms for glycosylation of modified viosamine, genes encoding dTDP-viosamine aminotransferase (vioA), dTDP-viosamine acetyltransferase (vioB), and viosamine-derivative transferase (vioT) were isolated and defective mutants were generated. MALDI-TOF-MS analysis of a lysyl endopeptidase-digested peptide including all six glycosylation sites from each flagellin indicated that the molecular masses of the three flagellin mutants were reduced with highly heterogeneous patterns at regular intervals of 146 Da in the mass range from m/z 13,819 to 15,732. The data indicated that the glycopeptides obtained from mutants had glycans consisting only of deoxyhexose instead of the flagellin glycans including the viosamine derivatives determined previously. The motility and virulence on host tobacco leaves were strongly impaired in the ΔvioA mutant and were weakly reduced in the ΔvioB and ΔvioT mutant strains. These results suggest that the genes vioA, vioB, and vioT are essential for glycosylation of flagellin, and accordingly are required for bacterial virulence.

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