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

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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.

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Acknowledgments

We thank the Leaf Tobacco Research Laboratory of Japan Tobacco Inc. for providing P. syringae pv. tabaci 6605. This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN).

Author information

Correspondence to Yuki Ichinose.

Additional information

The nucleotide sequence of the viosamine-related gene cluster has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB499894.

Communicated by G. Klug.

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Nguyen, L.C., Yamamoto, M., Ohnishi-Kameyama, M. et al. Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci . Mol Genet Genomics 282, 595–605 (2009). https://doi.org/10.1007/s00438-009-0489-8

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Keywords

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