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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Alexeyev MF, Shokolenko IN, Croughan TP (1995) New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in gram-negative bacteria. Can J Microbiol 41:1053–1055
Arora SK, Bangera M, Lory S, Ramphal R (2001) A genomic island in Pseudomonas aeruginosa carries the determinants of flagellin glycosylation. Proc Natl Acad Sci USA 98:9342–9347
Arora SK, Wolfgang MC, Lory S, Ramphal R (2004) Sequence polymorphism in the glycosylation island and flagellins of Pseudomonas aeruginosa. J Bacteriol 186:2115–2122
Eguchi H, Kaya S, Araki Y (1992a) Occurrence of 2,4-dihydroxy-3,3,4-trimethylpyroglutamic acid as an N-acyl substituent in the O-polysaccharide chain of the lipopolysaccharide of Vibrio anguillarum V-123. Carbohydr Res 231:147–158
Eguchi H, Kaya S, Araki Y, Kojima N, Yokota S (1992b) Structure of the O-polysaccharide chain of the lipopolysaccharide of Vibrio anguillarum V-123. Carbohydr Res 231:159–169
Feng L, Tao J, Guo H, Xu J, Li Y, Rezwan F, Reeves P, Wang L (2004) Structure of the Shigella dysenteriae 7 O antigen gene cluster and identification of its antigen specific genes. Microb Pathog 36:109–115
Fujiwara N, Nakata N, Maeda S, Naka T, Doe M, Yano I, Kobayashi K (2007) Structural characterization of a specific glycopeptidolipid containing a novel N-acyl-deoxy sugar from Mycobacterium intracellulare serotype 7 and genetic analysis of its glycosylation pathway. J Bacteriol 189:1099–1108
Ishii T, Kaneko S (1998) Oligosaccharides generated by partial hydrolysis of the borate-rhamnogalacturonan II complex from sugar beet. Phytochemistry 49:1195–1202
Knirel YA, Dashunin VV, Shashkov AS, Kochetkov NK, Dmitriev BA, Hofman IL (1988) Somatic antigens of Shigella: structure of the O-specific polysaccharide chain of the Shigella dysenteriae type 7 lipopolysaccharide. Carbohydr Res 179:51–60
Konishi T, Taguchi F, Iwaki M, Ohnishi-Kameyama M, Yamamoto M, Maeda I, Nishida Y, Ichinose Y, Yoshida M, Ishii T (2009) Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin. Carbohydr Res (in press)
L’vov VL, Shashkov AS, Dmitriev BA, Kochetkov NK, Jann B, Jann K (1984) Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7. Carbohydr Res 126:249–259
Logan SM (2006) Flagellar glycosylation—a new component of the motility repertoire? Microbiology 156:1249–1262
Marolda CL, Feldman MF, Valvano MA (1999) Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1). Microbiology 145:2485–2495
Poon KKH, Westman EL, Vinogradov E, Jin S, Lam JS (2008) Functional characterization of MigA and WapR: putative rhamnosyltransferases involved in outer core oligosaccharide biosynthesis of Pseudomonas aeruginosa. J Bacteriol 190:1857–1865
Power PM, Jennings MP (2003) The genetics of glycosylation in Gram-negative bacteria. FEMS Microbioi Lett 218:211–222
Schäfer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73
Schirm M, Arora SK, Verma A, Vinogradov E, Thibault P, Ramphal R, Logan SM (2004) Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa. J Bacteriol 186:2523–2531
Shimizu R, Taguchi F, Marutani M, Mukaihara T, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003) The ΔfliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics 269:21–30
Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1:784–791
Taguchi F, Shimizu R, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003a) Post-translational modification of flagellin determines the specificity of HR induction. Plant Cell Physiol 44:342–349
Taguchi F, Shimizu R, Nakajima R, Toyoda K, Shiraishi T, Ichinose Y (2003b) Differential effects of flagellins from Pseudomonas syringae pv. tabaci, tomato and glycinea on plant defense response. Plant Physiol Biochem 41:165–174
Taguchi F, Ogawa Y, Takeuchi K, Suzuki T, Toyoda K, Shiraishi T, Ichinose Y (2006a) A homologue of the 3-oxoacyl-(acyl carrier protein) synthase III gene located in the glycosylation island of Pseudomonas syringae pv. tabaci regulates virulence factors via N-acyl homoserine lactone and fatty acid synthesis. J Bacteriol 188:8376–8384
Taguchi F, Takeuchi K, Katoh E, Murata K, Suzuki T, Marutani M, Kawasaki T, Eguchi M, Katoh S, Kaku H, Yasuda C, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2006b) Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci. Cell Microbiol 8:923–938
Takeuchi K, Taguchi F, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003) Flagellin glycosylation island in Pseudomonas syringae pv. glycinea and its role in host specificity. J Bacteriol 185:6658–6665
Takeuchi K, Ono H, Yoshida M, Ishii T, Kato E, Taguchi F, Miki R, Murata K, Kaku H, Ichinose Y (2007) Flagellin glycans from two pathovars of Pseudomonas syringae contain rhamnose in d and l configurations in different ratios and modified 4-amino-4,6-dideoxyglucose. J Bacteriol 189:6945–6956
Wang Y, Xu Y, Perepelov AV, Qi Y, Knirel YA, Wang L, Feng L (2007) Biochemical characterization of dTDP-D-Qui4N and dTDP-D-Qui4NAc biosynthetic pathways in Shigella dysenteriae type 7 and Escherichia coli O7. J Bacteriol 189:8626–8635
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).
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.
About this article
Cite this article
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
- Flagellin glycosylation