Abstract

Previously we revealed that flagellin proteins in Pseudomonas syringae pv. tabaci 6605 (Pta 6605) were glycosylated with a trisaccharide, modified viosamine (mVio)-rhamnose-rhamnose and that glycosylation was required for virulence. We further identified some glycosylation-related genes, including vioA, vioB, vioT, fgt1, and fgt2. In this study, we newly identified vioR and vioM in a so-called viosamine island as biosynthetic genes for glycosylation of mVio in Pta 6605 by the mass spectrometry (MS) of flagellin glycan in the respective mutants. Furthermore, characterization of the mVio-related genes and MS analyses of flagellin glycans in other pathovars of P. syringae revealed that mVio-related genes were essential for mVio biosynthesis in flagellin glycans, and that P. syringae pv. syringae B728a, which does not possess a viosamine island, has a different structure of glycan in its flagellin protein.

Highlights

  • Protein glycosylation is found in eukaryotes and in prokaryotes

  • These results indicate that both flagellins from pv. tabaci (Pta) 6605 and P. aeruginosa PAK strain may possess similar glycan

  • We found that the viosamine aminotransferase (vioA), vioB, and vioT genes and four other genes may be involved in the glycosylation of flagellin by the synthesis of dTDP-modified viosamine (mVio)

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Summary

Introduction

Protein glycosylation is found in eukaryotes and in prokaryotes. bacterial glycoproteins were found in various animal pathogenic bacteria, including Pseudomonas aeruginosa, Campylobacter coli, C. jejuni, Helicobacter pylori, Aeromonas caviae, Escherichia coli, and Neisseria meningitides, the reports of glycoproteins in phytopathogenic bacteria were restricted to P. syringae and Acidovorax avenae [1,2,3,4]. Glycosylation of flagellins was reported to be important for virulence in animal pathogenic bacteria such as P. aeruginosa and C. jejuni [5,6] and in plant pathogenic bacteria P. syringae pv. We previously reported that flagellin glycans from Pta 6605 and Pgl race 4 were composed of two or three rhamnosyl residues and one D-Quip4N(3-hydroxy-1-oxobutyl)2Me residue (modified viosamine, mVio) [9,10,11,12]. We determined molecular masses of flagellin glycan of each strain and compared the organization of various viosamine islands. Tomato T1 [20], NCPPB1108 [21] and. FF5 [22] enable us to compare the genomic organization of various viosamine islands. Structural analyses of genomic organization and biochemical analyses of flagellin glycans revealed that flagellin glycans have both universality and diversity

Results and Discussion
Putative Biosynthetic Pathway of dTDP-mVio
Bacteria and Culture Conditions
DNA Cloning and Generation of Mutant Strains
Purification of Flagellin Proteins
Preparation of Glycosylated Peptides
MALDI-TOF Mass Spectrometry
LC-ESI Mass Spectrometry
Conclusions

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