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Identification of Genes Involved in the Glycosylation of Modified Viosamine of Flagellins in Pseudomonas syringae by Mass Spectrometry.

Yamamoto M, Ohnishi-Kameyama M, Nguyen CL, Taguchi F, Chiku K, Ishii T, Ono H, Yoshida M, Ichinose Y - Genes (Basel) (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. ymasanob@affrc.go.jp.

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.

No MeSH data available.


Related in: MedlinePlus

Extracted ion chromatograms (XIC) and mass spectra of P. syringae pv. syringae B728a. Asp-N digested glycopeptides were analyzed by LC-ESI/MS. XICs are of calculated m/z values in Table 2 corresponding to four glycopeptides, D139-F167 (a), D168-A188 (b), E189-I199 (c), and D200-S211 (d). Blue Ser residues have glycans. The dominant peak at 60.4 min in XIC (a) gave the mass spectrum (e), which showed [M+2H]2+ at m/z 1,935 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 2 (two protons)]/2 (double charge)) and [M+3H]3+ at m/z 1,291 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 3 (triple protons)] / 3 (triple charge)). The peak at m/z 1,834 is the fragment ion losing the terminal saccharide (orange circle). Other peaks at m/z 1,753, 1,680, 1,579, 1,499, and 1,426 are the fragment ions of a cascade of deglycosylation. The peaks in the XICs (b–d) also gave the mass spectra showing [M+H]+, [M+2H]2+, or [M+3H]3+ (the m/z values are underlined) accompanied by deglycosylated fragment ions (f–h, respectively). All glycopeptides have glycans composed of saccharides with masses of 203 (N-acetylated hexosamine, orange circle), 160 (methylated deoxyhexose, blue pentagon), and 146 (deoxyhexose, blue square) from the distal end. The symbol “+” or “2+” means the ion singly charged or doubly charged, respectively.
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f7-genes-02-00788: Extracted ion chromatograms (XIC) and mass spectra of P. syringae pv. syringae B728a. Asp-N digested glycopeptides were analyzed by LC-ESI/MS. XICs are of calculated m/z values in Table 2 corresponding to four glycopeptides, D139-F167 (a), D168-A188 (b), E189-I199 (c), and D200-S211 (d). Blue Ser residues have glycans. The dominant peak at 60.4 min in XIC (a) gave the mass spectrum (e), which showed [M+2H]2+ at m/z 1,935 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 2 (two protons)]/2 (double charge)) and [M+3H]3+ at m/z 1,291 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 3 (triple protons)] / 3 (triple charge)). The peak at m/z 1,834 is the fragment ion losing the terminal saccharide (orange circle). Other peaks at m/z 1,753, 1,680, 1,579, 1,499, and 1,426 are the fragment ions of a cascade of deglycosylation. The peaks in the XICs (b–d) also gave the mass spectra showing [M+H]+, [M+2H]2+, or [M+3H]3+ (the m/z values are underlined) accompanied by deglycosylated fragment ions (f–h, respectively). All glycopeptides have glycans composed of saccharides with masses of 203 (N-acetylated hexosamine, orange circle), 160 (methylated deoxyhexose, blue pentagon), and 146 (deoxyhexose, blue square) from the distal end. The symbol “+” or “2+” means the ion singly charged or doubly charged, respectively.

Mentions: Psy B728a flagellin conserves six serine residues, which are glycosylated in Pta 6605, although the identity of the amino acid sequence is lower among pathovars of P. syringae (Figure 5). However, the molecular mass of flagellin glycopeptide (N136-R210) of Psy B728a (Group IV) was obviously smaller than those of other pathovars/strains (Figure 6 and Table 2). The molecular mass of one glycan is 509 (Table 2), indicating the glycan structure is different from those in Groups I and II and that there is no viosamine-related saccharide, as indicated by the analysis of the gene cluster of the vioamine island. Analysis of Asp-N-digested flagellin with LC-ESI/MS revealed that Psy 728a also had six glycans at the 143, 164, 176, 183, 193, and 201 Ser residues (Figure 7). The glycan was composed of saccharides with the mass values of 203, 160, and 146 from the non-reducing end, which corresponded to N-acetylhexosamine, methylated deoxyhexose, and deoxyhexose, respectively. In Pta 6605 and Pgl race 4, fgt1 and fgt2, located upstream of fliC, encode flagellin glycosyltransferase, and Fgt1 transfers rhamnose to a serine residue [8]. Because all pathovars/strains of P. syringae investigated, including Psy 728a, conserve fgt1 and fgt2, the proximal deoxyhexose of the glycan chain seems to be rhamnose. However, the detailed structural analyses regarding the kinds of deoxyhexose and hexosamine, the positions of modifications, and the linkage types between saccharides are under investigation.


Identification of Genes Involved in the Glycosylation of Modified Viosamine of Flagellins in Pseudomonas syringae by Mass Spectrometry.

Yamamoto M, Ohnishi-Kameyama M, Nguyen CL, Taguchi F, Chiku K, Ishii T, Ono H, Yoshida M, Ichinose Y - Genes (Basel) (2011)

Extracted ion chromatograms (XIC) and mass spectra of P. syringae pv. syringae B728a. Asp-N digested glycopeptides were analyzed by LC-ESI/MS. XICs are of calculated m/z values in Table 2 corresponding to four glycopeptides, D139-F167 (a), D168-A188 (b), E189-I199 (c), and D200-S211 (d). Blue Ser residues have glycans. The dominant peak at 60.4 min in XIC (a) gave the mass spectrum (e), which showed [M+2H]2+ at m/z 1,935 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 2 (two protons)]/2 (double charge)) and [M+3H]3+ at m/z 1,291 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 3 (triple protons)] / 3 (triple charge)). The peak at m/z 1,834 is the fragment ion losing the terminal saccharide (orange circle). Other peaks at m/z 1,753, 1,680, 1,579, 1,499, and 1,426 are the fragment ions of a cascade of deglycosylation. The peaks in the XICs (b–d) also gave the mass spectra showing [M+H]+, [M+2H]2+, or [M+3H]3+ (the m/z values are underlined) accompanied by deglycosylated fragment ions (f–h, respectively). All glycopeptides have glycans composed of saccharides with masses of 203 (N-acetylated hexosamine, orange circle), 160 (methylated deoxyhexose, blue pentagon), and 146 (deoxyhexose, blue square) from the distal end. The symbol “+” or “2+” means the ion singly charged or doubly charged, respectively.
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Related In: Results  -  Collection

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f7-genes-02-00788: Extracted ion chromatograms (XIC) and mass spectra of P. syringae pv. syringae B728a. Asp-N digested glycopeptides were analyzed by LC-ESI/MS. XICs are of calculated m/z values in Table 2 corresponding to four glycopeptides, D139-F167 (a), D168-A188 (b), E189-I199 (c), and D200-S211 (d). Blue Ser residues have glycans. The dominant peak at 60.4 min in XIC (a) gave the mass spectrum (e), which showed [M+2H]2+ at m/z 1,935 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 2 (two protons)]/2 (double charge)) and [M+3H]3+ at m/z 1,291 ([3,869 (monoisotopic mass of glycosylated D139-F167) + 3 (triple protons)] / 3 (triple charge)). The peak at m/z 1,834 is the fragment ion losing the terminal saccharide (orange circle). Other peaks at m/z 1,753, 1,680, 1,579, 1,499, and 1,426 are the fragment ions of a cascade of deglycosylation. The peaks in the XICs (b–d) also gave the mass spectra showing [M+H]+, [M+2H]2+, or [M+3H]3+ (the m/z values are underlined) accompanied by deglycosylated fragment ions (f–h, respectively). All glycopeptides have glycans composed of saccharides with masses of 203 (N-acetylated hexosamine, orange circle), 160 (methylated deoxyhexose, blue pentagon), and 146 (deoxyhexose, blue square) from the distal end. The symbol “+” or “2+” means the ion singly charged or doubly charged, respectively.
Mentions: Psy B728a flagellin conserves six serine residues, which are glycosylated in Pta 6605, although the identity of the amino acid sequence is lower among pathovars of P. syringae (Figure 5). However, the molecular mass of flagellin glycopeptide (N136-R210) of Psy B728a (Group IV) was obviously smaller than those of other pathovars/strains (Figure 6 and Table 2). The molecular mass of one glycan is 509 (Table 2), indicating the glycan structure is different from those in Groups I and II and that there is no viosamine-related saccharide, as indicated by the analysis of the gene cluster of the vioamine island. Analysis of Asp-N-digested flagellin with LC-ESI/MS revealed that Psy 728a also had six glycans at the 143, 164, 176, 183, 193, and 201 Ser residues (Figure 7). The glycan was composed of saccharides with the mass values of 203, 160, and 146 from the non-reducing end, which corresponded to N-acetylhexosamine, methylated deoxyhexose, and deoxyhexose, respectively. In Pta 6605 and Pgl race 4, fgt1 and fgt2, located upstream of fliC, encode flagellin glycosyltransferase, and Fgt1 transfers rhamnose to a serine residue [8]. Because all pathovars/strains of P. syringae investigated, including Psy 728a, conserve fgt1 and fgt2, the proximal deoxyhexose of the glycan chain seems to be rhamnose. However, the detailed structural analyses regarding the kinds of deoxyhexose and hexosamine, the positions of modifications, and the linkage types between saccharides are under investigation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. ymasanob@affrc.go.jp.

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.

No MeSH data available.


Related in: MedlinePlus