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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

Viosamine island in Pta 6605 and generation of vioM and vioR mutant strains. The nucleotide sequences for the glycosylation and viosamine islands of Pta 6605 and P. aeruginoas PAK were deposited in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AB499894, AB061230 and AF332547, respectively. Each gene is named by the putative function of the product: transporter (tp), glutamine synthetase (gs), dTDP-viosamine aminotransferase (vioA), methyltransferase in modified viosamine (mVio) (vioM), mVio transferase (vioT), 3-oxoacyl-(acyl-carrier-protein) synthase III (vioS), dTDP-viosamine acetyltransferase (vioB), 3-oxoacyl-(acyl-carrier-protein) reductase (vioR), and acyl-carrier protein (acp). Primers used for PCR are indicated by arrows. Each primer sequence is described in the Experimental Section. The flagellar gene cluster from flgL to fliD in Pta 6605 and the P. aeruginosa PAK strain is also shown with a comparison of homologous genes. Percentages show amino acid identity.
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f1-genes-02-00788: Viosamine island in Pta 6605 and generation of vioM and vioR mutant strains. The nucleotide sequences for the glycosylation and viosamine islands of Pta 6605 and P. aeruginoas PAK were deposited in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AB499894, AB061230 and AF332547, respectively. Each gene is named by the putative function of the product: transporter (tp), glutamine synthetase (gs), dTDP-viosamine aminotransferase (vioA), methyltransferase in modified viosamine (mVio) (vioM), mVio transferase (vioT), 3-oxoacyl-(acyl-carrier-protein) synthase III (vioS), dTDP-viosamine acetyltransferase (vioB), 3-oxoacyl-(acyl-carrier-protein) reductase (vioR), and acyl-carrier protein (acp). Primers used for PCR are indicated by arrows. Each primer sequence is described in the Experimental Section. The flagellar gene cluster from flgL to fliD in Pta 6605 and the P. aeruginosa PAK strain is also shown with a comparison of homologous genes. Percentages show amino acid identity.

Mentions: A viosamine island was previously isolated from Pta 6605 by PCR methods [11]. A detailed homology search of seven ORFs in this island revealed that all ORFs had corresponding homologous genes in a glycosylation island in the P. aeruginosa PAK strain [25] as shown in Figure 1. These results indicate that both flagellins from Pta 6605 and P. aeruginosa PAK strain may possess similar glycan. Major components of flagellin glycan were reported to be rhamnose, mannose, glucose and 4-amino-4,6-dideoxyglucose (viosamine) in P. aeruginosa PAK strain, although its whole structure was not elucidated yet [26]. It is not known whether each gene in viosamine cluster of P. aeruginosa has the same function to the corresponding homologous gene, because each amino acid identity was relatively low. However, the genes in viosamine cluster in P. aeruginosa seem to also contribute the modification of flagellin glycan. We found that not only the vioA, vioB, and vioT genes but also four other genes may be involved in the glycosylation of flagellin by the synthesis of dTDP-mVio. Therefore, we redesignated the gene homologous to methyltransferase as vioM, 3-oxoacyl-(acyl-carrier protein) synthase III as vioS, 3-oxoacyl-(acyl-carrier protein) reductase as vioR, and hp2 (hypothetical protein gene 2) as acp. Similarities of the deduced amino acid sequences of VioM vs. OrfG, VioS vs. OrfC, VioR vs. OrfD, and ACP vs. OrfB are 12%, 35%, 28%, and 19%, respectively (Table 1). Sequence-specific deletion of vioM or vioR was confirmed by genomic PCR and sequencing.


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)

Viosamine island in Pta 6605 and generation of vioM and vioR mutant strains. The nucleotide sequences for the glycosylation and viosamine islands of Pta 6605 and P. aeruginoas PAK were deposited in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AB499894, AB061230 and AF332547, respectively. Each gene is named by the putative function of the product: transporter (tp), glutamine synthetase (gs), dTDP-viosamine aminotransferase (vioA), methyltransferase in modified viosamine (mVio) (vioM), mVio transferase (vioT), 3-oxoacyl-(acyl-carrier-protein) synthase III (vioS), dTDP-viosamine acetyltransferase (vioB), 3-oxoacyl-(acyl-carrier-protein) reductase (vioR), and acyl-carrier protein (acp). Primers used for PCR are indicated by arrows. Each primer sequence is described in the Experimental Section. The flagellar gene cluster from flgL to fliD in Pta 6605 and the P. aeruginosa PAK strain is also shown with a comparison of homologous genes. Percentages show amino acid identity.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927599&req=5

f1-genes-02-00788: Viosamine island in Pta 6605 and generation of vioM and vioR mutant strains. The nucleotide sequences for the glycosylation and viosamine islands of Pta 6605 and P. aeruginoas PAK were deposited in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AB499894, AB061230 and AF332547, respectively. Each gene is named by the putative function of the product: transporter (tp), glutamine synthetase (gs), dTDP-viosamine aminotransferase (vioA), methyltransferase in modified viosamine (mVio) (vioM), mVio transferase (vioT), 3-oxoacyl-(acyl-carrier-protein) synthase III (vioS), dTDP-viosamine acetyltransferase (vioB), 3-oxoacyl-(acyl-carrier-protein) reductase (vioR), and acyl-carrier protein (acp). Primers used for PCR are indicated by arrows. Each primer sequence is described in the Experimental Section. The flagellar gene cluster from flgL to fliD in Pta 6605 and the P. aeruginosa PAK strain is also shown with a comparison of homologous genes. Percentages show amino acid identity.
Mentions: A viosamine island was previously isolated from Pta 6605 by PCR methods [11]. A detailed homology search of seven ORFs in this island revealed that all ORFs had corresponding homologous genes in a glycosylation island in the P. aeruginosa PAK strain [25] as shown in Figure 1. These results indicate that both flagellins from Pta 6605 and P. aeruginosa PAK strain may possess similar glycan. Major components of flagellin glycan were reported to be rhamnose, mannose, glucose and 4-amino-4,6-dideoxyglucose (viosamine) in P. aeruginosa PAK strain, although its whole structure was not elucidated yet [26]. It is not known whether each gene in viosamine cluster of P. aeruginosa has the same function to the corresponding homologous gene, because each amino acid identity was relatively low. However, the genes in viosamine cluster in P. aeruginosa seem to also contribute the modification of flagellin glycan. We found that not only the vioA, vioB, and vioT genes but also four other genes may be involved in the glycosylation of flagellin by the synthesis of dTDP-mVio. Therefore, we redesignated the gene homologous to methyltransferase as vioM, 3-oxoacyl-(acyl-carrier protein) synthase III as vioS, 3-oxoacyl-(acyl-carrier protein) reductase as vioR, and hp2 (hypothetical protein gene 2) as acp. Similarities of the deduced amino acid sequences of VioM vs. OrfG, VioS vs. OrfC, VioR vs. OrfD, and ACP vs. OrfB are 12%, 35%, 28%, and 19%, respectively (Table 1). Sequence-specific deletion of vioM or vioR was confirmed by genomic PCR and sequencing.

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