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Dieter Spiteller - One of the best experts on this subject based on the ideXlab platform.
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identification of the biosynthetic gene cluster for 3 methylarginine a toxin produced by Pseudomonas syringae pv syringae 22d 93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, Annette Wensing, Helge Weingart, Matthias S. Ullrich, J Hofmann, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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3 methylarginine from Pseudomonas syringae pv syringae 22d 93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-methylarginine biosynthesis to be identified.
Beate Volksch - One of the best experts on this subject based on the ideXlab platform.
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identification of the biosynthetic gene cluster for 3 methylarginine a toxin produced by Pseudomonas syringae pv syringae 22d 93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, Annette Wensing, Helge Weingart, Matthias S. Ullrich, J Hofmann, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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3 methylarginine from Pseudomonas syringae pv syringae 22d 93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-methylarginine biosynthesis to be identified.
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biological control of Pseudomonas syringae pv glycinea by epiphytic bacteria under field conditions
Microbial Ecology, 2001Co-Authors: Beate Volksch, R MayAbstract:The efficacy of a bacterial strain as a biocontrol agent in the field may be related to the ecological similarity between the biocontrol agent and the target pathogen. Therefore, a number of different Pseudomonas syringae strains were evaluated for their antagonistic activities in vitro (agar-diffusion assay) and in planta (greenhouse assay) against the target pathogen, Pseudomonas syringae pv. glycinea. Six strains of five different pathovars were found to be antagonistic in vitro as well as in planta. The epiphytic fitness of the antagonistic Pseudomonas syringae strain 22d/93 and its two antibiotic-resistant mutants were examined on soybean plants in the fields. After adaptation the parental strain and its mutants had the ability to establish and maintain large epiphytic populations (about 106 cfu/g FW) over the whole growing season after a single spray inoculation. The epiphytic behaviors of the mutants and the parent were not significantly different. The introduced bacteria did not influence the total bacterial population size. When the antagonist was coinoculated with the pathogen, the development of the pathogen was significantly reduced during the whole growing season. When the antagonistic strain was inoculated 4 weeks in advance of the pathogen, this antagonistic effect could be markedly enhanced. The final population size of the pathogen reached just 104 cfu/g FW and was significantly reduced to 0.12% compared to the pathogen alone. This study demonstrates that biological control of foliar pathogens through colonization of the host plants with near isogenic or ecologically similar antagonistical strains seems to be a realistic goal.
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characterization of two epiphytic bacteria from soybean leaves with antagonistic activities against Pseudomonas syringae pv glycinea
Journal of Basic Microbiology, 1996Co-Authors: Beate Volksch, Jorg Nuske, R MayAbstract:The strains 48b/90 and 22d/93 are naturally occurring ephiphytes which were isolated from soybean leaves. On the basis of pheno- and genotypic characteristics 48b/90 was identified as Erwinia herbicola and 22d/93 as Pseudomonas syringae. These two isolates produced biological active substances against different indicator organisms. The E. herbicola strain showed clear antagonistic properties against Escherichia coli and Pseudomonas syringae pv. glycinea, but not against Geotrichum candidum. 22d/93 was active against P. glycinea and G. candidum, but not against E. coli. Strain 48b/90 produced at least two different inhibitors: an antibiotic substance and an inhibitor of the alginate synthesis. Strain 22d/93 produced at least three different compounds inhibitory to P. glycinea and one to G. candidum. Their activities against the bacterial blight pathogen, P. glycinea, can be observed in planta, too. Under the influences of the antagonists the pathogen multiplied at lower rates and to lower stationary phase population levels. The development of bacterial blight symptoms was suppressed.
Sam Cartinhour - One of the best experts on this subject based on the ideXlab platform.
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Genomic Plasticity Enables Phenotypic Variation of Pseudomonas syringae pv. tomato DC3000
2014Co-Authors: Zhongmeng Bao, Sam Cartinhour, Christopher R. Myers, Brian H Kvitko, Alan Collmer, Paul V. Stodghill, Hanh Lam, Hai-lei Wei, Suma Chakravarthy, Peter SchweitzerAbstract:Whole genome sequencing revealed the presence of a genomic anomaly in the region of 4.7 to 4.9 Mb of the Pseudomonas syringae pv. tomato (Pst) DC3000 genome. The average read depth coverage of Pst DC3000 whole genome sequencing results suggested that a 165 kb segment of the chromosome had doubled in copy number. Further analysis confirmed the 165 kb duplication and that the two copies were arranged as a direct tandem repeat. Examination of the corresponding locus in Pst NCPPB1106, the parent strain of Pst DC3000, suggested that the 165 kb duplication most likely formed after the two strains diverged via transposition of an ISPsy5 insertion sequence (IS) followed by unequal crossing over between ISPsy5 elements at each end of the duplicated region. Deletion of one copy of the 165 kb region demonstrated that the duplication facilitated enhanced growth in some culture conditions, but did not affect pathogenic growth in host tomato plants. These types of chromosomal structures are predicted to be unstable and we have observed resolution of the 165 kb duplication to single copy and its subsequent re-duplication. These data demonstrate the role of IS elements in recombination events that facilitate genomic reorganization in P. syringae.
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regulons of three Pseudomonas syringae pv tomato dc3000 iron starvation sigma factors
Applied and Environmental Microbiology, 2013Co-Authors: Bronwyn G Butcher, Sam Cartinhour, Paul Stodghill, Christopher R. Myers, Bryan Swingle, Eric MarkelAbstract:Pseudomonas syringae pv. tomato DC3000 contains genes for 15 sigma factors. The majority are members of the extracytoplasmic function class of sigma factors, including five that belong to the iron starvation subgroup. In this study, we identified the genes controlled by three iron starvation sigma factors. Their regulons are composed of a small number of genes likely to be involved in iron uptake.
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Genomic Plasticity Enables Phenotypic Variation of Pseudomonas syringae pv. tomato DC3000
2013Co-Authors: Zhongmeng Bao, Sam Cartinhour, Christopher R. Myers, Brian H Kvitko, Alan Collmer, Paul V. Stodghill, Hanh Lam, Hai-lei Wei, Suma Chakravarthy, Peter SchweitzerAbstract:Whole genome sequencing revealed the presence of a genomic anomaly in the region of 4.7 to 4.9 Mb of the Pseudomonas syringae pv. tomato (Pst) DC3000 genome. The average read depth coverage of Pst DC3000 whole genome sequencing results suggested that a 165 kb segment of the chromosome had doubled in copy number. Further analysis confirmed the 165 kb duplication and that the two copies were arranged as a direct tandem repeat. Examination of the corresponding locus in Pst NCPPB1106, the parent strain of Pst DC3000, suggested that the 165 kb duplication most likely formed after the two strains diverged via transposition of an ISPsy5 insertion sequence (IS) followed by unequal crossing over between ISPsy5 elements at each end of the duplicated region. Deletion of one copy of the 165 kb region demonstrated that the duplication facilitated enhanced growth in some culture conditions, but did not affect pathogenic growth in host tomato plants. These types of chromosomal structures are predicted to be unstable and we have observed resolution of the 165 kb duplication to single copy and its subsequent re-duplication. These data demonstrate the role of IS elements i
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substrate and target sequence length influence rectepsy recombineering efficiency in Pseudomonas syringae
PLOS ONE, 2012Co-Authors: Sam Cartinhour, Bryan SwingleAbstract:We are developing a new recombineering system to assist experimental manipulation of the Pseudomonas syringae genome. P. syringae is a globally dispersed plant pathogen and an important model species used to study the molecular biology of bacteria-plant interactions. We previously identified orthologs of the lambda Red bet/exo and Rac recET genes in P. syringae and confirmed that they function in recombineering using ssDNA and dsDNA substrates. Here we investigate the properties of dsDNA substrates more closely to determine how they influence recombineering efficiency. We find that the length of flanking homologies and length of the sequences being inserted or deleted have a large effect on RecTEPsy mediated recombination efficiency. These results provide information about the design elements that should be considered when using recombineering.
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recombineering using recte from Pseudomonas syringae
Applied and Environmental Microbiology, 2010Co-Authors: Sam Cartinhour, Bryan Swingle, Eric Markel, Alan H ChambersAbstract:In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.
Bryan Swingle - One of the best experts on this subject based on the ideXlab platform.
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regulons of three Pseudomonas syringae pv tomato dc3000 iron starvation sigma factors
Applied and Environmental Microbiology, 2013Co-Authors: Bronwyn G Butcher, Sam Cartinhour, Paul Stodghill, Christopher R. Myers, Bryan Swingle, Eric MarkelAbstract:Pseudomonas syringae pv. tomato DC3000 contains genes for 15 sigma factors. The majority are members of the extracytoplasmic function class of sigma factors, including five that belong to the iron starvation subgroup. In this study, we identified the genes controlled by three iron starvation sigma factors. Their regulons are composed of a small number of genes likely to be involved in iron uptake.
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substrate and target sequence length influence rectepsy recombineering efficiency in Pseudomonas syringae
PLOS ONE, 2012Co-Authors: Sam Cartinhour, Bryan SwingleAbstract:We are developing a new recombineering system to assist experimental manipulation of the Pseudomonas syringae genome. P. syringae is a globally dispersed plant pathogen and an important model species used to study the molecular biology of bacteria-plant interactions. We previously identified orthologs of the lambda Red bet/exo and Rac recET genes in P. syringae and confirmed that they function in recombineering using ssDNA and dsDNA substrates. Here we investigate the properties of dsDNA substrates more closely to determine how they influence recombineering efficiency. We find that the length of flanking homologies and length of the sequences being inserted or deleted have a large effect on RecTEPsy mediated recombination efficiency. These results provide information about the design elements that should be considered when using recombineering.
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recombineering using recte from Pseudomonas syringae
Applied and Environmental Microbiology, 2010Co-Authors: Sam Cartinhour, Bryan Swingle, Eric Markel, Alan H ChambersAbstract:In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.
Sascha D Braun - One of the best experts on this subject based on the ideXlab platform.
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identification of the biosynthetic gene cluster for 3 methylarginine a toxin produced by Pseudomonas syringae pv syringae 22d 93
Applied and Environmental Microbiology, 2010Co-Authors: Sascha D Braun, Beate Volksch, Annette Wensing, Helge Weingart, Matthias S. Ullrich, J Hofmann, Dieter SpitellerAbstract:The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km, 7 mM; kcat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.
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3 methylarginine from Pseudomonas syringae pv syringae 22d 93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv glycinea
ChemBioChem, 2008Co-Authors: Sascha D Braun, Beate Volksch, Jorg Nuske, Dieter SpitellerAbstract:: The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-methylarginine biosynthesis to be identified.
