Progress 04/15/01 to 04/15/05
Outputs
Microarray technology was used to define important regulatory genes that control expression of the syr-syp gene clusters in P. syringae pv. syringae B301D. A 96-well, 70mer oligonucleotide array was designed to include all of the ORFs within the syr-syp genomic island along with representative genes associated with virulence and other important processes. The salA regulon was characterized, and results confirmed that syringomycin and syringopeptin biosynthesis genes are members of this key regulon. Genes associated with the Hrp type III system, phytohormone production, and diverse global regulators were not identified as members of the salA regulon. A 70mer oligonucleotide microarray was used to define the SalA regulon and to evaluate the influence of plant signal molecules on expression of the syr-syp genomic island. Transcriptional profiling was conducted for both a salA mutant and the wild type strain B301D. Expression of 23 open-reading frames was observed to be
over 2-fold higher in B3201D as compared to the salA mutant. The maximum change ranged up to 15 fold for some toxin biosynthesis genes. Except for the sylD synthetase gene for syringolin production, all of the ORFs controlled by SalA were located in the syr-syp genomic island and were associated with biosynthesis, secretion and regulation of syringomycin and syringopeptin. Recent microarray analysis of a syrF mutant demonstrated the same regulatory effect on the syr and syp genes as observed for a salA mutant. Progress was also made in the overexpression of the SalA and SyrF regulatory proteins for functional studies in controlling expression of the syrB1 and sypA synthetase genes. The genes/operons of the syr-syp genomic island are controlled by a sigma70-like factor that binds to the -10 promoter region. Gel shift experiments demonstrated that purified SyrF protein binds to the promoter region of syrB1 to activate transcription. Real-time PCR was used to look at the expression of
syr and syp genes during infection of bean leaves by strain B728a. Both the syrB1 and sypA genes were induced to high levels and their expression levels corresponded with disease severity. The RND efflux system encoded by the pseABC operon located at the left border of the syp gene cluster was further characterized. Nonpolar RND mutant strains were reduced significantly in secretion of both syringomycin and syringopeptin. Expression of a pseA-GUS reporter fusion was controlled by the GacA regulon. A total of 12 synthetase modules representative of the predicted substrate specificity of the Syr and Syp synthetases were overexpressed, and several of these were functionally defined for amino acid specificity. The results advance our fundamental understanding of bacterial virulence and toxigenesis relative to the plant environment.
Impacts
A large genomic island was characterized that is dedicated to phytotoxin production by Pseudomonas syringae, a plant pathogenic bacterium that causes serious diseases of a broad range of field crop and ornamental plants. We cloned and characterized key genes involved in the biosynthesis, secretion and regulation of lipodepsipeptide toxin production and this has generated valuable information about toxigenesis in disease development. An oligonucleotide microarray was used to define and characterize the salA regulon, and results determined that both syringomycin and syringopeptin biosynthesis genes are coordinately controlled by this key regulon. The results expand our understanding of bacterial disease processes that will lead to development of improved methods for disease control in agronomically important crops.
Publications
- Lu, S.-E., N. Wang, J. Wang, Z. J. Chen, and D. C. Gross. 2005. Oligonucleotide microarray analysis of the SalA regulon controlling phytotoxin production by Pseudomonas syringae pv. syringae. Mol. Plant-Microbe Interact. 18:324-333.
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Progress 01/01/04 to 12/31/04
Outputs
A 70mer oligonucleotide microarray was used to define the SalA regulon and to evaluate the influence of plant signal molecules on expression of the syr-syp genomic island. Transcriptional profiling was conducted for both a salA mutant and the wild type strain B301D. Expression of 23 open-reading frames was observed to be over 2-fold higher in B3201D as compared to the salA mutant. The maximum change ranged up to 15 fold for some toxin biosynthesis genes. Except for the sylD synthetase gene for syringolin production, all of the ORFs controlled by SalA were located in the syr-syp genomic island and were associated with biosynthesis, secretion and regulation of syringomycin and syringopeptin. Recent microarray analysis of a syrF mutant demonstrated the same regulatory effect on the syr and syp genes as observed for a salA mutant. The genes/operons of the syr-syp genomic island are controlled by a sigma70-like factor that binds to the -10 promoter region. Gel shift
experiments demonstrated that purified SyrF protein binds to the promoter region of syrB1 to activate transcription. Real-time PCR was used to look at the expression of syr and syp genes during infection of bean leaves by strain B728a. Both the syrB1 and sypA genes were induced to high levels and their expression levels corresponded with disease severity. The results advance our fundamental understanding of bacterial virulence and toxigenesis relative to the plant environment.
Impacts
We continue to characterize a large genomic island that is dedicated to phytotoxin production by Pseudomonas syringae, a plant pathogenic bacterium that causes serious diseases of a broad range of field crop and ornamental plants. We have cloned and characterized key genes involved in the biosynthesis, secretion and regulation of lipodepsipeptide toxin production, and this has generated valuable information about toxigenesis in disease development. An oligonucleotide microarray was used to define and characterize the salA regulon, and results determined that both syringomycin and syringopeptin biosynthesis genes are coordinately controlled by this key regulon.
Publications
- Wang, N., S.-E. Lu, and D. C. Gross. 2004. Characterization of the regulatory mechanism involved in co-regulation of the syringomycin synthetase genes of Pseudomonas syringae pv. syringae. Phytopathology 94:S107.
- Lu, S.-E., N. Wang, and D. C. Gross. 2004. Oligonucleotide microarray analysis of the SalA regulon controlling phytotoxin production by Pseudomonas syringae pv. syringae. Phytopathology 94:S63.
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Progress 01/01/03 to 12/31/03
Outputs
Microarray technology was used to define important regulatory genes that control expression of the syr-syp gene clusters in P. syringae pv. syringae B301D. A 96-well, 70mer oligonucleotide array was designed to include all of the ORFs within the syr-syp genomic island along with representative genes associated with virulence and other important processes. The salA regulon was characterized, and results confirmed that syringomycin and syringopeptin biosynthesis genes are members of this key regulon. The syr and syp synthetase genes were highly expressed up to 15-fold in the parental strain as compared to the salA mutant. Genes associated with the Hrp type III system, phytohormone production, and diverse global regulators were not identified as members of the salA regulon. Progress was also made in the overexpression of the SalA and SyrF regulatory proteins for functional studies in controlling expression of the syrB1 and sypA synthetase genes. The RND efflux system
encoded by the pseABC operon located at the left border of the syp gene cluster was further characterized. Nonpolar RND mutant strains were reduced significantly in secretion of both syringomycin and syringopeptin. Expression of a pseA-GUS reporter fusion was controlled by the GacA regulon. A total of 12 synthetase modules representative of the predicted substrate specificity of the Syr and Syp synthetases were overexpressed, and several of these were functionally defined for amino acid specificity. Analysis of a transcriptional fusion between the sypA synthetase gene and a GUS reporter demonstrated that plant signal molecules activate syringopeptin biosynthesis, as originally described for syringomycin. The results advance our fundamental understanding of bacterial virulence and toxigenesis relative to the plant environment.
Impacts
We continue to characterize a large genomic island that is dedicated to phytotoxin production by Pseudomonas syringae, a plant pathogenic bacterium that causes serious diseases of a broad range of field crop and ornamental plants. We have cloned and characterized key genes involved in the biosynthesis, secretion and regulation of lipodepsipeptide toxin production, and this has generated valuable information about toxigenesis in disease development. An oligonucleotide microarray was used to define and characterize the salA regulon, and results determined that both syringomycin and syringopeptin biosynthesis genes are coordinately controlled by this key regulon.
Publications
- Scholz-Schroeder, B. K., J. D. Soule, and D. C. Gross. 2003. The sypA, sypB, and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv. syringae B301D. Mol. Plant-Microbe Interact. 16:271-280.
- Lu, S.-E., J. D. Soule, and D. C. Gross. 2003. Characterization of the argA gene required for arginine biosynthesis and syringomycin production by Pseudomonas syringae pv. syringae. Appl. Enivorn. Microbiol. 69:7273-7280.
- Gross, D. C., I. Grgurina, B. K. Scholz-Schroeder, and S.-E. Lu. 2003. Characteristics of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. Pages 137-145 in Pseudomonas syringae Pathovars and Related Pathogens, edited by N. S. Iacobellis et al. Kluwer Academic Publishers, The Netherlands.
- Kang, H., and D. C. Gross. 2003. Characterization of an RND transporter located within the syr-syp genomic island of Pseudomonas syringae pv. syringae. Phytopathology 93:S43 (Abstr.).
- Wang, N., S.-E. Lu, and D. C. Gross. 2003. Analysis of genetic and environmental factors that regulate production of syringopeptin by Pseudomonas syringae pv. syringae. Phytopathology 93:S88.
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Progress 01/01/02 to 12/31/02
Outputs
The cloning and sequencing of the right and left border regions of the syr-syp gene clusters was completed and we have identified the corresponding region in the genome of strain B728a. The genomic island is conserved in strain B301D from pear and strain B728a from bean. The genes and ORFs identified from the genomic islands of the two strains exhibited approximately 98% identity at the nucleotide level. A DNA region at the left border of the genomic island of strain B301D was not identified in the B728a genomic data, and investigations of the unique region on toxin production and pathogenesis are in progress. The entire 155-kb syr-syp region was not present in the genome of strain DC3000 of the related pathovar, P. syringae pv. tomato. Microarray studies of expression of ORFs in the syr-syp genomic island are underway to resolve key regulatory elements. Progress was made in characterizing an RND type of efflux system located at the left border of the syp gene
cluster. Mutants of the RND gene that encodes an inner membrane protein were defective in production of both lipodepsipeptide toxins as well as increased sensitivity to the beta-lactam antibiotic, aztreonam. A total of 12 synthetase modules representative of the predicted substrate specificity of the Syr and Syp synthetases were cloned into pMEKm12 for expression in P. syringae. These modules were overexpressed and are being analyzed biochemically for amino acid specificity of acyladenylation domains to confirm predicted enzymatic functions. A transcriptional fusion between the sypA synthetase gene and the uidA gene expressing GUS activity was constructed to evaluate the role of plant signal molecules on activation of syringopeptin production. The results advance our fundamental understanding of bacterial virulence and toxigenesis relative to the plant environment.
Impacts
We continue to characterize a large genomic island that is dedicated to phytotoxin production by Pseudomonas syringae, a plant pathogenic bacterium that causes serious diseases of a broad range of crop and ornamental plants. We have now cloned and characterized regions flanking the syr and syp gene clusters, and this has generated valuable information about genomic relatedness to pseudomonads that infect plants or animals. We developed an expression vector called pMEKm12 that advances our ability to study the function of proteins important to pathogenesis, and investigators worldwide are now using this vector to study important biological processes in pseudomonads.
Publications
- Lu, S.-E., B.K. Scholz-Schroeder, and D. C. Gross. 2002. Construction of pMEKm12, an expression vector for protein production in Pseudomonas syringae. FEMS Microbiol. Lett. 210:115-121.
- Lu, S. E., B. K. Scholz-Schroeder, and D. C. Gross. 2002. Construction of pMEKm12 for expressing Syr proteins in Pseudomonas syringae required for syringomycin production. Phytopathology 92:S49 (Abstr.).
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Progress 01/01/01 to 12/31/01
Outputs
A translational fusion between the syrB1 synthetase gene and the uidA gene expressing GUS activity was used to determine the relationship among the salA, syrF, and syrG regulatory loci. Expression of the syrB1::uidA reporter required functional salA and syrF genes. Expression of a syrF::uidA fusion was dependent on the salA gene, demonstrating that salA is located upstream of syrF in the regulatory hierarchy controlling syringomycin production. The salA and syrF genes were cloned into the overexpression vector pMEKm12 to generate fusions to a maltose-binding protein. The proteins were purified and then evaluated in gel mobility shift assays for binding to the promoter region of the syrB operon. Results did not demonstrate binding interactions despite efforts to optimize assay conditions. The cloning and sequencing of the right and left border regions of the syr-syp gene clusters was nearly completed with the identification of several new ORFs with putative regulatory
functions. Marker exchange mutagenesis of loci flanking the syr-syp synthetase genes is being used to systematically analyze effects on production of phytotoxins. Several representative synthetase modules were cloned into pMEKm12 for expression in P. syringae. Purified proteins are being characterized for amino acid specificity of acyladenylation domains for definitive proof of biochemical function in toxin synthesis.
Impacts
We have defined key biosynthetic and regulatory genes dedicated to phytotoxin production by Pseudomonas syringae, an economically important plant pathogenic bacterium. The syringomycin and syringopeptin gene clusters encompass an approximately 145-kb genomic island, representing about 2% of the bacterial genome. The results advance our fundamental understanding of bacterial virulence in relation to toxigenesis.
Publications
- Scholz-Schroeder, B. K., Soule, J. D., Lu, S., Grgurina, I. and Gross, D. C. 2001. A physical map of the syringomycin and syringopeptin gene clusters localized to an approximately 145-kb DNA region of Pseudomonas syringae pv. syringae strain B301D. Mol. Plant-Microbe Interact. 14:1426-1435.
- Lu, S.-E., Scholz-Schroeder, B.K. and Gross, D.C. 2002. Characterization of the salA, syrF, and syrG regulatory genes located at the right border of the syringomycin gene cluster of Pseudomonas syringae pv. syringae. Mol. Plant-Microbe Interact. 15:43-53.
- Scholz-Schroeder, B. K., Hutchison, M. L., Grgurina, I. and Gross, D. C. 2001. The contribution of syringopeptin and syringomycin to virulence of Pseudomonas syringae pv. syringae strain B301D on the basis of sypA and syrB1 biosynthesis mutant analysis. Mol. Plant-Microbe Interact. 14:366-348.
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