Characterization of the regulatory mechanism controlling phytotoxin production by Pseudomonas syringae pv. syringae /
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| Other Authors: | |
| Format: | Thesis eBook |
| Language: | English |
| Published: |
[College Station, Tex.] :
[Texas A&M University],
[2007]
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| Subjects: | |
| Online Access: | Link to OAK Trust copy |
| Abstract: | Syringopeptin (syp) and syringomycin (syr) are major necrosis-inducing lipodepsipeptide phytotoxins produced by P. syringae pv. syringae. This report demonstrates that syringopeptin production is activated by plant signal molecules. Syringopeptin production by BR132 was increased two-fold by addition of arbutin (100 [mu]M) and D-fructose (0.1%) to syringomycin minimal medium (SRM). Subgenomic analysis of transcriptional expression with a 70-mer oligonucleotide microarray demonstrated that the syr-syp genes are induced 2.5- to 10.5-fold by arbutin and D-fructose. The syr-syp genomic island was found to be organized into 12 transcriptional units based on reverse transcriptional PCR (RT-PCR) and computer analysis. The transcriptional start sites of the salA gene and operons III and IV were located 63, 75, and 104-bp upstream of the start codons of salA, syrP, and syrB1, respectively, using primer extension analysis. The predicted -10/-35 promoter region of operon IV was confirmed based on mutagenesis analyses of the syrB1::uidA reporter with [beta]-glucuronidase (GUS) assays. A 20-bp conserved sequence (TGTCccgN4cggGACA) with dyad symmetry around the -35 region was identified via computer analysis for the syr-syp genes/operons responsible for biosynthesis and secretion of syringomycin and syringopeptin. Expression of the syrB1::uidA fusion was decreased 59% when 6-bp was deleted from the 5' end of the syr-syp box in the promoter region of operon IV. These results demonstrate that the conserved promoter sequences of the syr-syp genes contribute to the co-regulation of syringomycin and syringopeptin production. Microarray analysis established that the syr-syp genes responsible for synthesis and secretion of syringomycin and syringopeptin belong to theSyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrFproteins fused to maltose-binding protein (MBP). Both MBP-SalA and MBP-SyrFfusion proteins were purified with maltose-affinity chromatography. Gel shift analysis revealed that the purified MBP-SyrF, but not the MBP-SalA fusion protein, bound to a 262-bp fragment containing the syr-syp box. Purified MBP-SalA caused the shift of a324-bp band containing the putative syrF promoter. Gel filtration analysis orcross-linking experiments indicated that both SalA and SyrF form dimers in vitro. This study may provide an important perspective on the regulation of syringomycin and syringopeptin production. |
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| Item Description: | "Major Subject: Plant Pathology" Title from author supplied metadata (automated record created on Apr. 27, 2007.) Vita. Abstract. Electronic resource. |
| Format: | Mode of access: World Wide Web. System requirements: World Wide Web access and Adobe Acrobat Reader. |
| Bibliography: | Includes bibliographical references. |