Characterization of the regulatory mechanism controlling phytotoxin production by Pseudomonas syringae pv. syringae

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 µM) 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 β-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 the SyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrF proteins fused to maltose-binding protein (MBP). Both MBP-SalA and MBP-SyrF fusion 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 a 324-bp band containing the putative syrF promoter. Gel filtration analysis or cross-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.