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.

An RND (resistance-nodulation-cell division) transporter, called the PseC protein, was identified at the left border of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. The PseC protein exhibited amino acid homology to a putative RND transporter of Ralstonia solanacearum with identities of 61% (i.e., PseC). The pseC mutant strain showed a larger reduction in syringopeptin secretion (67%) than syringomycin secretion (41%). A β-glucuronidase assay with a pseA::uidA reporter construct indicated that the GacS/A two-component system controls expression of the pseA gene. Expression of the sypA gene by mutant strain B301D-HK4 corresponded to approximately 13% of that by parental strain B301D, whereas the syrB1 gene expression by mutant strain B301D-HK4 was nearly 61%. Mutant strain B301D-HK4 was reduced in virulence by about 58% as compared to parental strain B301D. A drug-supersensitive acrB mutant of E. coli showed increased resistance to acriflavine and tetracycline upon heterologous expression of the pseA, pseB, and pseC genes. Thus, the PseC protein, an RND transporter, has an important role in secretion of syringomycin and syringopeptin. An ATP-binding cassette (ABC) transporter, called the PseF protein, was identified at the left border of the syr-syp genomic island. The PseF protein exhibited amino acid homology to a putative ABC transporter of E. coli W3104 with identities of 57.6% (i.e., PseF to MacB). The pseF mutant strain showed significant reduction in secretion of syringomycin (74%) and syringopeptin (71%). Expression of the sypA gene by mutant strain B301D-HK7 was approximately 6.9% as compared to that of parental strain B301D, while the syrB1 gene expression by mutant strain B301D-HK7 was nearly 14.6%. Mutant strain B301D-HK7 was less virulent by approximately 67% than parental strain B301D. Expression of the pseF gene was induced approximately six times by strain B301D grown on SRMAF, as compared to that of strain B301D grown on SRM. During infection of bean plants by P. syringae pv. syringae strain B728a, expression of the pseF gene increased 3 days after inoculation. Thus, the PseF protein, an ABC transporter, responsible for secretion of syringomycin and syringopeptin is required for full virulence.