Oligonucleotide Microarray Analysis of the SalA Regulon Controlling Phytotoxin Production by Pseudomonas syringae pv. syringae

Abstract

The salA gene is a key regulatory element for syringomycin production by Pseudomonas syringae pv. syringae and encodes a member of the LuxR regulatory protein family. Previous studies revealed that salA, a member of the GacS/GacA signal transduction system, was required for bacterial virulence, syringomycin production, and expression of the syrB1 synthetase gene. To define the SalA regulon, the spotted oligonucleotide microarray was constructed using gene-specific 70-mer oligonucleotides of all open reading frames (ORFs) predicted in the syringomycin (syr) and syringopeptin (syp) gene clusters along with representative genes important to bacterial virulence, growth, and survival. The microarray containing 95 oligos was used to analyze transcriptional changes in a salA mutant (B301DSL07) and its wild-type strain, B301D. Expression of 16 genes was significantly higher (> twofold) in B301D than in the salA mutant; the maximum change in expression was 15-fold for some toxin biosynthesis genes. Except for the sylD synthetase gene for syringolin production, all ORFs controlled by SalA were located in the syr-syp genomic island and were associated with biosynthesis, secretion, and regulation of syringomycin and syringopeptin. The positive regulatory effect of SalA on transcription of sypA, syrB1, syrC, and sylD was verified by reporter fusions or real-time polymerase chain reaction analysis. None of the genes or ORFs was significantly down-regulated by the salA gene. These results demonstrated that a subgenomic oligonucleotide microarray is a powerful tool for defining the SalA regulon and its relationship to other genes important to plant pathogenesis.