Characterization of the Transcriptional Activators SalA and SyrF, Which Are Required for Syringomycin and Syringopeptin Production byPseudomonas syringaepv. syringae

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Abstract
Production of the phytotoxins syringomycin and syringopeptin byPseudomonas syringaepv. syringae is controlled by the regulatory genessalAandsyrF. Analysis with 70-mer oligonucleotide microarrays established that thesyr-sypgenes 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 a maltose-binding protein (MBP) inEscherichia coliandP. syringaepv. syringae. Both the MBP-SalA and MBP-SyrF fusion proteins were purified by 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 of thesyrB1promoter region containing thesyr-sypbox. Purified MBP-SalA caused a shift of a 324-bp band containing the putativesyrFpromoter. Gel filtration analysis and cross-linking experiments indicated that both SalA and SyrF form homodimers in vitro. Overexpression of the N-terminal regions of SalA and SyrF resulted in decreased syringomycin production by strain B301D and reduced levels of β-glucuronidase activities of thesypA::uidAandsyrB1::uidAreporters by 59% to 74%. The effect of SalA on the expression of thesyr-sypgenes is mediated by SyrF, which activates thesyr-sypgenes by directly binding to the promoter regions. Both SalA and SyrF resemble other LuxR family proteins in dimerization and interaction with promoter regions of target genes.

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