Characterization of a resistance-nodulation-cell division transporter system associated with the syr-syp genomic island of Pseudomonas syringae pv. syringae

A tripartite resistance-nodulation-cell division (RND) transporter system, called the PseABC efflux system, was identified at the left border of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. The PseABC efflux system was located within a 5.7-kb operon that encodes an outer membrane protein (PseA), a periplasmic membrane fusion protein (PseB), and an RND-type cytoplasmic membrane protein (PseC). The PseABC efflux system exhibited amino acid homology to a putative RND efflux system of Ralstonia solanacearum, with identities of 48% for PseA, 51% for PseB, and 61% for PseC. A nonpolar mutation within the pseC gene was generated by nptII insertional mutagenesis. The resultant mutant strain showed a larger reduction in syringopeptin secretion (67%) than in syringomycin secretion (41%) compared to parental strain B301D (P < 0.05). A β-glucuronidase assay with a pseA::uidA reporter construct indicated that the GacS/GacA two-component system controls expression of the pseA gene. Quantitative real-time reverse transcription-PCR was used to determine transcript levels of the syringomycin (syrB1) and syringopeptin (sypA) synthetase genes in strain B301D-HK4 (a pseC mutant). The 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% (P < 0.05). In addition, the virulence of mutant strain B301D-HK4 for immature cherry fruits was reduced by about 58% compared to parental strain B301D (P < 0.05). Although the resistance of mutant strain B301D-HK4 to any antibiotic used in this study was not reduced compared to parental strain B301D, a drug-supersensitive acrB mutant of Escherichia coli showed two- to fourfold-increased resistance to acriflavine, erythromycin, and tetracycline upon heterologous expression of the pseA, pseB, and pseC genes (pseABC efflux genes). The PseABC efflux system is the first RND transporter system described for P. syringae, and it has an important role in secretion of syringomycin and syringopeptin.