Characterization of a Resistance-Nodulation-Cell Division Transporter System Associated with the syr-syp Genomic Island of Pseudomonas syringae pv. syringae

Abstract

ABSTRACT 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.