Anne E. Marsden, Peter J. Intile, Kayley H. Schulmeyer, Ethan R. Simmons-Patterson, Mark L. Urbanowski, Matthew C. Wolfgang, Timothy L. Yahr

The Pseudomonas aeruginosa cAMP-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in a vfr mutant wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive feedback loops, we tested the hypothesis that Vfr regulates the expression of exsA. ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is encoded as the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (PexsA) located immediately upstream of exsA. PexsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a PexsA promoter probe was demonstrated by electrophoretic mobility shift assays and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and PexsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the PexsA promoter, which is internal to the previously characterized exsCEBA operon. Vfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa. Regulation by Vfr allows for the coordinate production of related virulence functions such type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA.