Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System

J Bacteriol. 2016 Apr 14;198(9):1442-50. doi: 10.1128/JB.00049-16. Print 2016 May.


The Pseudomonas aeruginosa cyclic AMP (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 avfr 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 virulence factor regulator (Vfr) regulates the expression of exsA ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is 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.

Importance: 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 as 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Cyclic AMP Receptor Protein / genetics
  • Cyclic AMP Receptor Protein / metabolism*
  • DNA Footprinting
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation, Bacterial
  • Operon
  • Promoter Regions, Genetic
  • Protein Binding
  • Pseudomonas aeruginosa / genetics*
  • Pseudomonas aeruginosa / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism
  • Type III Secretion Systems / genetics*
  • Type III Secretion Systems / metabolism*
  • Virulence Factors / genetics


  • Bacterial Proteins
  • Cyclic AMP Receptor Protein
  • DNA-Binding Proteins
  • ExsA protein, Pseudomonas aeruginosa
  • Recombinant Fusion Proteins
  • Trans-Activators
  • Type III Secretion Systems
  • Vfr protein, Pseudomonas aeruginosa
  • Virulence Factors