Microbial populations typically contain a small fraction of transiently and non-inheritably multidrug-resistant 'persister' cells. These cells are largely responsible for biofilm tolerance to antibiotics. Recently, persistence was shown to increase in late Pseudomonas aeruginosa isolates from chronically infected lungs of cystic fibrosis patients receiving long-term antibiotic therapy. No significant increase in stable, inheritable antibiotic resistance was observed. This has led to the proposition that persistence is the main cause for the recalcitrance of chronic infections that do not respond to antibiotic therapy even in the absence of detectable drug resistance. In this research proposal, we will focus on two genetic determinants responsible for transient multidrug tolerance in P. aeruginosa that were identified during a preceding FWO research project. The functional characterization of these genes will generate detailed insight in the mechanistic basis of persistence. This will be achieved by combining the extensive expertise of the Michiels group in the genetic analysis of persistence with biochemical and biophysical in vitro studies enabled by the expertise of the Versées group in the analysis of the structure/function relationship of proteins and enzymes. Ultimately, this collaboration is expected to result in a detailed and complete mechanistic understanding of the biological principles underlying persistence.