Abstract
The gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.