Challenging the paradigm: non-canonical exoprotease cheating in clinical Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa is a model organism for studying social behaviors in bacteria, such as the exploitation of exoprotease by social cheaters. The current paradigm holds that continuous culture of exoprotease-producing individuals with protein as the sole carbon source selects for exoprotease non-producers mutants with an impaired quorum-sensing regulator, LasR, which controls exoprotease expression. However, recent studies reveal that some isolates lacking functional LasR still produce exoproteases under the control of another regulator, RhlR. Here, we extended this study to two clinical strains, AUS 411 and AUS 531, isolated from cystic fibrosis patients and harboring functional LasR. Surprisingly, in AUS 411, exoprotease-non-producers appeared from the first growth passage, but most cells lost exoprotease production only transiently, with stable non-producers isolated only in late passages. In contrast, AUS 531 slowly selected stable non-producers with limited cheating ability, which neither accumulated to high proportions nor caused population collapses. Contrary to the paradigm, these non-producers had no inactivating mutations in lasR yet were more fit than laboratory-derived lasR deletion mutants in both casein and casamino acid media. Our findings demonstrate that social behavior can differ significantly from that in reference strains, suggesting that some P. aeruginosa strains evolve quorum-sensing networks with robust resistance to exploitation.