Inactivation of MexT in Pseudomonas aeruginosa destabilizes cooperation and favors the emergence of a unique quorum sensing variant

Pseudomonas aeruginosa , an opportunistic gram-negative pathogen, uses a cell-cell signaling system called quorum sensing to coordinate group behaviors. Quorum sensing in P. aeruginosa is a model to study cooperative behaviors in populations. Several studies of cooperation have been conducted using strain PAO1. Wildtype PAO1 harbors a mutation in mexS , which encodes a negative regulator of the transcription factor MexT, which in turn activates many genes, including the efflux pump MexEF-OprN. We hypothesized that the PAO1 mexS mutation might affect cooperative behaviors. When P. aeruginosa is passaged daily on casein as a sole carbon source, quorum sensing is required to induce synthesis of the extracellular proteases needed to acquire carbon and energy. During growth on casein, individuals with inactivating mutations in the quorum sensing regulator LasR are reproducibly enriched in the population. These LasR mutants are cheaters that benefit from cooperatively-produced proteases and have a fitness advantage over cooperators. We passaged wildtype PAO1, PAO1 with a gene-corrected version of mexS , or PAO1 with a null mutation of mexT on casein as the sole carbon and energy source. We found that correcting the mexS mutation resulted in unstable cooperation: bacterial cultures failed to propagate after about 15 days, whereas the wildtype propagated for the duration of our 30-day experiment. The MexS-corrected and MexT-deficient populations also reproducibly exhibited emergence of a particular quorum sensing variant, LasR-V226I. This variant activated a subset of quorum-sensing regulated genes, suggesting an evolutionary pathway to alter P. aeruginosa quorum sensing regulons.