Inactivation of MexT in Pseudomonas aeruginosa PAO1 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. Wild-type 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. When PAO1 is grown 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 wild-type PAO1, PAO1 with a gene-corrected version of mexS, or PAO1 with a null mutant 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 wild type 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.IMPORTANCEQuorum sensing governs cooperative activities and has been used as a model for studying cooperative behaviors in bacterial populations. Pseudomonas aeruginosa has two interlinked acyl-homoserine lactone quorum-sensing circuits, LasR-I and RhlR-I; the relationship between these circuits is influenced by the regulator MexT. In the well-studied strain PAO1, inactivation of MexT results in enhanced activity of the quorum-sensing transcription factor, RhlR, and we found that it also destabilized cooperative behaviors in this bacterium. MexT-inactive strains also reproducibly support the emergence of a specific LasR variant, V226I, of P. aeruginosa, offering insight into evolutionary pressures that select for mutations in quorum-sensing transcription factors and, by extension, a method for bacteria to alter the cohort of genes that are quorum-controlled.