Modulation of the <i>Pseudomonas aeruginosa</i> quorum sensing cascade by MexT-regulated factors

<i>Pseudomonas aeruginosa (Pa</i>) uses quorum sensing (QS), a cell-cell communication system that enables it to sense cell density and alter gene expression. <i>Pa</i> has three complete QS circuits controlled by the regulators LasR, RhlR, and PqsR, which together activate the transcription of hundreds of genes. In the well-studied strain PAO1, QS is organized hierarchically, with PqsR and RhlR activity dependent on LasR. This hierarchy depends on the transcription factor MexT; deletion of <i>mexT</i> allows for RhlR activity in the absence of LasR. We aimed to identify how MexT modulates <i>Pa</i> QS architecture. We compared the transcriptome of PAO1 to that of PAO1Δ<i>mexT</i> and determined a MexT regulon. We identified two MexT-regulated operons that may affect the QS hierarchy: the efflux pump genes <i>mexEF-oprN</i> and the <i>Pseudomonas</i> quinolone signal (PQS) synthesis genes <i>pqsABCDE</i>. We tested whether the products of these genes affected the QS hierarchy. A <i>mexEF</i> knockout mutant, like a <i>mexT</i> deletion mutant, exhibited RhlR activity earlier and to a higher magnitude than wild-type PAO1. MexEF-OprN is known to export quinolones, and we found that exogenous addition of PQS also resulted in earlier and higher magnitude of RhlR activity, through PqsE, compared with wild-type PAO1. We also discovered alternate QS architectures in clinical isolates, where RhlR activity is not fully dependent on LasR. In these isolates, MexT does not influence the relationship between LasR and RhlR. Our work reveals a new suite of factors that regulate QS in <i>Pa</i>, with implications for bacterial behaviors in environmental and clinical settings.IMPORTANCEBacteria interact with both abiotic and biotic factors in their environment. Quorum sensing (QS) is one mechanism that bacteria use to communicate with other bacteria and coordinate behaviors in the population. QS regulates a wide variety of processes ranging from the production of light to the modulation of virulence factors; some bacteria use single QS circuits, whereas others have several. The opportunistic pathogen <i>Pseudomonas aeruginosa</i> uses QS to control some virulence functions and has three complete QS circuits. Our study explores why bacteria might have multiple QS circuits. We show how a non-QS regulated factor, MexT, influences QS regulators in <i>P. aeruginosa,</i> and we uncover the diversity of QS architectures in clinical isolates. These studies begin to reveal the benefits (or disadvantages) of multiple QS circuits, allowing us to understand the behaviors of bacteria that have a range of implications in health, agriculture, and bioremediation.