Modulation of the Pseudomonas aeruginosa quorum sensing cascade by MexT-regulated factors
Pseudomonas aeruginosa (Pa) is a ubiquitous bacterium that uses quorum sensing (QS), a cell-cell communication system that enables it to sense cell density and to alter gene expression. Pa has three complete QS circuits controlled by the transcriptional regulators LasR, RhlR, and PqsR (MvfR), that together control hundreds of genes, including virulence factors. In the well-described strain PAO1, QS is organized hierarchically, with PqsR and RhlR activity dependent on LasR. In PAO1, this hierarchy depends on the non-QS transcription factor MexT; by an unknown mechanism, deletion of mexT allows for RhlR activity in the absence of LasR. We aimed to identify how regulators such as MexT modulate the QS architecture in Pa. We compared the transcriptome of PAO1 to that of PAO1?mexT and identified 152 differentially expressed genes. MexT does not appear to regulate rhlR or pqsR directly; however, we identified two MexT-regulated operons that may affect the hierarchy in PAO1. These operons encode the drug efflux pump genes mexEF-oprN and the Pseudomonas quinolone signal (PQS) synthesis genes pqsABCDE. We performed genetic experiments to test whether the products of these genes affected the QS hierarchy. As with the mexT knockout mutant, we found that a PAO1 mexEF knockout mutant exhibited RhlR activity earlier, and to a higher magnitude, than wild-type PAO1. MexEF-OprN is known to export the PQS precursor HHQ, and we found that exogenous addition of PQS to PAO1 partially affects RhlR activity, resulting in earlier timing and higher magnitude compared to wild-type PAO1. We further elucidated that this is likely due to positive regulation by PqsE. These data link both the drug efflux pump MexEF-OprN and PQS QS to the regulation of the QS hierarchy in PAO1. We wondered if the same applied to QS architectures in Pa clinical isolates. We discovered that there are alternate QS architectures in clinical isolates, where RhlR activity is not fully dependent on LasR. In these isolates, surprisingly, MexT does not influence the relationship between LasR and RhlR, and this is indicative of a different QS architecture in the clinical isolates. Overall, we further elucidated the regulation of QS architecture in PAO1 and identified unique QS architectures in clinical isolates. Importantly, our work reveals a new suite of factors that regulate QS in Pa, with implications for a variety of Pa behaviors both in the laboratory and clinical settings. Overall design: Bacteria from wild-type PAO1 and PAO1?mexT were harvested at mid log and early stationary phase.  RNA was isolated and gene expresion quantified by sequencing.