Discovery of unknown quorum-sensing reprogramming factors in Pseudomonas aeruginosa using a dual-acting-gene-blocking approach

LasR-null, QS-active mutants commonly appear in Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lung patients. However, underlying QS-reprogramming factors in CF isolates have yet to be uncovered. Here, we developed an unconventional screening approach, named dual-acting-gene-blocking (DAGB), to identify unknown QS-reprogramming determinants. Mutations in mexT have been linked to QS-reprogramming in the laboratory PAO1 LasR-null mutant. To block the inactivation of MexT, we engineered a recombinant LasR-null strain by introducing another copy of wild-type mexT into the neutral site of the genome. This strategy presumably ensures at least one copy of mexT is functioning if another one is inactive during the QS-required evolution, which thus blocks the inactivating MexT-mediated QS activation. Using DAGB, we identified a point mutation in rpoA (T262->A, RpoA*), encoding the α-subunit of RNA polymerase (RNAP), which could rewire QS activity in the LasR-null background. Like the MexT mutant, RpoA* also activated QS by suppressing the expression of the MexEF-OprN efflux pump. In contrast to the MexT mutant, RpoA* caused milder repression of the MexEF-OprN pump, resulting in an intermediate QS-active phenotype and a slower decline rate of growth fitness when treated with chloramphenicol. With iterative applications of the DAGB-based approach, we further discovered other non- mexT determinants capable of rewiring QS. Our study first reports non-MexT QS-reprogramming regulators in the LasR-null mutant, shedding light on understanding the QS activation mechanism in those P. aeruginosa LasR-null, QS-active clinical isolates. Our findings demonstrate that DAGB is a productive avenue to rapidly identify unknown genetic determinants in P. aeruginosa . Most importantly, the established DAGB-based framework is generalizable and could potentially be extended to uncover untapped pathways in other research areas as well as other organisms. ### Competing Interest Statement The authors have declared no competing interest.