RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE

The ability of Pseudomonas aeruginosa to rapidly modulate its response to antibiotic stress and persist in the presence of antibiotics is closely associated with the process of cell-to-cell signaling. The alternative sigma factor RpoN (sigma(54)) is involved in the regulation of quorum sensing (QS) and plays an important role in the survival of stationary-phase cells in the presence of carbapenems. Here, we demonstrate that a Delta rpoN mutant grown in nutrient-rich medium has increased expression of pqsA, pqsH, and pqsR throughout growth, resulting in the increased production of the Pseudomonas quinolone signal (PQS). The link between pqsA and its role in carbapenem tolerance was studied using a Delta rpoN Delta pqsA mutant, in which the carbapenem-tolerant phenotype of the Delta rpoN mutant was abolished. In addition, we demonstrate that another mechanism leading to carbapenem tolerance in the Delta rpoN mutant is mediated through pqsE. Exogenously supplied PQS abolished the biapenem-sensitive phenotype of the Delta rpoN Delta pqsA mutant, and overexpression of pqsE failed to alter the susceptibility of the Delta rpoN Delta pqsA mutant to biapenem. The mutations in the Delta rpoN Delta rhlR mutant and the Delta rpoN Delta pqsH mutant led to susceptibility to biapenem. Comparison of the changes in the expression of the genes involved in QS in wild-type PAO1 with their expression in the Delta rpoN mutant and the Delta rpoN mutant-derived strains demonstrated the regulatory effect of RpoN on the transcript levels of rhlR, vqsR, and rpoS. The findings of this study demonstrate that RpoN negatively regulates the expression of PQS in nutrient-rich medium and provide evidence that RpoN interacts with pqsA, pqsE, pqsH, and rhlR in response to antibiotic stress.