Regulation of the mexAB-oprM efflux operon of Pseudomonas aeruginosa

Multidrug efflux systems are important determinants of antimicrobial resistance in the human pathogen Pseudomonas aeruginosa where they compromise anti-pseudomonal chemotherapy. The major multidrug efflux system in P. aeruginosa is encoded by the mexAB-oprM operon, expression of which is regulated by a MexR repressor whose activity is negatively modulated by the anti-repressor protein, ArmR. armR occurs as part of a two-gene operon, PA3720-armR, that is regulated by the product of the divergently-transcribed nalC repressor gene, with nalC mutants showing elevated PA3720-armR expression and, so, elevated mexAB-oprM expression and multidrug resistance. The function of the PA3720 protein is unknown. We show here that PA3720 functions as a non-specific RNA binding protein that binds to and destabilizes the adjacent armR-bearing mRNA. Expression of armR is also shown to be translationally coupled to PA3720. In an earlier study, aminoglycosides were shown to induce expression of PA3720-armR, suggesting that these drugs may also promote mexAB-oprM expression dependent on ArmR. Consistent with this, aminoglycosides promoted expression of mexAB-oprM; however, this was independent of ArmR. Instead, the aminoglycoside-responsive AmgRS two-component system mediated aminoglycoside induction of this efflux system. Consistent with this, mutational activation of the AmgS sensor kinase yielded elevated levels of mexAB-oprM expression, and purified AmgR bound specifically to the mexAB-oprM promoter region. Screening of P. aeruginosa strain PAO1 transposon mutants for increased sensitivity to carbenicillin- carbenicillin is a MexAB-OprM substrate and carbenicillin sensitivity is a surrogate for loss of mexAB-oprM expression - led to the identification of several genes with probable roles in mexAB-oprM expression or activity. The gene disrupted in one of these mutants, bamB, encodes for a component of the outer membrane β-barrel protein assembly machinery and was assessed for its role in facilitating the assembly of OprM, a β-barrel outer membrane protein. Western immunoblotting revealed that BamB is not required for OprM insertion into the outer membrane. These studies provide additional insights into the regulation of mexAB-oprM expression in P. aeruginosa.