Assignment of the substrate-selective subunits of the MexEF-OprN multidrugefflux pump of Pseudomonas aeruginosa

Pseudomonas aeruginosa expresses a low level of the MexAB-OprM efflux pump and shows natural resistance to many structurally and functionally diverse antibiotics. The mutation that has been referred to previously as nfxC expr esses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluor oquinolones, imipenem, and chloramphenicol and hypersusceptibility to beta- lactam antibiotics. To address the antibiotic specificity of the MexEF-OprN efflux pump, we introduced a plasmid carrying the mexEF-oprN operon into P , aeruginosa lacking the mexAB-oprM operon, The transformants exhibited res istance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show beta-lactam hypersusceptibility. The t ransformants exhibited additional resistance to tetracycline, In the next e xperiment, we analyzed the MexEF-OprN pump subunit(s) responsible for subst rate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacki ng MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/Delta MexAB tr ansformants exhibited MexEF-OprN-type pump function that rendered the strai ns resistant to fluoroquinolones and chloramphenicol but did not change sus ceptibility to beta-lactam antibiotics compared with the host strain. The M exAB-OprN/Delta OprM, MexAF-OprM/Delta MexB, and MexEB-OprM/Delta MexA muta nts exhibited antibiotic susceptibility indistinguishable from that in the mutant lacking both types of efflux pumps. The results imply that the MexEF -OprM pump selects substrates by a MexEF functional unit. Interestingly, Op rN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.