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Antimicrobial Agents and Chemotherapy, March 2000, p. 658-664, Vol. 44, No. 3
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Assignment of the Substrate-Selective Subunits of the MexEF-OprN Multidrug Efflux Pump of Pseudomonas aeruginosa

Hideaki Maseda, Hiroshi Yoneyama, and Taiji Nakae*

Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan

Received 1 July 1999/Returned for modification 28 September 1999/Accepted 20 December 1999

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 expresses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluoroquinolones, 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 resistance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show beta -lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEF-OprN pump subunit(s) responsible for substrate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacking MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/Delta MexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to beta -lactam antibiotics compared with the host strain. The MexAB-OprN/Delta OprM, MexAF-OprM/Delta MexB, and MexEB-OprM/Delta MexA mutants 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, OprN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.

* Corresponding author. Mailing address: Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan. Phone: 81-463-93-5436. Fax: 81-463-93-5437. E-mail: nakae{at}is.icc.u-tokai.ac.jp

Antimicrobial Agents and Chemotherapy, March 2000, p. 658-664, Vol. 44, No. 3
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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