Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa

• Published in: Molecular microbiology 1997-01, Vol.23 (2), p.345-354
• Main Authors: Ko¨hler, Thilo, Michéa‐Hamzehpour, Mehri, Henze, Uta, Gotoh, Naomasa, Kocjancic Curty, Lasta, Pechère, Jean‐Claude
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Outer Membrane Proteins - genetics; Blotting, Western; Cloning, Molecular; Drug Resistance, Multiple - genetics; Membrane Proteins - genetics; Molecular Sequence Data; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; Sequence Alignment; Sequence Analysis, DNA
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.1997.2281594.x

Antibiotic‐resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB–oprM efflux operon. Antibiotic‐accumulation studies indicated that OprN was part of an energy‐dependent antibiotic‐efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB–oprM and mexCD–oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug‐resistance phenotype in an OprN‐overexpressing strain, but did not affect the susceptibility profile of the wild‐type strain. Expression of the mexEF–oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild‐type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF–oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.