<b>Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of <i>Pseudomonas aeruginosa</i></b>

TLDR

The study used chloramphenicol and ciprofloxacin selection to generate resistant mutants, isolated a cosmid complementing an OprM‑deficient strain, and sequenced a 6180‑bp fragment revealing three ORFs (MexE, MexF, OprN) homologous to known efflux components. The mutants overexpressed OprN, functioning in an energy‑dependent efflux system; deletion of mexE abolished the multidrug resistance, and the mexEF‑oprN operon, positively regulated by a LysR‑type activator, conferred resistance and may export pyocyanin biosynthetic intermediates.

Abstract

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 .