Transcriptional regulation of the mexEF-oprN multidrug efflux pump operon by MexT and an unidentified repressor in nfxC-type mutant of Pseudomonas aeruginosa

Abstract

Expression of the MexEF-OprN efflux pump in Pseudomonas aeruginosa seems to be upregulated by MexT. The region upstream of the mexEF-oprN operon contains mexT arranged in tandem with mexEF-oprN and separated by 230 bp of mexT-mexE intergenic DNA. Therefore, it is likely that this intergenic DNA contains the promoter-operator element of mexEF-oprN. To characterize how the expression of the mexEF-oprN operon was controlled, we analyzed the mexT-mexE intergenic DNA by constructing a series of intergenic DNA deletions connected with the mexEClacZ reporter and made two important discoveries. The first was that the central region of the DNA contained two nod boxes. The mexT-proximal nod box was identified as the MexT-binding site by gel-shift assays using purified MexT. The mexT-distal nod box was required for the transcription of the mexEF-oprN operon, but not for the binding of MexT, suggesting that this region accommodates the binding of the RNA polymerase. The second observation is that there is a 13 bp inverted repeat sequence separated by 10 bp immediately upstream of the mexE gene. Deletion of this region caused a sudden rise in MexEF-OprN production, suggesting that this region accommodates the binding of a putative repressor protein. © 2010 Federation of European Microbiological Societies.