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

Maseda, Hideaki, Uwate, Maki, Nakae, Taiji
FEMS microbiology letters 2010 v.311 no.1 pp. 36-43
DNA-directed RNA polymerase, Pseudomonas aeruginosa, intergenic DNA, mutants, operon, terminal repeat sequences, transcription (genetics), transporters
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 mexE:lacZ 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.