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, 181 (20), 6300-5

Characterization of MexT, the Regulator of the MexE-MexF-OprN Multidrug Efflux System of Pseudomonas Aeruginosa

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Characterization of MexT, the Regulator of the MexE-MexF-OprN Multidrug Efflux System of Pseudomonas Aeruginosa

T Köhler et al. J Bacteriol.

Abstract

We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed in nfxC-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the mexEF-oprN operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in Escherichia coli from a plasmid carrying the mexT gene. Expression of a mexE::lacZ fusion was 10-fold higher in nfxC-type mutants than in the wild-type strain; however, transcription of mexT as well as the mexT DNA region was unchanged. Located adjacent to mexT but transcribed in opposite direction, the beginning of an ORF termed qrh (quinone oxidoreductase homologue) was identified. Expression of a qrh::lacZ fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of nfxC-type mutants to carbapenem antibiotics.

Figures

FIG. 1
FIG. 1
Labeling of E. coli cells with [35S]Met and [35S]Cys carrying either the control T7 expression vector pWSK29 or the mexT-carrying plasmid pWNC5. The molecular masses (in kilodaltons) of standard protein markers are indicated on the right.
FIG. 2
FIG. 2
(A) β-Galactosidase activity in P. aeruginosa wild-type strain PAO1 and nfxC-type mutant PT149 carrying either the control plasmid pMP220 or the mexE::lacZ fusion plasmid pNFZ4. Data are the results from one representative experiment. Error bars indicate standard deviations for triplicate determinations of LacZ activity. (B) Expression of mexT::lacZ and qrh::lacZ fusions in wild-type strain PAO1 in the absence or presence of plasmid-encoded copies of MexT and in the nfxC-type mutant PT149. Samples were taken during the mid-exponential growth phase.
FIG. 3
FIG. 3
Effect of mexT on expression of mexE::lacZ (pNFZ4), mexT::lacZ (pTZ4), and qrh::lacZ (pQRZ4) fusions in E. coli MC1061. The presence and absence of the mexT-carrying plasmid pMEXT are indicated by + and − symbols, respectively.

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