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

Abstract

We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed innfxC-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 amexE::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 termedqrh (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.

Variation of the mexT gene, a regulator of the MexEF-OprN efflux pump expression in wild-type strains of Pseudomonas aeruginosa

“…These mexT genes were unimpaired because KH4014a-vmexT transformed with mexT(8380) exhibited the nfxC-phenotype (Table 3). The results are consistent with the sequence analysis of mexT in the nfxC-type mutant derived from PAO1-Geneva suggesting that the nfxCtype mutant must have an additional mutation as indicated previously [14].…”

Mechanism of action of and resistance to quinolones

“…This efflux pump, which can extrude fluoroquinolones, chloramphenicol, trimethoprim and tetracycline (Vila and Martínez, 2008), has a particular genetic structure: upstream from the mexE there is no repressor gene transcribed divergently, but there is an ORF, called mexT, encoding a protein, which belongs to the LysR family of transcriptional activators, and is transcribed in the same direction as the other three genes. MexT is essential for the mexEF-oprN activation (Kohler et al, 1999). In addition, adjacent to and activated by mexT is a further gene, transcribed divergently, termed mexS [previously qrh (Kohler et al, 1999)], which does encode a regulatory protein, as it is, in fact, an oxidoreductase, but can confer the MAR phenotype by enhancement of MexEF-OprN overexpression (Sobel et al, 2005b).…”

“…MexT is essential for the mexEF-oprN activation (Kohler et al, 1999). In addition, adjacent to and activated by mexT is a further gene, transcribed divergently, termed mexS [previously qrh (Kohler et al, 1999)], which does encode a regulatory protein, as it is, in fact, an oxidoreductase, but can confer the MAR phenotype by enhancement of MexEF-OprN overexpression (Sobel et al, 2005b). The mexEF-oprN is not expressed under standard growth in laboratory conditions.…”

Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates

“…We therefore suspect that, in these two isolates, loss of OprD was caused by an OprD repression mechanism acting at the post-transcriptional level. Recently, Köhler et al (1999) showed that co-regulation of OprD and the MexEF-OprN efflux system occurred at the transcriptional but also at the post-transcriptional level. Ochs et al (1999) also suggested that OprD is influenced by more than one mechanism of repression.…”