Article

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

Department of Biological Science & Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan.
FEMS Microbiology Letters (impact factor: 2.04). 10/2010; 311(1):36-43. DOI:10.1111/j.1574-6968.2010.02063.x pp.36-43
Source: PubMed

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 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.

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    Article: Evidence of MexT-independent overexpression of MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa in presence of metabolic stress.
    Ayush Kumar, Herbert P Schweizer
    [show abstract] [hide abstract]
    ABSTRACT: The Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins. We have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive. Our results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions.
    PLoS ONE 01/2011; 6(10):e26520. · 4.09 Impact Factor
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Keywords

central region
 
gel-shift assays
 
intergenic DNA
 
intergenic DNA deletions
 
mexE gene
 
mexEF-oprN
 
MexEF-OprN efflux pump
 
mexEF-oprN operon
 
MexEF-OprN production
 
MexT-binding site
 
mexT-distal nod box
 
mexT-mexE intergenic DNA
 
mexT-proximal nod box
 
promoter-operator element
 
Pseudomonas aeruginosa
 
purified MexT
 
putative repressor protein
 
region accommodates
 
region upstream
 
second observation
 

Hideaki Maseda