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J Bacteriol. 2007 Aug;189(15):5441-51. Epub 2007 Jun 1.

Protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa.

Author information

1
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6. poolek@post.queensu.ca.

Abstract

nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [K(D)], 158.0 +/- 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative "noninteracting" mutant MexR protein, MexR(I104F), was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; K(D), 853.2 +/- 151.1 nM). Consistent with this, MexR(I104F) repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal alpha-helix of the protein that alone is sufficient for interaction with MexR.

PMID:
17545281
PMCID:
PMC1951821
DOI:
10.1128/JB.00543-07
[Indexed for MEDLINE]
Free PMC Article

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