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The multidrug efflux regulator TtgV recognizes a wide range of structurally different effectors in solution and complexed with target DNA: evidence from isothermal titration calorimetry.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2005 May 27; Vol. 280 (21), pp. 20887-93. Date of Electronic Publication: 2005 Mar 13. - Publication Year :
- 2005
-
Abstract
- TtgV modulates the expression of the ttgGHI operon, which encodes an efflux pump that extrudes a wide variety of chemicals including mono- and binuclear aromatic hydrocarbons, aliphatic alcohols, and antibiotics of dissimilar chemical structure. Using a 'lacZ fusion to the ttgG promoter, we show that the most efficient in vivo inducers were 1-naphthol, 2,3-dihydroxynaphthalene, 4-nitrotoluene, benzonitrile, and indole. The thermodynamic parameters for the binding of different effector molecules to purified TtgV were determined by isothermal titration calorimetry. For the majority of effectors, the interaction was enthalpy-driven and counterbalance by unfavorable entropy changes. The TtgV-effector dissociation constants were found to vary between 2 and 890 mum. There was a relationship between TtgV affinity for the different effectors and their potential to induce gene expression in vivo, indicating that the effector binding constant is a major determinant for efficient efflux pump gene expression. Equilibrium dialysis and isothermal titration calorimetry studies indicated that a TtgV dimer binds one effector molecule. No evidence for the simultaneous binding of multiple effectors to TtgV was obtained. The binding of TtgV to a 63-bp DNA fragment containing its cognate operator was tight and entropy-driven (K(D) = 2.4 +/- 0.35 nm, DeltaH = 5.5 +/- 0.04 kcal/mol). The TtgV-DNA complex was shown to bind 1-napthol with an affinity comparable with the free soluble TtgV protein, K(D) = 4.8 +/- 0.19 and 3.0 +/- 0.15 mum, respectively. The biological relevance of this finding is discussed.
- Subjects :
- Bacterial Proteins genetics
Escherichia coli genetics
Gene Expression Regulation, Bacterial drug effects
Indoles metabolism
Indoles pharmacology
Membrane Transport Proteins genetics
Naphthols metabolism
Naphthols pharmacology
Nitriles metabolism
Nitriles pharmacology
Operon genetics
Promoter Regions, Genetic genetics
Protein Binding
Pseudomonas putida chemistry
Pseudomonas putida genetics
Recombinant Fusion Proteins
Thermodynamics
Toluene metabolism
Toluene pharmacology
Transcription, Genetic
beta-Galactosidase genetics
Bacterial Proteins metabolism
Calorimetry methods
DNA, Bacterial metabolism
Membrane Transport Proteins metabolism
Toluene analogs & derivatives
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 280
- Issue :
- 21
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15767250
- Full Text :
- https://doi.org/10.1074/jbc.M500783200