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.
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.
Guazzaroni
María-Eugenia
ME
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda, 1, E-18008 Granada, Spain.
Krell
Tino
T
Felipe
Antonia
A
Ruiz
Raquel
R
Meng
Cuixiang
C
Zhang
Xiaodong
X
Gallegos
María-Trinidad
MT
Ramos
Juan L
JL
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism / Q000494:pharmacology; D009570:Nitriles / Q000378:metabolism / Q000494:pharmacology; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D016958:Pseudomonas putida / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins; D013816:Thermodynamics; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007211:Indoles; D026901:Membrane Transport Proteins; D009284:Naphthols; D009570:Nitriles; D011993:Recombinant Fusion Proteins; C430335:TtgG protein, Pseudomonas putida; C430336:TtgH protein, Pseudomonas putida; C029350:1-naphthol; D014050:Toluene; C030374:indole; C014356:benzonitrile; C017572:4-nitrotoluene; D001616:beta-Galactosidase; C053499:2,3-dihydroxynaphthalene