Abstract
The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.
Highlights
Pumps have recently become the object of special interest, because they are a cause of concern in the emergence of multidrug resistant pathogens such as Pseudomonas aeruginosa [8], Escherichia coli [9], Stenotrophomonas maltophilia [10], or Neisseria gonorrhoeae [11, 12], and for their importance in bacterial tolerance to toxic xenobiotics like organic solvents [7]
The DOT-T1E18 mutant still showed increased susceptibilities to these compounds as compared with its parental strain. This indicates, first, that coumestrol is, in addition to phloretin, quercetin, and naringenin, a substrate of the TtgABC pump and, second, that the TtgABC efflux pump is not inhibited by MC207110. This suggests the existence of other pump(s) that are impaired by MC207110 and expel the same compounds, accounting for the high level of resistance of P. putida DOT-T1E to these natural antimicrobials
The low minimal inhibitory concentration (MIC) values obtained for coumestrol (0.78 g/ml) or quercetin (4.2 g/ml) are comparable with those found for clinically relevant antibiotics [35, 37], which points to the antibacterial potential of these natural compounds against Pseudomonas sp. strains when used in combination with MDR inhibitors
Summary
As P. putida is a very abundant soil bacterium found in close association with plant roots, we hypothesized that the TtgABC/TtgR system might be involved in the resistance to plant defense metabolites. To address this issue, two complementary approaches were used. Initial in vivo studies aimed at exploring the TtgABC-mediated resistance to plant defense metabolites, as well as the role of TtgR in the expression of the ttgABC operon in response to these compounds. The binding of different effectors to DNA-bound TtgR is explored, which is an innovative approach we judge of general relevance for the study of transcriptional repressors
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