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Members of the IclR family of regulators exhibit a highly conserved effector recognition domain and interact with a limited number of effectors. In contrast with most IclR family members, TtgV, the transcriptional repressor of the TtgGHI efflux pump, exhibits multidrug recognition properties. A three-dimensional model of the effector domain of TtgV was generated based on the available three-dimensional structure of several IclR members, and a series of point mutants was created. Using isothermal titration calorimetry, we determined the binding parameters of the most efficient effectors for TtgV and its mutant variants. All mutants bound biaromatic compounds with higher affinity than the wild-type protein, whereas monoaromatic compounds were bound with lower affinity. This tendency was particularly pronounced for mutants F134A and H200A. TtgVF134A bound 4-nitrotoluene with an affinity 13-fold lower than that of TtgV (17.4+/-0.6 microM). This mutant bound 1-naphthol with an affinity of 5.7 microM, which is seven times as great as that of TtgV (40 microM). The TtgVV223A mutant bound to DNA with the same affinity as the wild-type TtgV protein, but it remained bound to the target operator in the presence of effectors, suggesting that Val-223 could be part of an intra-TtgV signal recognition pathway. Thermodynamic analyses of the binding of effectors to TtgV and to its mutants in complex with their target DNA revealed that the binding of biaromatic compounds resulted in a more efficient release of the repressor protein than the binding of monoaromatics. The physiological significance of these findings is discussed.

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.

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.

The final sigma(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that final sigma(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that final sigma(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that final sigma(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in final sigma(54) to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to final sigma(54) interactions and final sigma(54)-DNA interactions occur in response to activator to allow final sigma(54) isomerization and the holoenzyme to progress from the closed complex to the open complex.

Control of transcription frequently involves the direct interaction of activators with RNA polymerase. In bacteria, the formation of stable open promoter complexes by the sigma(54) RNA polymerase is critically dependent on sigma(54) amino Region I sequences. Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator. Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted sigma(54), we have explored the involvement of Region I in transcription activation. Results show that Region I in trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization. Evidence is presented indicating that the holoenzyme (and not the promoter DNA per se) is one interacting target used by Region I in preventing polymerase isomerization. Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis. Region I in trans is able to inhibit activated transcription by the holoenzyme containing full-length sigma(54). Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I. Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNA-polymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the -40 to -70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position -42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/RNA polymerase interactions. Methylation of T-42 was highest in cells bearing XylS and in the presence of an effector. Gs in the -47 to -61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between -41 and -78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN6GGNCA. These motifs cover the -70 to -56 and the -49 to -35 regions. Single point mutations revealed that nucleotides located at -49 to -46 and at -59, -60, -62, and -70 are the most critical for appropriate XylS-Pm interactions.

The XylS protein is the positive regulator of the promoter controlling the meta-cleavage pathway (Pm) for catabolism of certain alkylbenzoates on the TOL plasmid of Pseudomonas. Transcription from Pm is mediated by XylS either in the presence of benzoate effectors or through XylS hyperproduction. Two regions of the NH2 terminus of XylS (residues 37-45) had been predicted to be involved in effector control of XylS transcriptional activation. Different methods were used to induce mutations in this region, including genetic selections (where Pm controlled a tetracycline resistance gene), bisulfite mutagenesis at a unique restriction site, and extensive oligonucleotide mutagenesis at residues 41 and 45. The mutants fell into four classes based on their phenotypes with respect to effector-mediated activation of a Pm-lacZ fusion: (a) effector profiles similar to wild type, (b) no Pm stimulation with benzoates, (c) altered effector specificity, and (d) higher basal Pm activities, in some cases including changes in effector specificity. In some mutants, higher basal Pm activity was apparently due to mutations that increased XylS stability. Substitutions at Arg-41 resulted in all four mutant phenotypes, indicating that this is a critical residue in XylS for effector stimulation of transcription activity.