Aromatic Effectors Research Articles

An updated structural model of the A domain of the Pseudomonas putida XylR regulator poses an atypical interplay with aromatic effectors.

A revised model of the aromatic binding A domain of the σ54 -dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR and DmpR. The resulting frame was instrumental for mapping a number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. Yet, several loop regions of the A domain could provide the flexibility required for widening such a tunnel for passage of aromatic ligands. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Structural and proteomic analyses confirmed the predicted in/out distribution of residues but also supported two additional possible scenarios of interaction of the A domain with aromatic effectors: a dynamic interaction of the fully structured yet flexible protein with the aromatic partner and/or inducer-assisted folding of the A domain.

The Y233 gatekeeper of DmpR modulates effector-responsive transcriptional control of σ54 -RNA polymerase.

DmpR is the obligate transcriptional activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR belongs to the AAA+ class of mechano-transcriptional regulators that employ ATP-hydrolysis to engage and remodel σ54 -RNA polymerase to allow transcriptional initiation. Previous work has established that binding of phenolic effectors by DmpR is a prerequisite to relieve interdomain repression and allow ATP-binding to trigger transition to its active multimeric conformation, and further that a structured interdomain linker between the effector- and ATP-binding domains is involved in coupling these processes. Here, we present evidence from ATPase and in vivo and in vitro transcription assays that a tyrosine residue of the interdomain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and aromatic effector-responsive transcriptional activation by DmpR. An alanine substitution of Y233A results in both increased ATPase activity and enhanced sensitivity to aromatic effectors. We propose a model in which effector-binding relocates Y233 to synchronize signal-reception with multimerisation to provide physiologically appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family and has implications for development of enhanced sensitivity of biosensor used to detect pollutants.

Expanding the Boolean Logic of the Prokaryotic Transcription Factor XylR by Functionalization of Permissive Sites with a Protease-Target Sequence

The σ54-dependent prokaryotic regulator XylR implements a one-input/one-output actuator that transduces the presence of the aromatic effector m-xylene into transcriptional activation of the cognate promoter Pu. Such a signal conversion involves the effector-mediated release of the intramolecular repression of the N-terminal A domain on the central C module of XylR. On this background, we set out to endow this regulator with additional signal-sensing capabilities by inserting a target site of the viral protease NIa in permissive protein locations that once cleaved in vivo could either terminate XylR activity or generate an effector-independent, constitutive transcription factor. To find optimal protein positions to this end, we saturated the xylR gene DNA with a synthetic transposable element designed for randomly delivering in-frame polypeptides throughout the sequence of any given protein. This Tn5-based system supplies the target gene with insertions of a selectable marker that can later be excised, leaving behind the desired (poly) peptides grafted into the protein structure. Implementation of such knock-in-leave-behind (KILB) method to XylR was instrumental to produce a number of variants of this transcription factor (TF) that could compute in vivo two inputs (m-xylene and protease) into a single output following a logic that was dependent on the site of the insertion of the NIa target sequence in the TF. Such NIa-sensitive XylR specimens afforded the design of novel regulatory nodes that entered protease expression as one of the signals recognized in vivo for controlling Pu. This approach is bound to facilitate the functionalization of TFs and other proteins with new traits, especially when their forward engineering is made difficult by, for example, the absence of structural data.

Three Dimensional Model for N-Terminal A Domain of DmpR (2-Dimethylphenol) Protein Based on Secondary Structure Prediction and Fold Recognition

DmpR (dimethylphenol regulatory protein) is a member of the NtrC family of transcriptional activators and controls the transcription of the dmp operons in response to aromatic effector compounds. Secondary structure and fold recognition prediction of N-terminal A domain of this protein (210 amino acid) was performed in Genesilico Metaserver and 3DJury. The consensus result from these servers suggested MJ_1460 as a template. Three dimensional structures were generated from the sequence structure alignments of the template and target protein with MODELLER. The results suggested that the N-terminal A domain of DmpR belongs to Muramoyl pentapeptide carboxypeptidase domain family. The binding interaction sites of the known effectors were predicted using protein-ligand docking. The proposed active site of N-terminal A domain of DmpR comprises of key residues such as Phe93, Glu127, Phe132, Ser160, Phe163, Met164, Arg166 and Pro189. The findings provide some direction to the experimental studies that aim to broaden the range of phenolic derivative which can be sensed by N-DmpR in order to improve the biodegradation potential.

The Sensor Kinase TodS Operates by a Multiple Step Phosphorelay Mechanism Involving Two Autokinase Domains

Expression of the Pseudomonas putida tod operon, which encodes enzymes for toluene metabolism, takes place from the P(todX) promoter and is mediated by the TodS/TodT two component system. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor- and an autokinase domain and separated by a receiver domain. Based on site-directed mutagenesis of phosphoaccepting His-190, Asp-500, and His-760 and in vitro transphosphorylation experiments with recombinant TodS fragments, we show that TodS uses a multiple step phosphorelay mechanism to activate TodT. Toluene binding stimulates exclusively phosphorylation of His-190, which is followed by phosphotransfer to Asp-500 and subsequently to His-760 prior to phosphorylation of TodT Asp-57. Mutation of His-190, Asp-500, and H760A prevented up-regulation of toluene-mediated stimulation of TodT transphosphorylation in vitro and reduced in vivo expression of P(todX) to the basal level. Calorimetric studies support that TodT binds to the C-terminal kinase module with a K(D) of approximately 200 nm and 1:1 stoichiometry. This is the first report of a multiple step phosphorelay mechanism of a sensor kinase that involves two autokinase domains.

Cooperativity and Selectivity in Chemomechanical Polyethylenimine Gels

Gel particles obtained from polyethylenimine (PEI) cross-linked with 10% ethylene glycol diglycidyl ether show considerable selectivity in size changes if exposed to solutions of different effector compounds. Monocarboxylic acids induce contractions of, e.g., -9% for benzoate and -40% for 2-naphthoate (all values in one dimension such as length). With diacids, the contractions increase to -29% for 1,4-benzene and -68% for 2,6-naphthalene disulfonate, indicating the significant contributions of noncovalent ionic cross-linking and of interactions with aromatic residues. A striking cooperativity is observed if aromatic compounds such as naphthoic acid are used simultaneously with amino acids as effectors. The combination with, e.g., 2-naphthoic acid and phenylalanine induces, e.g., 69% contraction, whereas the single effector compounds induce only 40% and 8%, respectively. As expected with contraction of chemomechanical polymers, the kinetics of effector absorption is significantly slower than that of the size changes, which take, e.g., 5 min to reach one-half of the final contraction; with smaller gel particles, the rates are significantly increased. Gravimetric determinations show that release of solvation water is largely responsible for the observed volume contractions. Copper or zinc ions induce small contractions, also in experiments with PEI solutions, but show no evidence of cooperativity in combination with amino acids or peptides. MAS NMR spectra of the gels induced by aromatic effector compounds exhibited moderate upfield shifts of the polymer backbone signals, as a result of ring current effects.

Dimension Changes in a Chemomechanical Polymer Containing Ethylenediamine and Alkyl Functions as Selective Recognition Units

AbstractTreatment of polymethyl methacrylate with dodecylamine and diethylenetriamine produces a chemomechanical polymer, which undergoes large macroscopic motions as a result of selective recognition of substrates (effectors) in the surrounding aqueous solution. The kinetics of these fully reversible expansions and contractions are compared to those of effector absorption and desorption. The expansion correlates with a polymer particle weight increase, which is in turn found to be largely a function of the uptake of water necessary for solvation of the effector molecules. The selectivity of the motions depends on the nature and placement of the effector ionic sites, and also on the size and nature of the organic residues. Ion pairing between the protonated amine functions and inorganic anions or anionic groups of organic effectors plays a decisive role. The large effects seen with aromatic effectors such as nucleotides, in contrast with saturated analogues, point to cation–π and C–H–π interactions as essential elements for the distinction between different effectors, including isomeric compounds. The material exhibits symmetric expansion/pH profiles, which depend distinctly on the ionic strength of the solution. The strong dependence of the motions not only on the applied pH, but also on the concentrations of two simultaneously active effectors, such as AMP and phosphate, present chemically induced logical gate functions; this is tentatively explained by a two‐site binding model. In contrast, the large expansions produced by simultaneous action of, for example, copper or zinc and amino acids or peptides are due to co‐complexation between the metal ions and known organic chelators. With such ternary complexes, volume expansions by factors of up to 15 are possible with effector concentrations as low as 0.25 mM. The sensitivity can be further increased by miniaturization of the polymer particles; the velocity of the response can be optimized by increasing the surface‐to‐volume ratio. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

A chitosan-based chemomechanical polymer triggered by stacking effects with aromatic effectors including aminoacid derivatives

Chitosan with a covalently attached anthrylunit is used as chemomechanical polymer, in which stacking and cation–π interactions allow aromatic effector compounds with positively charged nitrogen centers to trigger macroscopic motions in aqueous surrounding. Thus, only protonated heterocycles such as imidazole or histamine lead at pH 5 to expansion, in contrast to toluenesulfonic acid, or pyrazole and pyrimidine. Inorganic salts and pH influence the polymer swelling, and must be taken into account for the calculation of net effects induced by organic effectors. Reversible volume expansions on the top of the swelling effect of water alone are observed as function of different effector structures, showing, for example, 45% net effect with imidazole and 66% with benzimidazole. Aminoacids, for solubility reasons measured in the form of their methylesters, yield smaller expansions, showing, however, a regular and selective increase with the lipophilicity of the residues. The kinetics of effector uptake, which relates to the velocity of expansion, are measured with histamine and follow first order, with t 1/2=2.7 min for 50% absorption.

Quantitative structure–activity relationship (QSAR) analysis of aromatic effector specificity in NtrC-like transcriptional activators from aromatic oxidizing bacteria

A quantitative structure–activity relationship (QSAR) approach was taken to provide mechanistic insights into the interaction between the chemical structure of inducing compounds and the transcriptional activation of aromatic monooxygenase operons among the XylR/DmpR subclass of bacterial NtrC-like transcriptional regulators. Compared to XylR and DmpR, a broader spectrum of effector compounds was observed for the TbuT system from Ralstonia pickettii PKO1. The results of QSAR analysis for TbuT suggested that a steric effect, rather than hydrophobic or electronic effects, may be the predominant factor in determining aromatic effector specificity, and the active site of the regulator may positively interact not only with the methyl moiety but also with the most electron-rich aryl side of an aromatic effector.

Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.

The Pseudomonas-derived sigma54-dependent regulator DmpR has an amino-terminal A-domain controlling the specificity of activation by aromatic effectors, a central C-domain mediating an ATPase activity essential for transcriptional activation and a carboxy-terminal D-domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the -24, -12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A-domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A-domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 microM. The A-domain is also shown to have specific affinity for the C-domain and to repress the C-domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A-domain from the remainder of the regulator results in a system that does not respond to aromatics by its normal derepression mechanism. The mechanistic implications of aromatic non-responsiveness of autonomously expressed A-domain, despite its demonstrated ability to bind phenol, are discussed.

Integration of Global Regulation of Two Aromatic-Responsive σ54-Dependent Systems: a Common Phenotype by Different Mechanisms

Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related sigma(54)-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding sigma(54) RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of sigma(54)-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.

The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation

The transcriptional promoting activity of DmpR is under the strict control of its aromatic effector ligands that are bound by its regulatory N-terminal domain. The positive control function of DmpR resides within the central C-domain that is highly conserved among activators of σ 54-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with σ 54-RNA polymerase that are essential for open-complex formation and thus initiation of transcription. Wild-type and loss-of-function derivatives of DmpR, which are defective in distinct steps in nucleotide catalysis, were used to address the consequences of nucleotide binding and hydrolysis with respect to the multimeric state of DmpR and its ability to promote in vitro transcription. Here, we show that DmpR derivatives deleted of the regulatory N-terminal domain undergo an aromatic-effector independent ATP-binding triggered multimerisation as detected by cross-linking. In the intact protein, however, aromatic effector activation is required before ATP-binding can trigger an apparent dimer-to-hexamer switch in subunit conformation. The data suggest a model in which the N-terminal domain controls the transcriptional promoting property of DmpR by constraining ATP-mediated changes in its oligomeric state. The results are discussed in the light of recent mechanistic insights from the AAA + superfamily of ATPases that utilise nucleotide hydrolysis to restructure their substrates.

Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins.

We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.

New insights into the activation of o-xylene biodegradation in Pseudomonas stutzeri OX1 by pathway substrates.

The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o-xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins. The set-up included the prokaryotic enhancer-binding protein TouR, the sigma54-dependent PToMO promoter and the sigma54-containing RNA polymerase. With this system we prove that direct binding of 2-methylphenol (o-cresol) to TouR is the only regulatory step for activation of PToMO in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing. In addition, we found that while TouR failed entirely to activate PToMO in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o-cresol addition. The results presented here support the view that TouR-like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation.

An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR.

The activities of many prokaryotic sigma54-dependent transcriptional activators are controlled by the N-terminal A-domain of the protein, which is linked to the central transcriptional activation domain via a short B-linker. It used to be thought that these B-linkers simply serve as flexible tethers. Here we show that the B-linker of the aromatic-responsive regulator DmpR and many other regulators of the family contain signature heptad repeats with regularly spaced hydrophobic amino acids. Mutant analysis of this region of DmpR demonstrates that B-linker function is dependent on the heptad repeats and is critical for activation of the protein by aromatic effectors. The phenotypes of DmpR mutants refute the existing model that the level of ATPase activity directly controls the level of transcription it promotes. The mutant analysis also shows that the B-linker is involved in repression of ATPase activity and that allosteric changes upon effector binding are transduced to alleviate both B-linker repression of ATP hydrolysis and A-domain repression of transcriptional activation. The mechanistic implications of these findings for DmpR and other family members are discussed.

Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils.

Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of the dmp operon genes encoding the (methyl)phenol degradative pathway of Pseudomonas sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to para-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways.

The role of the interdomain B linker in the activation of the XylR protein of Pseudomonas putida.

In the presence of toluene and other structural analogues, the enhancer binding protein XylR activates the sigma54 promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida. Introduction of amino acid changes Val-219Asp and Ala-220Pro, which enter a proline kink at the interdomain region (B linker) between the A (signal reception) module and the central portion of XylR, originated a protein with unforeseen properties. These included a minor ability to activate Pu in the absence of aromatic effectors, a much higher responsiveness to m-xylene and a significant response to a large collection of aromatic inducers. Such changes could not be attributed to variations in XylR expression levels or to the fortuitous creation of a novel promoter, but to a genuine change in the properties of the activator. Structural predictions suggested that the mutation entirely disrupted an otherwise probable coiled-coil structure. A second directed mutant within the same region consisting of a major replacement of amino acids A220-N221 by the peptide HHHR produced an even more exacerbated phenotype. These data support a model in which the linker B region influences the effector profile by modifying at a distance the operative shape of the effector pocket and fixing the protein in an intermediate step of the activation process.

Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling.

The Pseudomonas derived sigma(54)-dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators. Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus. Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles. Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenol-binding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR. Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription. Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression.

Novel Effector Control through Modulation of a Preexisting Binding Site of the Aromatic-responsive ς54-Dependent Regulator DmpR

The Pseudomonas derived sigma(54)-dependent DmpR activator regulates transcription of the (methyl)phenol catabolic dmp-operon. DmpR is constitutively expressed, but its transcriptional promoting activity is positively controlled in direct response to the presence of multiple aromatic effectors. Previous work has led to a model in which effector binding by the amino-terminal region of the protein relieves repression of an intrinsic ATPase activity essential for its transcriptional promoting property. Here, we address whether the observed differences in the potencies of the multiple effectors (i) reside at the level of different aromatic binding sites, or (ii) are mediated through differential binding affinities; furthermore, we address whether binding of distinct aromatic effectors has different functional consequences for DmpR activity. These questions were addressed by comparing wild type and an effector specificity mutant of DmpR with respect to effector binding characteristics and the ability of aromatics to elicit ATPase activity and transcription. The results demonstrate that six test aromatics all share a common binding site on DmpR and that binding affinities determine the concentration at which DmpR responds to the presence of the effector, but not the magnitude of the responses. Interestingly, this analysis reveals that the novel abilities of the effector specificity mutant are not primarily due to acquisition of new binding abilities, but rather, they reside in being able to productively couple ATPase activity to transcriptional activation. The mechanistic implications of these findings in terms of aromatic control of DmpR activity are discussed.

Modulation of the function of the signal receptor domain of XylR, a member of a family of prokaryotic enhancer-like positive regulators.

The XylR protein controls expression from the Pseudomonas putida TOL plasmid upper pathway operon promoter (Pu) in response to aromatic effectors. XylR-dependent stimulation of transcription from a Pu::lacZ fusion shows different induction kinetics with different effectors. With toluene, activation followed a hyperbolic curve with an apparent K of 0.95 mM and a maximum beta-galactosidase activity of 2,550 Miller units. With o-nitrotoluene, in contrast, activation followed a sigmoidal curve with an apparent K of 0.55 mM and a Hill coefficient of 2.65. m-Nitrotoluene kept the XylR regulator in an inactive transcriptional form. Therefore, upon binding of an effector, the substituent on the aromatic ring leads to productive or unproductive XylR forms. The different transcriptional states of the XylR regulator are substantiated by XylR mutants. XylRE172K is a mutant regulator that is able to stimulate transcription from the Pu promoter in the presence of m-nitrotoluene; however, its response to m-aminotoluene was negligible, in contrast with the wild-type regulator. These results illustrate the importance of the electrostatic interactions in effector recognition and in the stabilization of productive and unproductive forms by the regulator upon aromatic binding. XylRD135N and XylRD135Q are mutant regulators that are able to stimulate transcription from Pu in the absence of effectors, whereas substitution of Glu for Asp135 in XylRD135E resulted in a mutant whose ability to recognize effectors was severely impaired. Therefore, the conformation of mutant XylRD135Q as well as XylRD135N seemed to mimic that of the wild-type regulator when effector binding occurred, whereas mutant XylRD135E seemed to be blocked in a conformation similar to that of wild-type XylR and XylRE172K upon binding to an inhibitor molecule such as m-nitrotoluene or m-aminotoluene.