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O'Neill, E., Wikström, P. & Shingler, V. (2001). An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR.. EMBO Journal, 20(4)
Open this publication in new window or tab >>An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR.
2001 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112325 (URN)10.1093/emboj/20.4.819 (DOI)11179226 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
Sze, C. C., Laurie, A. D. & Shingler, V. (2001). In vivo and in vitro effects of integration host factor at the DmpR-regulated sigma(54)-dependent Po promoter.. Journal of Bacteriology, 183(9)
Open this publication in new window or tab >>In vivo and in vitro effects of integration host factor at the DmpR-regulated sigma(54)-dependent Po promoter.
2001 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, no 9Article in journal (Refereed) Published
Abstract [en]

Transcription from the Pseudomonas CF600-derived sigma(54)-dependent promoter Po is controlled by the aromatic-responsive activator DmpR. Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro. In vivo, the Po promoter exhibits characteristics that typify many sigma(54)-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output. IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites. In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription. This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at sigma(54) promoters such as aiding binding of the regulator or recruitment of sigma(54)-RNA polymerase via UP element-like DNA. The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA. Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112324 (URN)10.1128/JB.183.9.2842-2851.2001 (DOI)11292804 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
Sarand, I., Skärfstad, E., Forsman, M., Romantschuk, M. & Shingler, V. (2001). Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils.. Applied and Environmental Microbiology, 67(1)
Open this publication in new window or tab >>Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils.
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2001 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 67, no 1Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112326 (URN)10.1128/AEM.67.1.162-171.2001 (DOI)11133441 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
Wikström, P., O'Neill, E., Ng, L. C. & Shingler, V. (2001). The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation.. Journal of Molecular Biology, 314(5)
Open this publication in new window or tab >>The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation.
2001 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 314, no 5Article in journal (Refereed) Published
Abstract [en]

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 sigma(54)-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with sigma(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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112323 (URN)10.1006/jmbi.2000.5212 (DOI)11743715 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
Skärfstad, E., O'Neill, E., Garmendia, J. & Shingler, V. (2000). Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling.. Journal of Bacteriology, 182(11)
Open this publication in new window or tab >>Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling.
2000 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 182, no 11Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112327 (URN)10809676 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
O'Neill, E., Sze, C. C. & Shingler, V. (1999). Novel effector control through modulation of a preexisting binding site of the aromatic-responsive sigma(54)-dependent regulator DmpR.. Journal of Biological Chemistry, 274(45)
Open this publication in new window or tab >>Novel effector control through modulation of a preexisting binding site of the aromatic-responsive sigma(54)-dependent regulator DmpR.
1999 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 45Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112328 (URN)10542286 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
Sze, C. C. & Shingler, V. (1999). The alarmone (p)ppGpp mediates physiological-responsive control at the sigma 54-dependent Po promoter.. Molecular Microbiology, 31(4)
Open this publication in new window or tab >>The alarmone (p)ppGpp mediates physiological-responsive control at the sigma 54-dependent Po promoter.
1999 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 31, no 4Article in journal (Refereed) Published
Abstract [en]

Transcription from the Pseudomonas-derived sigma 54-dependent Po promoter of the dmp operon is mediated by the aromatic-responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR-mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non-permissive conditions. Conversely (p)ppGpp-deficient Escherichia coli strains were found to be severely defective in DmpR-mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the beta, beta' and sigma 70 subunits of RNA polymerase, which confer prototrophy on ppGpp0 E. coli, was also found to restore specific DmpR-mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the sigma 54-RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from sigma 54 promoters.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112329 (URN)10096088 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
O'Neill, E., Ng, L. C., Sze, C. C. & Shingler, V. (1998). Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.. Molecular Microbiology, 28(1)
Open this publication in new window or tab >>Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.
1998 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 28, no 1Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112330 (URN)9593302 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
Powlowski, J., Sealy, J., Shingler, V. & Cadieux, E. (1997). On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.. Journal of Biological Chemistry, 272(2)
Open this publication in new window or tab >>On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.
1997 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 2Article in journal (Refereed) Published
Abstract [en]

DmpK from Pseudomonas sp. strain CF600 represents a group of proteins required by phenol-degrading bacteria that utilize a multicomponent iron-containing phenol hydroxylase. DmpK has been overexpressed in Escherichia coli and purified to homogeneity; it lacks redox cofactors and was found to strongly inhibit phenol hydroxylase in vitro. Chemical cross-linking experiments established that DmpK binds to the two largest subunits of the oxygenase component of the hydroxylase; this may interfere with binding of the hydroxylase activator protein, DmpM, causing inhibition. Since expression of DmpK normally appears to be much lower than that of the components of the oxygenase, inhibition may not occur in vivo. Hence, the interaction between DmpK and the oxygenase manifested in the inhibition and cross-linking results prompted construction of E. coli strains in which the oxygenase component was expressed in the presence and absence of a low molar ratio of DmpK. Active oxygenase was detected only when expressed in the presence of DmpK. Furthermore, inactive oxygenase could be activated in vitro by adding ferrous iron, in a process that was dependent on the presence of DmpK. These results indicate that DmpK plays a role in assembly of the active form of the oxygenase component of phenol hydroxylase.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112331 (URN)8995386 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
Qian, H., Edlund, U., Powlowski, J., Shingler, V. & Sethson, I. (1997). Solution structure of phenol hydroxylase protein component P2 determined by NMR spectroscopy.. Biochemistry, 36(3)
Open this publication in new window or tab >>Solution structure of phenol hydroxylase protein component P2 determined by NMR spectroscopy.
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1997 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 3Article in journal (Refereed) Published
Abstract [en]

Phenol hydroxylase from Pseudomonas sp. CF600 is a member of a family of binuclear iron-center-containing multicomponent oxygenases, which catalyzes the conversion of phenol and some of its methyl-substituted derivatives to catechol. In addition to a reductase component which transfers electrons from NADH, optimal turnover of the hydroxylase requires P2, a protein containing 90 amino acids which is readily resolved from the other components. The three-dimensional solution structure of P2 has been solved by 3D heteronuclear NMR spectroscopy. On the basis of 1206 experimental constraints, including 1060 distance constraints obtained from NOEs, 70 phi dihedral angle constraints, 42 psi dihedral angle constraints, and 34 hydrogen bond constraints, a total of 12 converged structures were obtained. The atomic root mean square deviation for the 12 converged structure with respect to the mean coordinates is 2.48 A for the backbone atoms and 3.85 A for all the heavy atoms. This relatively large uncertainty can be ascribed to conformational flexibility and exchange. The molecular structure of P2 is composed of three helices, six antiparallel beta-strands, one beta-hairpin, and some less ordered regions. This is the first structure among the known multicomponent oxygenases. On the basis of the three-dimensional structure of P2, sequence comparisons with similar proteins from other multicomponent oxygenases suggested that all of these proteins may have a conserved structure in the core regions.

National Category
Natural Sciences
Identifiers
urn:nbn:se:umu:diva-112332 (URN)10.1021/bi9619233 (DOI)9012665 (PubMedID)
Available from: 2015-12-06 Created: 2015-12-06 Last updated: 2018-06-07
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