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  • 1.
    Bartilson, M
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nordlund, I
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600.1990In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 220, no 2Article in journal (Refereed)
    Abstract [en]

    The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and metacleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pVI150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.

  • 2.
    Bartilson, M
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nucleotide sequence and expression of the catechol 2,3-dioxygenase-encoding gene of phenol-catabolizing Pseudomonas CF600.1989In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 85, no 1Article in journal (Refereed)
    Abstract [en]

    Pseudomonas CF600 degrades phenol and some of its methylated derivatives via a plasmid-encoded catabolic pathway. The catechol 2,3-dioxygenase (C23O) enzyme of this pathway catalyses the conversion of catechol to 2-hydroxymuconic semialdehyde. We have determined the nucleotide (nt) sequence of the dmpB structural gene for this enzyme, and expressed and identified its polypeptide product in Escherichia coli. The xylE gene of TOL plasmid pWWO and the nahH gene of plasmid NAH7 encode analogous C23O enzymes. Comparison of these three genes shows homology of 78-81% on the nt level and 83-87% homology on the amino acid level.

  • 3. Fernández, S
    et al.
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    De Lorenzo, V
    Cross-regulation by XylR and DmpR activators of Pseudomonas putida suggests that transcriptional control of biodegradative operons evolves independently of catabolic genes.1994In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 176, no 16Article in journal (Refereed)
    Abstract [en]

    The Pu promoter of the toluene degradation plasmid pWW0 of Pseudomonas putida drives expression of an operon involved in the sequential oxidation of toluene and m- and p-xylenes to benzoate and toluates, respectively. Similarly, the Po promoter of plasmid pVI150 controls expression of an operon of Pseudomonas sp. strain CF600 which is required for the complete catabolism of phenol and cresols. These promoters, which both belong to the sigma 54-dependent class, are regulated by their cognate activators, XylR and DmpR, respectively. XylR and DmpR are homologous proteins, and both require aromatic compounds as effector molecules for activity. However, these two proteins respond to different profiles of aromatic compounds. The activity of each promoter in the presence of the heterologous regulator was monitored using lacZ and luxAB reporter systems. Genetic evidence is presented that the two activators can functionally substitute each other in the regulation of their corresponding promoters by binding the same upstream DNA segment. Furthermore, when coexpressed, the two proteins appear to act simultaneously on each of the promoters, expanding the responsiveness of these systems to the presence of effectors of both proteins. Potential mechanisms for the occurrence of evolutionary divergence between XylR and DmpR are discussed in view of the DNA sequence similarities among Pu, Po, and a third XylR-responsive promoter, Ps.

  • 4. Gullberg, M
    et al.
    Noreus, K
    Brattsand, G
    Friedrich, B
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and characterization of a 19-kilodalton intracellular protein. An activation-regulated putative protein kinase C substrate of T lymphocytes.1990In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 265, no 29Article in journal (Refereed)
    Abstract [en]

    Activation of protein kinase C in T cells results in rapid phosphorylation of a 19-kDa intracellular protein termed 19K. We report the purification of 19K from human peripheral T cells and an internal 20-amino acid sequence determined from this protein. It is shown that 19K is a novel cytoplasmatic protein which is phosphorylated in vitro by partially purified protein kinase C. 19K-specific antibodies, raised by immunizing rabbits with purified protein, were used to show that the 19K is expressed, and phosphorylated in response to protein kinase C activation, in several cellular systems. These antibodies were also used to precipitate 19K from both [35S]methionine and 32Pi-labeled T cells. The data showed that 15 min of phorbol ester treatment has no effect on the rate of 19K synthesis but results in induction of 19K phosphorylation. However, we demonstrate, by Western blot analysis, that expression of 19K in primary peripheral T cells increased at least 10-fold over a period of 4 days after activation. The increase in 19K expression correlates with initiation of DNA synthesis, and in proliferating T cells 19K comprises approximately 0.2% of total cytoplasmatic protein. Thus, 19K is a novel putative protein kinase C substrate which is subject to activation associated up-regulation in human T cells.

  • 5. Marklund, U
    et al.
    Brattsand, G
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Gullberg, M
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Serine 25 of oncoprotein 18 is a major cytosolic target for the mitogen-activated protein kinase.1993In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 268, no 20Article in journal (Refereed)
    Abstract [en]

    Oncoprotein 18 (Op18) is an 18-19-kDa cytoplasmic phosphoprotein, of unknown function, that is frequently up-regulated in transformed cells. Stimulation of various cell-surface receptors results in extensive phosphorylation of Op18 and this protein has, therefore, previously been implicated in intracellular signaling. In the present study, by expression of specific Op18 cDNA mutant constructs and phosphopeptide mapping, we have identified in vivo phosphorylation sites. In conjunction with in vitro phosphorylation experiments, using purified wild-type and mutant Op18 proteins in combination with a series of kinases, these results have identified two distinct proline-directed kinase families that phosphorylate Op18 with overlapping but distinct site preference. These two kinase families, mitogen activated protein (MAP) kinases and cyclin dependent cdc2 kinases, are involved in receptor and cell cycle-regulated phosphorylation events, respectively. Therefore, Op18 may reside at a junction where receptor and cell cycle-regulated kinase families interact with the same substrate. The present study shows that the MAP kinase has a 20-fold preference for Ser25 as opposed to Ser38 of Op18, while cdc2 kinases have a 5-fold preference for the Ser38 residue. Only a minor fraction of the 4.5 x 10(6) Op18 molecules/cell in a leukemic T-cell line are normally in their Ser25 phosphorylated form. However, antigen receptor stimulation of this cell line is shown to result in a rapid conversion of 35-45% of all Op18 molecules to the Ser25 phosphorylated form. These results suggest that Ser25 of Op18 may be a major cytoplasmic target for the MAP kinase in cells with high expression of Op18.

  • 6.
    Ng, L C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    O'Neill, E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Genetic evidence for interdomain regulation of the phenol-responsive final sigma54-dependent activator DmpR.1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 29Article in journal (Refereed)
    Abstract [en]

    The final sigma54-dependent DmpR activator regulates transcription of the dmp operon that encodes the enzymes for catabolism of (methyl)phenols. DmpR is expressed constitutively, but its transcriptional promoting activity is controlled positively in direct response to the presence of aromatic pathway substrates (effectors). DmpR has a distinct domain structure with the amino-terminal A-domain controlling the specificity of activation of the regulator by aromatic effectors (signal reception), a central C-domain mediating an ATPase activity essential for transcriptional activation, and a carboxyl-terminal D-domain involved in DNA binding. Deletion of the A-domain has been shown previously to result in an effector-independent transcriptional activator with constitutive ATPase activity. These results, in conjunction with the location of mutations within the A- and C-domains which exhibit an effector-independent (semiconstitutive) property, have led to a working model in which the A-domain serves to mask the ATPase and transcriptional promoting activity of the C-domain in the absence of effectors. To investigate the mechanism by which the A-domain exerts its repressive effect, we developed a genetic system to select positively for intramolecular second site revertants of DmpR. The results demonstrate (i) that mutations within the A-domain can suppress the semiconstitutive activity of C-domain located mutations and vice versa; (ii) that the C-domain located mutations do not influence the intrinsic ATPase and transcriptional promoting property of the C-domain in the absence of the A-domain; and (iii) that semiconstitutive mutations of the A- and C-domain have an additive effect. Taken together these results support a model in which the A-domain represses the function(s) of the C-domain by direct interactions between residues of the two domains.

  • 7.
    Ng, L C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Poh, C L
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aromatic effector activation of the NtrC-like transcriptional regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it controls.1995In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 177, no 6Article in journal (Refereed)
    Abstract [en]

    Pseudomonas putida P35X (NCIB 9869) metabolizes phenol and monomethylphenols via a chromosomally encoded meta-cleavage pathway. We have recently described a 13.4-kb fragment of the chromosome that codes for the first eight genes of the catabolic pathway and a divergently transcribed positive regulator, phhR. The eight structural genes lie in an operon, the phh operon, downstream of a -24 TGGC, -12 TTGC promoter sequence. Promoters of this class are recognized by RNA polymerase that utilizes the alternative sigma 54 factor encoded by rpoN (ntrA) and are positively regulated by activators of the NtrC family. In this study, we have identified the coding region for the 63-kDa PhhR gene product by nucleotide sequencing of a 2,040-bp region and polypeptide analysis. PhhR was found to have homology with the NtrC family of transcriptional activators, in particular with DmpR, the pVI150-encoded regulator of (methyl)phenol catabolism by Pseudomonas sp. strain CF600. By using a luciferase reporter system, PhhR alone was shown to be sufficient to activate transcription from the phh operon promoter in an RpoN+ background but not an RpoN- background. Luciferase reporter systems were also used to directly compare the aromatic effector profiles of PhhR and DmpR. Evidence that the difference in the growth substrate ranges of strains P35X and CF600 is due to the effector activation specificities of the regulators of these systems rather than the substrate specificities of the catabolic enzymes is presented.

  • 8.
    Ng, L C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sze, C C
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Poh, C L
    Cloning and sequences of the first eight genes of the chromosomally encoded (methyl) phenol degradation pathway from Pseudomonas putida P35X.1994In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 151, no 1-2Article in journal (Refereed)
    Abstract [en]

    Pseudomonas putida P35X (NCIB 9869) metabolises phenol and cresols via a chromosomally encoded meta-cleavage pathway. A 13.4-kb fragment of the chromosome involved in encoding phenol catabolism was cloned and characterized. Deletion analysis and nucleotide sequencing of a 6589-bp region, in conjunction with enzyme assays, were used to identify the phhKLMNOP genes encoding the phenol hydroxylase, the phhB gene encoding catechol 2,3-dioxygenase (EC 1.13.11.2) and the phhQ gene that encodes a small ferredoxin-like protein. The genes are organised in an operon-like structure, in the order phhKLMNOPQB, and the deduced amino-acid sequences share high homology (68.3-99.7%) with those of the plasmid-encoded genes dmpKLMNOPQB of Pseudomonas sp. strain CF600. Genetic evidence is presented that the difference in the growth substrate ranges of Pseudomonas P35X and CF600 are due to the effector activation specificities of the regulators of these systems, rather than the substrate specificities of the catabolic enzymes.

  • 9.
    Nordlund, I
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Powlowski, J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hagström, A
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Conservation of regulatory and structural genes for a multi-component phenol hydroxylase within phenol-catabolizing bacteria that utilize a meta-cleavage pathway.1993In: Journal of general microbiology, ISSN 0022-1287, Vol. 139, no 11Article in journal (Refereed)
    Abstract [en]

    Pseudomonas sp. strain CF600 can degrade phenol and some of its methylated derivatives via a plasmid (pVI150)-encoded pathway. The metabolic route involves hydroxylation by a multi-component phenol hydroxylase and a subsequent meta-cleavage pathway. All 15 structural genes involved are clustered in an operon that is regulated by a divergently transcribed transcriptional activator. The multi-component nature of the phenol hydroxylase is unusual since reactions of this type are usually accomplished by single component flavoproteins. We have isolated and analysed a number of marine bacterial isolates capable of degrading phenol and a range of other aromatic compounds as sole carbon and energy sources. Southern hybridization and enzyme assays were used to compare the catabolic pathways of these strains and of the archetypal phenol-degrader Pseudomonas U, with respect to known catabolic genes encoded by Pseudomonas CF600. All the strains tested that degraded phenol via a meta-cleavage pathway were found to have DNA highly homologous to each of the components of the multicomponent phenol hydroxylase. Moreover, DNA of the same strains also strongly hybridized to probes specific for pVI150-encoded meta-pathway genes and the specific regulator of its catabolic operon. These results demonstrate conservation of structural and regulatory genes involved in aromatic catabolism within strains isolated from diverse geographical locations (UK, Norway and USA) and a range of habitats that include activated sludge, sea water and fresh-water mud.

  • 10.
    Nordlund, I
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Powlowski, J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.1990In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 172, no 12Article in journal (Refereed)
    Abstract [en]

    Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.

  • 11.
    Nordlund, I
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nucleotide sequences of the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas CF600.1990In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1049, no 2Article in journal (Refereed)
    Abstract [en]

    The nucleotide sequence of a 2493 base pair (bp) region, spanning the coding regions for the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), was determined. The deduced protein sequence for HMSD is 486 amino acid residues long with an Mr of 51,682. HMSD has homology with a number of aldehyde dehydrogenases from various eukaryotic sources. The deduced protein sequence for HMSH is 283 amino acids long with an Mr of 30,965. The amino acid composition of this enzyme is similar to that of isofunctional enzymes from toluene and m-cresol catabolic pathways.

  • 12.
    O'Neill, E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ng, L C
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sze, C C
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.1998In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 28, no 1Article in journal (Refereed)
    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.

  • 13.
    O'Neill, E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sze, C C
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Novel effector control through modulation of a preexisting binding site of the aromatic-responsive sigma(54)-dependent regulator DmpR.1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, no 45Article in journal (Refereed)
    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.

  • 14.
    O'Neill, E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikström, P
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR.2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4Article in journal (Refereed)
    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.

  • 15.
    Pavel, H
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsman, M
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An aromatic effector specificity mutant of the transcriptional regulator DmpR overcomes the growth constraints of Pseudomonas sp. strain CF600 on para-substituted methylphenols.1994In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 176, no 24Article in journal (Refereed)
    Abstract [en]

    The pVI150 catabolic plasmid of Pseudomonas sp. strain CF600 carries the dmp system, which comprises the divergently transcribed dmpR gene and the dmp operon coding for the catabolic enzymes required for growth on (methyl)phenols. The constitutively expressed DmpR transcriptional activator positively controls the expression of the RpoN-dependent dmp operon promoter in the presence of the aromatic effector in the growth medium. However, the magnitude of the transcriptional response differs depending on the position of the methyl substituent on the aromatic ring. Experiments involving an elevated copy number of the dmp system demonstrate that growth on para-substituted methylphenols is limited by the level of the catabolic enzymes. An effector specificity mutant of DmpR, DmpR-E135K, that responded to the presence of 4-ethylphenol, a noneffector of the wild-type protein, was isolated by genetic selection. The single point mutation in DmpR-E135K, which results in a Glu-to-Lys change in residue 135, also results in a regulator with enhanced recognition of para-substituted methylphenols. The DmpR-E135K mutation, when introduced into the wild-type strain, confers enhanced utilization of the para-substituted methylphenols. These experiments demonstrate that the aromatic effector activation of wild-type DmpR by the para-substituted methylphenols is a major factor limiting the catabolism of these compounds.

  • 16. Platt, A
    et al.
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Taylor, S C
    Williams, P A
    The 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahM and nahO genes of the naphthalene catabolic plasmid pWW60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences.1995In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 141 ( Pt 9)Article in journal (Refereed)
    Abstract [en]

    We report the complete nucleotide sequence and over-expression of the nahOM genes for the acetaldehyde dehydrogenase (acylating) and the 4-hydroxy-2-oxovalerate aldolase from the meta pathway operon of the naphthalene catabolic plasmid pWW60-22 from Pseudomonas sp. NCIMB9816. Additional partial sequence analysis of adjacent DNA shows the gene order within the operon to be nahNLOMK, identical to the order found for the isofunctional genes in the meta pathway operons in the toluene/xylene pathway of TOL plasmid pWW0 and the phenol/methylphenol pathway of pVI150. The deduced amino acid sequences of NahO and NahM were significantly homologous to the equivalent enzymes encoded by other Pseudomonas meta pathways, although both were the most divergent in each comparison. The nahOM genes were inserted downstream of the T7 promoter in the expression vector pET3a and similar constructs were also made of the isofunctional regions from pVI150 (dmpFG) and TOL plasmid pDK1 (xyIQK). High expression of all three gene pairs was detected by enzyme assays and by SDS-PAGE.

  • 17.
    Powlowski, J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sahlman, L
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600.1993In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 175, no 2Article in journal (Refereed)
    Abstract [en]

    The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp. strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) [acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10]. A procedure for purifying these two enzyme activities to homogeneity is reported here. The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa. Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes. The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex. In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol. 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures. The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.

  • 18.
    Powlowski, J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sealy, J
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cadieux, E
    On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 2Article in journal (Refereed)
    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.

  • 19.
    Powlowski, J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600.1994In: Biodegradation, ISSN 0923-9820, E-ISSN 1572-9729, Vol. 5, no 3-4Article in journal (Refereed)
    Abstract [en]

    Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid located dmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-known meta-cleavage pathway for catechol, and include the recently discovered meta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of these meta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to the meta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations between meta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

  • 20.
    Powlowski, J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.1990In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 172, no 12Article in journal (Refereed)
    Abstract [en]

    An in vitro study of the multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600 was performed. Phenol-stimulated oxygen uptake from crude extracts was strictly dependent on the addition of NAD(P)H and Fe2+ to assay mixtures. Five of six polypeptides required for growth on phenol were necessary for in vitro activity. One of the polypeptides was purified to homogeneity and found to be a flavin adenine dinucleotide containing iron-sulfur protein with significant sequence homology, at the amino terminus, to plant-type ferredoxins. This component, as in other oxygenase systems, probably functions to transfer electrons from NAD(P)H to the iron-requiring oxygenase component. Phenol hydroxylase from this organism is thus markedly different from bacterial flavoprotein monooxygenases commonly used for hydroxylation of other phenolic compounds, but bears a number of similarities to multicomponent oxygenase systems for unactivated compounds.

  • 21. Qian, H
    et al.
    Edlund, U
    Powlowski, J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sethson, I
    Solution structure of phenol hydroxylase protein component P2 determined by NMR spectroscopy.1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 3Article in journal (Refereed)
    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.

  • 22. Roper, D I
    et al.
    Subramanya, H S
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wigley, D B
    Preliminary crystallographic analysis of 4-oxalocrotonate tautomerase reveals the oligomeric structure of the enzyme.1994In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 243, no 4Article in journal (Refereed)
    Abstract [en]

    Crystals of recombinant 4-oxalocrotonate tautomerase from Pseudomonas sp. strain CF600 have been obtained in a form suitable for X-ray analysis. The enzyme is a highly efficient catalyst and is unusual in that it consists of subunits of only 62 amino acids. It crystallises in the triclinic space group, P1, with unit cell dimensions a = 39.6 A, b = 51.5 A, c = 51.6 A, alpha = 60.0 degrees, beta = 81.4 degrees, gamma = 69.6 degrees. The crystals diffract to beyond 1.9 A resolution and are stable to irradiation with X-rays. Preliminary crystallographic data are not consistent with the previously suggested pentameric structure, but indicate that the complex is in fact a hexamer with 32 symmetry.

  • 23.
    Sarand, I
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsman, M
    Romantschuk, M
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils.2001In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 67, no 1Article in journal (Refereed)
    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.

  • 24.
    Shinger, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Thomas, C M
    Transcription in the trfA region of broad host range plasmid RK2 is regulated by trfB and korB.1984In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 195, no 3Article in journal (Refereed)
    Abstract [en]

    Transcription at various points in the trf A region of broad host range plasmid RK2 has been analysed by measuring expression of the galK gene inserted at EcoRI sites introduced previously by TB1723 transposition mutagenesis. Rightward transcription (anti-clockwise on RK2) probably from a single promoter, proceeds across two open reading frames coding for a 13 kD polypeptide of unknown function, and the trf A gene, which provides a protein(s) essential for plasmid replication. This transcription is not auto-regulated by the products of either open reading frame and is also not subject to significant attenuation prior to the end of the trfA open reading frame. Leftward transcription appears to be directed by at least two well separated promoters, the more leftward being three to four times stronger than the more rightward. Rightward, but not leftward, transcription is repressed about 9-fold by the trfB locus of RK2 alone (so far not separable from the loci korA and korD) in trans while the combination of the korB and trfB loci in trans represses both rightward transcription (about 100-fold) and leftward transcription (the stronger activity by 10 to 15-fold). Regulation of these operons is therefore qualitatively different. The kilD locus in the trfA region, which is suppressed by korD (trfB) is thus probably part of the rightward (trfA) operon, while leftward transcription may represent the start of an operon containing kilB. The results suggest that RK2kor loci act by repressing transcription of kil loci and that the kil and kor control circuits may be part of an interlocking system of RK2 genes involved in replication and stable maintenance.

  • 25.
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Signal sensing by sigma 54-dependent regulators: derepression as a control mechanism.1996In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 19, no 3Article in journal (Refereed)
    Abstract [en]

    Transcription by RNA polymerase utilizing the alternative sigma factor beta 54 is regulated by a distinct class of positive activators designated the sigma 54-dependent family. The activities of these regulators are themselves modulated in response to a wide variety of environmental signals. Factors that modulate the expression or the activity of the regulatory protein in response to chemical and metabolic changes are ultimately responsible for determining the level of expression of sigma 54-dependent genes and hence the diverse bacterial functions that they encode. Many members of the sigma 54-dependent family are part of two-component sensor-response systems. This MicroReview emphasizes recent data concerning the activities of a distinct subgroup of the sigma 54-dependent regulators that directly sense and respond with transcriptional activation to the presence of small effector molecules in their environment. The functional consequences of effector activation in terms of regulation of the enzymatic (ATPase) activity of these transcriptional activators and interdomain interactions are discussed.

  • 26.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bartilson, M
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moore, T
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators.1993In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 175, no 6Article in journal (Refereed)
    Abstract [en]

    The catabolic plasmid pVI150 of Pseudomonas sp. strain CF600 encodes all the genetic information required for the regulated metabolism of phenol and some of its methyl-substituted derivatives. The structural dmp genes of the pathway are clustered in a single operon that lies just downstream of a -24 TGGC, -12 TTGC nif/ntr-like promoter sequence. Promoters of this class are recognized by a minor form of RNA polymerase utilizing sigma 54 (NtrA, RpoN). Primer extension analysis demonstrated that the dmp operon transcript initiates downstream of the -24, -12 promoter. Transposon insertion mutants, specifically defective in the regulation of the dmp operon, were isolated, and complementation of a phenol-utilization regulatory mutant was used to identify the regulatory locus, dmpR. The 67-kDa dmpR gene product alone was shown to be sufficient for activation of transcription from the dmp operon promoter. Nucleotide sequence determination revealed that DmpR belongs to the NtrC family of transcriptional activators that regulate transcription from -24, -12 promoters. The deduced amino acid sequence of DmpR has high homology (40 to 67% identity) with the central and carboxy-terminal regions of these activators, which are believed to be involved in the interaction with the sigma 54 RNA polymerase and in DNA binding, respectively. The amino-terminal region of DmpR was found to share 64% identity with the amino-terminal region of XylR, which is also a member of this family of activators. This region has been implicated in effector recognition of aromatic compounds that is required for the regulatory activity of XylR.

  • 27.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Franklin, F C
    Tsuda, M
    Holroyd, D
    Bagdasarian, M
    Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600.1989In: Journal of general microbiology, ISSN 0022-1287, Vol. 135, no 5Article in journal (Refereed)
    Abstract [en]

    Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1.2 kb region of DNA encoding a 39.5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39.5 kDa polypeptide as one component.

  • 28.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moore, T
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600.1994In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 176, no 6Article in journal (Refereed)
    Abstract [en]

    The dmp operon of the pVI150 catabolic plasmid of Pseudomonas sp. strain CF600 encodes the enzymes involved in the catabolism of phenol and methylphenols. The regulator of this dmp pathway, DmpR, is a member of the NtrC family of transcriptional activators and controls transcription of the dmp operon in response to aromatic effector compounds (V. Shingler, M. Bartilson, and T. Moore, J. Bacteriol. 175:1596-1604, 1993). Using a lux gene fusion reporter system, in which the DmpR-regulated operon promoter controls the expression of luciferase activity, we have shown in the study reported here that DmpR is activated by, but responds differentially to, the presence of a wide range of aromatic compounds. In many microbial regulatory systems, including some members of the NtrC family, the response to environmental fluctuations involves information transfer from surface sensory proteins to transcriptional regulators. However, DmpR-mediated activation of phenol metabolism in response to aromatic compounds occurs in the absence of a specific sensory protein. We used hybrids between DmpR and XylR, a structurally related regulator of toluene and xylene metabolism, to demonstrate that it is the amino-terminal domains of these regulators that determine the specificity of transcriptional activation. The results suggest that it is the direct interaction of aromatic compounds with the DmpR and XylR proteins that regulates their transcriptional promoting activity.

  • 29.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Pavel, H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds.1995In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 17, no 3Article in journal (Refereed)
    Abstract [en]

    The NtrC-like regulator DmpR controls transcription from the dmp operon that encodes the enzymes for catabolism of phenol and some related aromatic compounds. DmpR activates transcription from the sigma 54-dependent dmp-operon promoter in the presence of pathway substrates or structural analogues in the growth medium. Using affinity-purified DmpR and a truncated derivative, we show here that aromatic compounds directly activate the ATPase activity of this protein in vitro, and that the amino-terminal domain represses this activity in the absence of an aromatic ligand. In order to dissect the activation process, derivatives of DmpR exhibiting single amino acid changes were isolated and their effector-dependence and specificity profiles were analysed in vivo. The mechanistic implications of the phenotypes of these mutants are discussed.

  • 30.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Powlowski, J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Marklund, U
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600.1992In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 174, no 3Article in journal (Refereed)
    Abstract [en]

    The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.

  • 31.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Thomas, C M
    Analysis of nonpolar insertion mutations in the trfA gene of IncP plasmid RK2 which affect its broad-host-range property.1989In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1007, no 3Article in journal (Refereed)
    Abstract [en]

    Replication of broad-host-range plasmid RK2 requires the protein product(s) of the plasmid-encoded trfA gene to initiate replication at oriV, the vegetative replication origin. The trfA gene contains two translational starts which direct translation of two polypeptides, of 382 and 285 amino acids, which differ by the 97 amino acids at their N-terminus. Nonpolar insertions which abolish expression of the larger TrfA polypeptide but otherwise retain the trfA gene's normal expression signals severely reduce plasmid replication efficiency in Pseudomonas aeruginosa and to a lesser extent in Pseudomonas putida, but have very little effect in Escherichia coli. This indicates that the organization of the trfA gene, producing two polypeptides products, plays an important part in the broad-host-range of plasmid RK2 by providing a degree of flexibility in the way the plasmid's replication system interacts with host biochemistry.

  • 32.
    Shingler, V
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Thomas, C M
    Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products.1984In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 175, no 3Article in journal (Refereed)
    Abstract [en]

    Broad host-range plasmid RK2 is a member of the Escherichia coli incompatibility group P. Unlike most other groups of plasmids, members of the P group are capable of efficient transfer between and maintenance in most gram-negative bacterial species. It is of interest whether this broad host-range results from differences between the mechanism of replication of broad and narrow host-range plasmids. The regions of RK2 required for replication in E. coli have previously been defined as an origin of vegetative replication, oriVRK2 , and a gene, trfA , specifying a positively required trans-acting product. In this study Tn1723 transposon insertions have been used to map the trfA gene and determine its functional gene product. The Tn1723 insertions define the outer limits of the gene, a promoter region, a "leader" region not essential for trfA activity and a coding region. Three polypeptides of 13 X 10(3), 43 X 10(3) and 32 X 10(3) molecular weight are produced from this region and the production of a 32 X 10(3) Mr polypeptide is shown to be correlated with trfA activity in E. coli. Analysis of polypeptides produced from transposon insertion derivatives in which all but 35 base-pairs of inserted DNA is deleted, along with the effect of these insertions on trfA activity, suggest that the 43 X 10(3) and 32 X 10(3) Mr polypeptide coding sequences overlap in the same reading frame and that all three polypeptides (13 X 10(3), 32 X 10(3) and 43 X 10(3) Mr) may be translated from the same initial transcript.

  • 33.
    Skärfstad, E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    O'Neill, E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Garmendia, J
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling.2000In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 182, no 11Article in journal (Refereed)
    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.

  • 34. Smith, C A
    et al.
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Thomas, C M
    The trfA and trfB promoter regions of broad host range plasmid RK2 share common potential regulatory sequences.1984In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 12, no 8Article in journal (Refereed)
    Abstract [en]

    The positions of the trfA and trfB promoters of broad host range IncP plasmid RK2 (identical to RP1, RP4, R68 and R18 ) were identified by RNA polymerase protection studies, and the nucleotide sequences of the promoter regions determined. A mutation within the trfA promoter sequence is associated with loss of kilD activity. In addition a probable promoter region for the kilB locus was identified. The three promoter regions share common palindromic sequences which may serve as sites for the coordinate regulation of replication and kil functions.

  • 35.
    Sze, C C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Laurie, A D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    In vivo and in vitro effects of integration host factor at the DmpR-regulated sigma(54)-dependent Po promoter.2001In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, no 9Article in journal (Refereed)
    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.

  • 36.
    Sze, C C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moore, T
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Growth phase-dependent transcription of the sigma(54)-dependent Po promoter controlling the Pseudomonas-derived (methyl)phenol dmp operon of pVI150.1996In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 178, no 13Article in journal (Refereed)
    Abstract [en]

    Transcription from Pseudomonas-derived -24, -12 Po promoter of the pVI150-encoded dmp operon is mediated by the sigma 54-dependent DmpR activator in response to the presence of aromatic pathway substrates in the medium. However, global regulatory mechanisms are superimposed on this regulatory system so that the specific response to aromatic effectors is absent in cultures until the stationary phase is reached. Here we genetically dissect the system to show that the growth phase response is faithfully mimicked by a minimal system composed of the dmpR regulatory gene and the Po promoter regulatory region and can be reproduced in heterologous Escherichia coli. Using this system, we show that the growth phase-dependent DmpR-mediated response to aromatic compounds is limited to fast-growing cultures. Thus, during exponential growth of cultures in minimal media containing different carbon sources, the response to aromatics is immediate, while the response is suppressed in cultures grown on rich media until the exponential-to-stationary phase transition. Elements known to be involved in the DmpR-mediated transcription from Po were analyzed for the ability to influence the growth phase response. Most dramatically, overexpression of DmpR was shown to completely abolish the growth phase response, suggesting that a negatively acting factor may mediate this level of regulation. The possible mechanism of action and integration (of the specific regulation of the dmp operon-encoded catabolic enzymes with the physiological status of the bacteria are discussed.

  • 37.
    Sze, C C
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The alarmone (p)ppGpp mediates physiological-responsive control at the sigma 54-dependent Po promoter.1999In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 31, no 4Article in journal (Refereed)
    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.

  • 38. Thomas, C M
    et al.
    Smith, C A
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cross, M A
    Hussain, A A
    Pinkney, M
    Regulation of replication and maintenance functions of broad host-range plasmid RK2.1985In: Basic life sciences, ISSN 0090-5542, Vol. 30Article in journal (Refereed)
    Abstract [en]

    Replication of broad host-range plasmid RK2 depends on a cisacting vegatative replication origin oriVRK2 and the polypeptide product(s) of the trans-acting gene trfA as well as on host-specified products. The trfA gene is the second cistron in a polycistronic unit whose first cistron may be kilD, one of 4 known RK2-specified kil loci (kilA, B, C, and D) which are inhibitory for bacterial host or plasmid vector in the absence of kor functions which suppress in trans the effect of their respective kil genes. Transcription of the operon containing trfA is negatively regulated by the products of both the trfB locus (alias korD and korA) and korB. The loci, trfB and korB, are expressed from a single transcriptional unit which we propose to be negatively autoregulated by the products of both loci, although an additional, weaker and unregulated transcript may also express korB. While deletions in the oriVRK2 region have indicated the presence of copy number control elements adjacent to and possibly overlapping with the minimal oriVRK2 segment, the overriding control of copy number seems to reside in the trfB and korB loci which in conjunction appear to reduce expression of the trfA gene to levels limiting for replication. Coregulation of trfA with kil genes may indicate that kil genes play a role in plasmid maintenance other than replication.

  • 39.
    Wikström, P
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    O'Neill, E
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ng, L C
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation.2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 314, no 5Article in journal (Refereed)
    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.

1 - 39 of 39
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