umu.sePublications
Change search
Refine search result
12 1 - 50 of 81
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1. Andres Valderrama, J
    et al.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Carmona, Manuel
    Diaz, Eduardo
    AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp CIB2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 4, p. 1892-1904Article in journal (Refereed)
    Abstract [en]

    Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp(60) phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N-6-TGCAT) present at two different locations within the P-N promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp(60) of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other -proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria.

  • 2.
    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.

  • 3.
    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.

  • 4. Beljantseva, Jelena
    et al.
    Kudrin, Pavel
    Andresen, Liis
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Vicky
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Atkinson, Gemma C.
    Tenson, Tanel
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
    Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 14, p. 3726-3731Article in journal (Refereed)
    Abstract [en]

    The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ: RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5 alpha. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.

    Download full text (pdf)
    fulltext
  • 5. Beljantseva, Jelena
    et al.
    Kudrin, Pavel
    Jimmy, Steffi
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ehn, Marcel
    Pohl, Radek
    Varik, Vallo
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). 1University of Tartu, Institute of Technology, Tartu, Estonia.
    Tozawa, Yuzuru
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tenson, Tanel
    Rejman, Dominik
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). 1University of Tartu, Institute of Technology, Tartu, Estonia.
    Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 41839Article in journal (Refereed)
    Abstract [en]

    The alarmone nucleotide (p) ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p) ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p) ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.

    Download full text (pdf)
    fulltext
  • 6. Bernardo, Lisandro
    et al.
    Johansson, Linda U M
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    σ54-promoter discrimination and regulation by ppGpp and DksA2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 2, p. 828-838Article in journal (Refereed)
    Abstract [en]

    The sigma(54)-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of sigma(54)-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of sigma(54)-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of sigma(54)-dependent transcription in combination with a series of sigma(54)-promoters with graded affinities for sigma(54)-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted sigma(54)-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous sigma(54)-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive sigma(54)-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity sigma(54)-promoters that is conferred by the strong DNA binding properties of the P. putida sigma(54)-RNA polymerase holoenzyme.

  • 7.
    Bernardo, Lisandro M D
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Linda U M
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Solera, Dafne
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ54-dependent transcription2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, no 3, p. 749-764Article in journal (Refereed)
    Abstract [en]

    The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from sigma70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo sigma54 transcription even though they do not have any major direct effects on sigma54 transcription in reconstituted in vitro transcription and sigma-factor competition assays, (ii) that previously defined mutations rendering the housekeeping sigma70 less effective at competing with sigma54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from sigma54 promoters in vivo reflects the innate affinity of the promoters for sigma54-RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of sigma54-dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated sigma70 promoters.

    Download full text (pdf)
    FULLTEXT01
  • 8. Carbera Rodrigues, Carlos
    et al.
    Ekström, Fredrik
    Wolf, Jana
    Seibt, Henrik
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stier, Gunter
    Grundström, Christin
    Hung, Shen-Hua
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The crystal structure of the DmpR AAA+ domain suggests a novel mode of ATP-dependent transcriptional controlManuscript (preprint) (Other academic)
  • 9. del Peso-Santos, Teresa
    et al.
    Bartolomé-Martín, David
    Fernández, Cristina
    Alonso, Sergio
    García, José Luis
    Díaz, Eduardo
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Perera, Julián
    Coregulation by phenylacetyl-coenzyme A-responsive PaaX integrates control of the upper and lower pathways for catabolism of styrene by Pseudomonas sp. strain Y2.2006In: J Bacteriol, ISSN 0021-9193, Vol. 188, no 13, p. 4812-21Article in journal (Refereed)
    Abstract [en]

    The P(styA) promoter of Pseudomonas sp. strain Y2 controls expression of the styABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of two paa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of the paa gene clusters of this strain. In silico analysis of the P(styA) promoter region revealed a sequence located just within styA that is similar to the reported PaaX binding sites of Escherichia coli and the proposed PaaX binding sites of the paa genes of Pseudomonas species. Here we show that protein extracts from some Pseudomonas strains that have paaX genes, but not from a paaX mutant strain, can bind and retard the migration of a P(styA) specific probe. Purified maltose-binding protein (MBP)-PaaX1 fusion protein specifically binds the P(styA) promoter proximal PaaX site, and this binding is eliminated by the addition of phenylacetyl-coenzyme A. The sequence protected by MBP-PaaX1 binding was defined by DNase I footprinting. Moreover, MBP-PaaX1 represses transcription from the P(styA) promoter in a phenylacetyl-coenzyme A-dependent manner in vitro. Finally, the inactivation of both paaX gene copies of Pseudomonas sp. strain Y2 leads to a higher level of transcription from the P(styA) promoter, while heterologous expression of the PaaX1 in E. coli greatly decreases transcription from the P(styA) promoter. These findings reveal a control mechanism that integrates regulation of styrene catabolism by coordinating the expression of the styrene upper catabolic operon to that of the paa-encoded central pathway and support a role for PaaX as a major regulatory protein in the phenylacetyl-coenzyme A catabolon through its response to the levels of this central metabolite.

  • 10.
    Del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bernardo, Lisandro M D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmfeldt, Linda
    Togneri, Peter
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A hyper-mutant of the unusual σ70-Pr promoter bypasses synergistic ppGpp/DksA co-stimulation2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 14, p. 5853-5865Article in journal (Refereed)
    Abstract [en]

    The activities of promoters can be temporally and conditionally regulated by mechanisms other than classical DNA-binding repressors and activators. One example is the inherently weak σ70-dependent Pr promoter that ultimately controls catabolism of phenolic compounds. The activity of Pr is up-regulated through the joint action of ppGpp and DksA that enhance the performance of RNA polymerase at this promoter. Here, we report a mutagenesis analysis that revealed substantial differences between Pr and other ppGpp/DksA co-stimulated promoters. In vitro transcription and RNA polymerase binding assays show that it is the T at the −11 position of the extremely suboptimal −10 element of Pr that underlies both poor binding of σ70-RNAP and a slow rate of open complex formation—the process that is accelerated by ppGpp and DksA. Our findings support the idea that collaborative action of ppGpp and DksA lowers the rate-limiting transition energy required for conversion between intermediates on the road to open complex formation.

  • 11.
    Del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Landfors, Mattias
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ryden, Patrik
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Pr is a member of a restricted class of σ70-dependent promoters that lack a recognizable -10 element2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 22, p. 11308-11320Article in journal (Refereed)
    Abstract [en]

    The Pr promoter is the first verified member of a class of bacterial σ(70)-promoters that only possess a single match to consensus within its -10 element. In its native context, the activity of this promoter determines the ability of Pseudomonas putida CF600 to degrade phenolic compounds, which provides proof-of-principle for the significance of such promoters. Lack of identity within the -10 element leads to non-detection of Pr-like promoters by current search engines, because of their bias for detection of the -10 motif. Here, we report a mutagenesis analysis of Pr that reveals strict sequence requirements for its activity that includes an essential -15 element and preservation of non-consensus bases within its -35 and -10 elements. We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads. However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440. Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.

    Download full text (pdf)
    fulltext
  • 12.
    del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Inter-sigmulon communication through topological promoter coupling2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 20, p. 9638-9649Article in journal (Refereed)
    Abstract [en]

    Divergent transcription from within bacterial intergenic regions frequently involves promoters dependent on alternative sigma-factors. This is the case for the non-overlapping sigma(70)- and sigma(54)-dependent promoters that control production of the substrate-responsive regulator and enzymes for (methyl) phenol catabolism. Here, using an array of in vivo and in vitro assays, we identify transcription-driven supercoiling arising from the sigma(54)-promoter as the mechanism underlying inter-promoter communication that results in stimulation of the activity of the sigma(70)-promoter. The non-overlapping 'back-to-back' configuration of a powerful sigma(54)-promoter and weak sigma(70)-promoter within this system offers a previously unknown means of inter-sigmulon communication that renders the sigma(70)-promoter subservient to signals that elicit sigma(54)-dependent transcription without it possessing a cognate binding site for the sigma(54)-RNA polymerase holoenzyme. This mode of control has the potential to be a prevalent, but hitherto unappreciated, mechanism by which bacteria adjust promoter activity to gain appropriate transcriptional control.

    Download full text (pdf)
    fulltext
  • 13.
    del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Perera, Julián
    The styrene-responsive StyS/StyR regulation system controls expression of an auxiliary phenylacetyl-coenzyme A ligase: implications for rapid metabolic coupling of the styrene upper- and lower-degradative pathways.2008In: Molecular microbiology, ISSN 1365-2958, Vol. 69, no 2, p. 317-30Article in journal (Refereed)
    Abstract [en]

    Pseudomonas sp. strain Y2 degrades styrene through oxidation to phenylacetic acid via the styABCD operon-encoded enzymes, whose expression is induced in response to styrene by the StyS/StyR two-component regulatory system. Further transformation of phenylacetic acid to tricarboxylic acid cycle intermediates is mediated by the enzymes of paa catabolic genes, whose expression is regulated by the PaaX repressor. The first step of this paa degradation pathway is catalysed by paaF-encoded phenylacetyl-coenzyme A ligases that produce phenylacetyl-coenzyme A. This metabolic intermediate, upon being bound by PaaX, inactivates PaaX-mediated repression of both the paa genes and the styABCD operon. Strain Y2 is unique in having three paaF genes located within two complete copies of the paa gene clusters. Expression of both paaF and paaF3 is controlled by the PaaX repressor. Here we use specific mutants in combination with in vivo and in vitro assays to demonstrate that paaF2, adjacent to the StyS/StyR regulatory genes, belongs to the StyR regulon and is not subject to repression by PaaX. We propose that this unexpected styrene-responsive regulatory strategy for the otherwise metabolically redundant PaaF2 auxiliary enzyme provides a system for rapid co-ordinate de-repression of the two sets of catabolic genes required for styrene degradation.

  • 14. Díaz-Salazar, Carlos
    et al.
    Calero, Patricia
    Espinosa-Portero, Rocío
    Jiménez-Fernández, Alicia
    Wirebrand, Lisa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Velasco-Domínguez, María G.
    López-Sánchez, Aroa
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Govantes, Fernando
    The stringent response promotes biofilm dispersal in Pseudomonas putida2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 18055Article in journal (Refereed)
    Abstract [en]

    Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response – (p)ppGpp sythetases [RelA and SpoT] and/or DksA – were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.

    Download full text (pdf)
    fulltext
  • 15. 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.

  • 16. 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.

  • 17. Jishage, Miki
    et al.
    Kvint, Kristian
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nyström, Thomas
    Regulation of sigma factor competition by the alarmone ppGpp2002In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 16, no 10, p. 1260-1270Article in journal (Refereed)
    Abstract [en]

    Many regulons controlled by alternative ç factors, including çs and ç32, are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of çs-dependent promoters because underproduction of ç70, specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-ç70) restored expression from çs-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type ç70 to compete with ç32 for core binding and the mutant ç70 proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type ç70. Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of çs and ç32 bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

  • 18.
    Johansson, Linda U M
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Solera, Dafne
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bernardo, Lisandro M D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moscoso, Joana A
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    sigma54-RNA polymerase controls sigma70-dependent transcription from a non-overlapping divergent promoter.2008In: Molecular microbiology, ISSN 1365-2958, Vol. 70, no 3, p. 709-23Article in journal (Refereed)
    Abstract [en]

    Divergent transcription of a regulatory gene and a cognate promoter under its control is a common theme in bacterial regulatory circuits. This genetic organization is found for the dmpR gene that encodes the substrate-responsive specific regulator of the sigma(54)-dependent Po promoter, which controls (methyl)phenol catabolism. Here we identify the Pr promoter of dmpR as a sigma(70)-dependent promoter that is regulated by a novel mechanism in which sigma(54)-RNA polymerase occupancy of the non-overlapping sigma(54)-Po promoter stimulates sigma(70)-Pr output. In addition, we show that DmpR stimulates its own production through Po activity both in vivo and in vitro. Hence, the demonstrated regulatory circuit reveals a novel role for sigma(54)-RNA polymerase, namely regulation of a sigma(70)-dependent promoter, and a new mechanism that places a single promoter under dual control of two alternative forms of RNA polymerase. We present a model in which guanosine tetra-phosphate plays a major role in the interplay between sigma(54)- and sigma(70)-dependent transcription to ensure metabolic integration to couple sigma(70)-Pr output to both low-energy conditions and the presence of substrate.

  • 19.
    Laurie, Andrew D.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bernardo, Lisandro M D
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sze, Chun Chau
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Szalewska-Palasz, Agnieszka
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nyström, Thomas
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 3, p. 1494-1503Article in journal (Refereed)
    Abstract [en]

    Some promoters, including the DmpR-controlled sigma(N)-dependent Po promoter, are effectively rendered silent in cells lacking the nutritional alarmone (p)ppGpp. Here we demonstrate that four mutations within the housekeeping sigma(D)-factor can restore sigma(N)-dependent Po transcription in the absence of (p)ppGpp. Using both in vitro and in vivo transcription competition assays, we show that all the four sigma(D) mutant proteins are defective in their ability to compete with sigma(N) for available core RNA polymerase and that the magnitude of the defect reflects the hierarchy of restoration of transcription from Po in (p)ppGpp-deficient cells. Consistently, underproduction of sigma(D) or overproduction of the anti-sigma(D) protein Rsd were also found to allow (p)ppGpp-independent transcription from the sigma(N)-Po promoter. Together with data from the direct effects of (p)ppGpp on sigma(N)-dependent Po transcription and sigma-factor competition, the results support a model in which (p)ppGpp serves as a master global regulator of transcription by differentially modulating alternative sigma-factor competition to adapt to changing cellular nutritional demands.

  • 20.
    Madhushani, Anjana
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    del Peso-Santos, Teresa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Moreno, Renata
    Rojo, Fernando
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Transcriptional and translational control through the 5 '-leader region of the dmpR master regulatory gene of phenol metabolism2015In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 1, p. 119-133Article in journal (Refereed)
    Abstract [en]

    Expression of pathways for dissimilation of toxic aromatic compounds such as (methyl)phenols interfaces both stress-response and carbon catabolite repression control cascades. In Pseudomonas putida, carbon catabolite repression is mediated by the protein Crc - a translational repressor that counteracts utilization of less-preferred carbon sources as growth substrates until they are needed. In this work we dissect the regulatory role of the 5-leader region (5-LR) of the dmpR gene that encodes the master regulator of (methyl)phenol catabolism. Using deletion and substitution mutants combined with artificial manipulations of Crc availability in P.putida, we present evidence that a DNA motif within the 5-leader region is critical for inhibition of the output from the Pr promoter that drives transcription of dmpR, while the RNA chaperone Hfq facilitates Crc-mediated translation repression through the 5-leader region of the dmpR mRNA. The results are discussed in the light of a model in which Hfq assists Crc to target a sequence within a loop formed by secondary structure of the 5-LR mRNA. Our results support the idea that Crc functions as a global translational inhibitor to co-ordinate hierarchical carbon utilization in Pseudomonads.

  • 21.
    Madhushani, Anjana
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wirebrand, Lisa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Vicky
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600Manuscript (preprint) (Other academic)
  • 22. 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.

  • 23. Moreno, Renata
    et al.
    Hernandez-Arranz, Sofia
    La Rosa, Ruggero
    Yuste, Luis
    Madhushani, Anjana
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rojo, Fernando
    The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs2015In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 1, p. 105-118Article in journal (Refereed)
    Abstract [en]

    The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putidaHfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation.

  • 24.
    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.

  • 25.
    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.

  • 26.
    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.

  • 27.
    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.

  • 28.
    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.

  • 29.
    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.

  • 30.
    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.

  • 31.
    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.

  • 32.
    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.

  • 33.
    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.

  • 34. 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.

  • 35. Porrúa, Odil
    et al.
    García-González, Vicente
    Santero, Eduardo
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Govantes, Fernando
    Activation and repression of a σN-dependent promoter naturally lacking upstream activation sequences2009In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 73, no 3, p. 419-433Article in journal (Refereed)
    Abstract [en]

    The Pseudomonas sp. strain ADP protein AtzR is a LysR-type transcriptional regulator required for activation of the atzDEF operon in response to nitrogen limitation and cyanuric acid. Transcription of atzR is directed by the σ(N)-dependent promoter PatzR, activated by NtrC and repressed by AtzR. Here we use in vivo and in vitro approaches to address the mechanisms of PatzR activation and repression. Activation by NtrC did not require any promoter sequences other than the sigma(N) recognition motif both in vivo and in vitro, suggesting that NtrC activates PatzR in an upstream activation sequences-independent fashion. Regarding AtzR-dependent autorepression, our in vitro transcription experiments show that the concentration of AtzR required for repression of the PatzR promoter in vitro correlates with AtzR affinity for its binding site. In addition, AtzR prevents transcription from PatzR when added to a preformed E-sigma(N)-PatzR closed complex, but isomerization to an open complex prevents repression. Gel mobility shift and DNase I footprint assays indicate that DNA-bound AtzR and E-σ(N) are mutually exclusive. Taken together, these results strongly support the notion that AtzR represses transcription from PatzR by competing with E-σ(N) for their overlapping binding sites. There are no previous reports of a similar mechanism for repression of σ(N)-dependent transcription.

  • 36. Porrúa, Odil
    et al.
    López-Sánchez, Aroa
    Platero, Ana I
    Santero, Eduardo
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Govantes, Fernando
    An A-tract at the AtzR binding site assists DNA binding, inducer-dependent repositioning and transcriptional activation of the PatzDEF promoter2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 90, no 1, p. 72-87Article in journal (Refereed)
    Abstract [en]

    The LysR-type regulator AtzR activates the Pseudomonas sp. ADP atzDEF operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the AtzR tetramer on the PatzDEF promoter and relaxation of an AtzR-induced DNA bend. Here we examine the in vivo and in vitro contribution of an A5 -tract present at the PatzDEF promoter region to AtzR binding and transcriptional activation. Substitution of the A-tract for the sequence ACTCA prevented PatzDEF activation and high-affinity AtzR binding, impaired AtzR contacts with the activator binding site and shifted the position of the AtzR-induced DNA bend. Analysis of a collection of mutants bearing different alterations in the A-tract sequence showed that the extent of AtzR-dependent activation does not correlate with the magnitude or orientation of the spontaneous DNA bend generated at this site. Our results support the notion that indirect readout of the A-tract-associated narrow minor groove is essential for the AtzR-DNA complex to achieve a conformation competent for activation of the PatzDEF promoter. Conservation of this motif in several binding sites of LysR-type regulators suggests that this mechanism may be shared by other proteins in this family.

  • 37.
    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.

  • 38.
    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.

  • 39.
    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.

  • 40.
    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.

  • 41. 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.

  • 42. Rodriguez-Herva, JJ
    et al.
    Duque, E
    Molina-Henares, MA
    Navarro-Aviles, G
    van Dillewijn, P
    de la Torre, J
    Molina-Henares, AJ
    Sanchez-de la Campa, A
    Ran, FA
    Segura, A
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ramos, JL
    Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT24402010In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 2, no 3, p. 373-380Article in journal (Refereed)
    Abstract [en]

    Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes sigma 28, is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putida sigma 28 recognizes a TCAAG-t-N-12-GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region.

  • 43. 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.

  • 44.
    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.

  • 45.
    Sarand, Inga
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Österberg, Sofia
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmqvist, Sofie
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmfeldt, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Parales, Rebecca E
    Section of Microbiology, 226 Briggs Hall, 1 Shields Ave., University of California, Davis, CA 95616, USA.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Metabolism-dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer-like proteins of Pseudomonas putida2008In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 10, no 5, p. 1320-1334Article in journal (Refereed)
    Abstract [en]

    Comparatively little is known about directed motility of environmental bacteria to common aromatic pollutants. Here, by expressing different parts of a (methyl)phenol-degradative pathway and the use of specific mutants, we show that taxis of Pseudomonas putida towards (methyl)phenols is dictated by its ability to catabolize the aromatic compound. Thus, in contrast to previously described chemoreceptor-mediated chemotaxis mechanisms towards benzoate, naphthalene and toluene, taxis in response to (methyl)phenols is mediated by metabolism-dependent behaviour. Here we show that P. putida differentially expresses three Aer-like receptors that are all polar-localized through interactions with CheA, and that inactivation of the most abundant Aer2 protein significantly decreases taxis towards phenolics. In addition, the participation of a sensory signal transduction protein composed of a PAS, a GGDEF and an EAL domain in motility towards these compounds is demonstrated. The results are discussed in the context of the versatility of metabolism-dependent coupling and the necessity for P. putida to integrate diverse metabolic signals from its native heterogeneous soil and water environments.

  • 46.
    Seibt, Henrik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aung, Kyaw Min
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ishikawa, Takahiko
    Sjöström, Annika
    Atkinson, Gemma C.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wai, Sun Nyunt
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elevated levels of VCA0117 in response to external signals activates type VI secretion in Vibrio cholerae A1552Manuscript (preprint) (Other academic)
  • 47.
    Seibt, Henrik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Y233 gatekeeper of DmpR modulates effector-responsive transcriptional control of δ54-RNA polymerase2019In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 21, no 4, p. 1321-1330Article in journal (Refereed)
    Abstract [en]

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

  • 48.
    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.

  • 49.
    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.

  • 50.
    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.

12 1 - 50 of 81
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf