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  • 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. 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.

  • 3. 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.

  • 4. 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.

  • 5.
    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.

  • 6. 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)
  • 7. 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.

  • 8.
    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.

  • 9.
    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.

  • 10.
    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.

  • 11.
    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.

  • 12. 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.

  • 13. 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.

  • 14.
    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.

  • 15.
    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.

  • 16.
    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.

  • 17.
    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)
  • 18. 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.

  • 19. 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.

  • 20. 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.

  • 21. 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.

  • 22.
    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.

  • 23.
    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)
  • 24.
    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.

  • 25.
    Shingler, Vicky
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals2016In: Aerobic utililization of hydrocarbons, oils and Lipids. Handbook of hydrocarbon and lipid microbiology / [ed] Fernando Rojo, Cham: Springer, 2016, p. 1-13Chapter in book (Refereed)
    Abstract [en]

    Bacterial transcriptional regulatory proteins that control catabolism of hydrocarbons and related chemicals have evolved (or are actively evolving) toward specifically detecting compounds that signal the presence of growth substrates. Laboratory evolution of the chemical-binding and response properties of sensory regulators has been achieved by a number of different techniques to generate novel derivatives with desired properties. Such manipulated and selected regulatory proteins are increasingly used in artificial genetic circuitry for improved biodegradation systems, biosensor construction, and in assembling regulatory cascades for synthetic biology within a wide range of biotechnological applications.

  • 26.
    Shingler, Vicky
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals2019In: Aerobic utililization of hydrocarbons, oils and Lipids. Handbook of hydrocarbon and lipid microbiology / [ed] Fernando Rojo, Cham: Springer, 2019, p. 737-749Chapter in book (Refereed)
    Abstract [en]

    Bacterial transcriptional regulatory proteins that control catabolism of hydrocarbons and related chemicals have evolved (or are actively evolving) toward specifically detecting compounds that signal the presence of growth substrates. Laboratory evolution of the chemical-binding and response properties of sensory regulators has been achieved by a number of different techniques to generate novel derivatives with desired properties. Such manipulated and selected regulatory proteins are increasingly used in artificial genetic circuitry for improved biodegradation systems, biosensor construction, and in assembling regulatory cascades for synthetic biology within a wide range of biotechnological applications.

  • 27.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals2010In: Handbook of hydrocarbon and lipid microbiology / [ed] Timmis, Kënneth. N; McGenity, Terry; Meer, Jan Roelof van der; Lorenzo, Victor de, Springer , 2010, 1, p. 1235-1245Chapter in book (Refereed)
    Abstract [en]

    Bacterial regulatory proteins that control catabolism of hydrocarbons and related chemicals have evolved (or are actively evolving) towards specifically detecting compounds that signal the presence of growth substrates. Laboratory evolution of the chemical-binding and response properties of sensory-regulators has been achieved by a number of different techniques to generate novel derivatives with desired properties. Such manipulated and selected regulatory proteins are increasingly used in artificial genetic circuitry for improved biodegradation systems, biosensor construction, and in assembling regulatory cascades for synthetic biology within a wide range of biotechnological applications

  • 28.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Integrated regulation in response to aromatic compounds: from signal sensing to attractive behaviour2003In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 5, no 12, p. 1226-1241Article in journal (Refereed)
    Abstract [en]

    Deciphering the complex interconnecting bacterial responses to the presence of aromatic compounds is required to gain an integrated understanding of how aromatic catabolic processes function in relation to their genome and environmental context. In addition to the properties of the catabolic enzymes themselves, regulatory responses on at least three different levels are important. At a primary level, aromatic compounds control the activity of specific members of many families of transcriptional regulators to direct the expression of the specialized enzymes for their own catabolism. At a second level, dominant global regulation in response to environmental and physiological cues is incorporated to subvert and couple transcription levels to the energy status of the bacteria. Mediators of these global regulatory responses include the alarmone (p)ppGpp, the DNA-bending protein IHF and less well-defined systems that probably sense the energy status through the activity of the electron transport chain. At a third level, aromatic compounds can also impact on catabolic performance by provoking behavioural responses that allow the bacteria to seek out aromatic growth substrates in their environment.

  • 29.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Signal sensory systems that impact σ(54)-dependent transcription2011In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 35, no 3, p. 425-440Article in journal (Refereed)
    Abstract [en]

    Alternative σ-factors of bacteria bind core RNA polymerase to program the specific promoter selectivity of the holoenzyme. Signal-responsive changes in the availability of different σ-factors redistribute the RNA polymerase among the distinct promoter classes in the genome for appropriate adaptive, developmental and survival responses. The σ(54) -factor is structurally and functionally distinct from all other σ-factors. Consequently, binding of σ(54) to RNA polymerase confers unique features on the cognate holoenzyme, which requires activation by an unusual class of mechano-transcriptional activators, whose activities are highly regulated in response to environmental cues. This review summarizes the current understanding of the mechanisms of transcriptional activation by σ(54) -RNA polymerase and highlights the impact of global regulatory factors on transcriptional efficiency from σ(54) -dependent promoters. These global factors include the DNA-bending proteins IHF and CRP, the nucleotide alarmone ppGpp, and the RNA polymerase-targeting protein DksA.

  • 30.
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Transcriptional regulation and catabolic strategies of phenol degradative pathways.2004In: The Pseudomonas Vol II: Virulence and Gene regulation / [ed] Ramos, J-L, Kluwer , 2004, p. 451-478Chapter in book (Refereed)
  • 31. Stec-Dziedzic, Ewa
    et al.
    Lyzen, Robert
    Skärfstad, Eleonore
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Szalewska-Palasz, Agnieszka
    Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein2013In: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms, ISSN 1874-9399, E-ISSN 1876-4320, Vol. 1829, no 2, p. 219-230Article in journal (Refereed)
    Abstract [en]

    RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of sigma-factors (sigma(70), sigma(S), sigma(54), sigma(32), sigma(E)) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the sigma(E)-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the sigma(E)-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency. 

  • 32. Szalewska-Palasz, Agnieszka
    et al.
    Bernardo, Lisandro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Linda
    Skärfstad, Eleonore
    Stec, Ewa
    Shingler, Victoria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dual by-pass mechanisms of RNA polymerase mutants: implications for ppGpp- and DksA-mediated control of σ54-dependent transcriptionManuscript (preprint) (Other academic)
  • 33. Szalewska-Palasz, Agnieszka
    et al.
    Johansson, Linda U M
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Bernardo, Lisandro M D
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Stec, Ewa
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54.2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 25, p. 18046-56Article in journal (Refereed)
    Abstract [en]

    The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain sigma70-dependent promoters. A number of promoters that are dependent on alternative sigma-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived sigma54-dependent Po promoter and several other sigma54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro sigma54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the beta- and beta'-subunits of core RNA polymerase that allow high levels of transcription from the sigma54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with sigma-factors to favor sigma54-holoenzyme formation over that with sigma70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent sigma70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of sigma54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent sigma70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative sigma-factors is controlled to meet cellular requirements.

  • 34. Sze, Chun Chau
    et al.
    Bernardo, Lisandro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Integration of global regulation of two aromatic-responsive sigma(54)-dependent systems: a common phenotype by different mechanisms2002In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 3, p. 760-770Article in journal (Refereed)
    Abstract [en]

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

  • 35.
    Wirebrand, Lisa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Madhushani, Anjana W. K.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Irie, Yasuhiko
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    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 CF6002018In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 1, p. 186-199Article in journal (Refereed)
    Abstract [en]

    The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.

  • 36.
    Wirebrand, Lisa
    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).
    López-Sánchez, Aroa
    Govantes, Fernando
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    PP4397/FlgZ provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 12205Article in journal (Refereed)
    Abstract [en]

    Bacteria swim and swarm using rotating fagella that are driven by a membrane-spanning motor complex. Performance of the fagella motility apparatus is modulated by the chemosensory signal transduction system to allow navigation through physico-chemical gradients – a process that can be fne-tuned by the bacterial second messenger c-di-GMP. We have previously analysed the Pseudomonas putida signalling protein PP2258 that has the capacity to both synthesize and degrade c-di-GMP. A PP2258 null mutant displays reduced motility, implicating the c-di-GMP signal originating from this protein in control of P. putida motility. In Escherichia coli and Salmonella, the PilZ-domain protein YcgR mediates c-di-GMP responsive control of motility through interaction with the fagellar motors. Here we provide genetic evidence that the P. putida protein PP4397 (also known as FlgZ), despite low sequence homology and a diferent genomic context to YcgR, functions as a c-di-GMP responsive link between the signal arising from PP2258 and alterations in swimming and swarming motility in P. putida.

  • 37.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Balsalobre, Carlos
    (p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB.2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, no 6, p. 1520-1533Article in journal (Refereed)
    Abstract [en]

    In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp-deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp(0) suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp(0) strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp-stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.

  • 38.
    Åberg, Anna
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Balsalobre, Carlos
    Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 67, no 6, p. 1223-1241Article in journal (Refereed)
  • 39.
    Österberg, Sofia
    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 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).
    Regulation of alternative sigma factor use2011In: Annual Review of Microbiology, ISSN 0066-4227, E-ISSN 1545-3251, Vol. 65, p. 37-55Article in journal (Refereed)
    Abstract [en]

    Alternative bacterial sigma factors bind the catalytic core RNA polymerase to confer promoter selectivity on the holoenzyme. The different holoenzymes are thus programmed to recognize the distinct promoter classes in the genome to allow coordinated activation of discrete sets of genes needed for adaptive responses. To form the holoenzymes, the different sigma factors must be available to compete for their common substrate (core RNA polymerase). This review highlights (a) the roles of antisigma factors in controlling the availability of alternative sigma factors and (b) the involvement of diverse regulatory molecules that promote the use of alternative sigma factors through subversion of the domineering housekeeping σ(70). The latter include the nucleotide alarmone ppGpp and small proteins (DksA, Rsd, and Crl), which directly target the transcriptional machinery to mediate their effects.

  • 40.
    Österberg, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skoog, Lisa
    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).
    PP4397 provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putidaManuscript (preprint) (Other academic)
  • 41.
    Österberg, Sofia
    et al.
    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). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The σ-factor FliA, ppGpp and DksA coordinate transcriptional control of the aer2 gene of Pseudomonas putida2010In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 12, no 6, p. 1439-1451Article in journal (Refereed)
    Abstract [en]

    Here the σ-factor requirement for transcription of three similar, but differentially regulated, aer genes of Pseudomonas putida KT2440 is investigated. Previous work has shown that the three Aer proteins, like chemoreceptors, colocalize to a single pole in a CheA-dependent manner. Lack of Aer2 - the most abundant of these three proteins - mediates defects in metabolism-dependent taxis and aerotaxis, while lack of Aer1 or Aer3 has no apparent phenotype. We show, using wild-type and mutant P. putida derivatives combined with P. putida reconstituted FliA- (σ28) and σ70-dependent in vitro transcription assays, that transcription of aer2 is coupled to motility through the flagella σ-factor FliA, while σ70 is responsible for transcription of aer1 and aer3. By comparing activities of the wild-type and mutant forms of the aer2 promoter, we present evidence (i) that transcription from FliA-dependent Paer2 is enhanced by changes towards the Escherichia coli consensus for FliA promoters rather than towards that of P. putida, (ii) that the nature of the AT-rich upstream region is important for both output and σ70 discrimination of this promoter, and (iii) that Paer2 output is directly stimulated by the bacterial alarmone ppGpp and its cofactor DksA.

  • 42.
    Österberg, Sofia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Åberg, Anna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Herrera Seitz, M. Karina
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Magnus
    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).
    Genetic dissection of a motility-associated c-di-GMP signalling protein of Pseudomonas putida2013In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 5, no 4, p. 556-565Article in journal (Refereed)
    Abstract [en]

    Lack of the Pseudomonas putidaPP2258 protein or its overexpression results in defective motility on solid media. The PP2258 protein is tripartite, possessing a PAS domain linked to two domains associated with turnover of c-di-GMP - a cyclic nucleotide that controls the switch between motile and sessile lifestyles. The second messenger c-di-GMP is produced by diguanylate cyclases and degraded by phosphodiesterases containing GGDEF and EAL or HD-GYP domains respectively. It is common for enzymes involved in c-di-GMP signalling to contain two domains with potentially opposing c-di-GMP turnover activities; however, usually one is degenerate and has been adopted to serve regulatory functions. Only a few proteins have previously been found to have dual enzymatic activities - being capable of both synthesizing and hydrolysing c-di-GMP. Here, using truncated and mutant derivatives of PP2258, we show that despite a lack of complete consensus in either the GGDEF or EAL motifs, the two c-di-GMP turnover domains can function independently of each other, and that the diguanylate cyclase activity is regulated by an inhibitory I-site within its GGDEF domain. Thus, motility-associated PP2258 can be added to the short list of bifunctional c-di-GMP signalling proteins.

1 - 42 of 42
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