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Shingler, V. (2019). Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals. In: Fernando Rojo (Ed.), Aerobic utililization of hydrocarbons, oils and Lipids. Handbook of hydrocarbon and lipid microbiology: (pp. 737-749). Cham: Springer
Open this publication in new window or tab >>Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals
2019 (English)In: 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.

Place, publisher, year, edition, pages
Cham: Springer, 2019
Series
Handbook of hydrocarbon and lipid microbiology
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-158264 (URN)10.1007/978-3-319-50418-6_34 (DOI)978-3-319-50417-9 (ISBN)978-3-319-50418-6 (ISBN)
Funder
Swedish Research CouncilThe Kempe Foundations
Available from: 2019-04-17 Created: 2019-04-17 Last updated: 2019-04-17Bibliographically approved
Seibt, H., Sauer, U. H. & Shingler, V. (2019). The Y233 gatekeeper of DmpR modulates effector-responsive transcriptional control of δ54-RNA polymerase. Environmental Microbiology, 21(4), 1321-1330
Open this publication in new window or tab >>The Y233 gatekeeper of DmpR modulates effector-responsive transcriptional control of δ54-RNA polymerase
2019 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 21, no 4, p. 1321-1330Article in journal (Refereed) Published
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.

National Category
Genetics
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-155401 (URN)10.1111/1462-2920.14567 (DOI)000464373000011 ()30773776 (PubMedID)
Note

Originally included in thesis in manuscript form 

Available from: 2019-01-15 Created: 2019-01-15 Last updated: 2019-06-13Bibliographically approved
Wirebrand, L., Madhushani, A. W. K., Irie, Y. & Shingler, V. (2018). Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600. Environmental Microbiology, 20(1), 186-199
Open this publication in new window or tab >>Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600
2018 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 1, p. 186-199Article in journal (Refereed) Published
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.

Keywords
catabolite repression, translational regulation, Crc, CrcZ, CrcY, Hfq, Phenol catabolism, IncP-2 plasmids
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-141902 (URN)10.1111/1462-2920.13966 (DOI)000419784100015 ()29076626 (PubMedID)
Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2018-06-09Bibliographically approved
Wirebrand, L., Österberg, S., López-Sánchez, A., Govantes, F. & Shingler, V. (2018). PP4397/FlgZ provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida. Scientific Reports, 8, Article ID 12205.
Open this publication in new window or tab >>PP4397/FlgZ provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 12205Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-141908 (URN)10.1038/s41598-018-29785-w (DOI)000441625500028 ()
Funder
Swedish Research Council, 2011-4791/2016-02047The Kempe Foundations
Note

Originally included in thesis in manuscript form

Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2018-09-05Bibliographically approved
Beljantseva, J., Kudrin, P., Jimmy, S., Ehn, M., Pohl, R., Varik, V., . . . Hauryliuk, V. (2017). Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor. Scientific Reports, 7, Article ID 41839.
Open this publication in new window or tab >>Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 41839Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-132129 (URN)10.1038/srep41839 (DOI)000393552300001 ()28157202 (PubMedID)
Available from: 2017-03-09 Created: 2017-03-09 Last updated: 2018-06-09Bibliographically approved
Beljantseva, J., Kudrin, P., Andresen, L., Shingler, V., Atkinson, G. C., Tenson, T. & Hauryliuk, V. (2017). Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA. Proceedings of the National Academy of Sciences of the United States of America, 114(14), 3726-3731
Open this publication in new window or tab >>Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA
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2017 (English)In: 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) Published
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.

Keywords
stringent response, (p)ppGpp, RNA-protein interaction, allosteric regulation, nucleotide signaling
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-133722 (URN)10.1073/pnas.1617868114 (DOI)000398159000059 ()
Available from: 2017-05-05 Created: 2017-05-05 Last updated: 2019-03-01Bibliographically approved
Díaz-Salazar, C., Calero, P., Espinosa-Portero, R., Jiménez-Fernández, A., Wirebrand, L., Velasco-Domínguez, M. G., . . . Govantes, F. (2017). The stringent response promotes biofilm dispersal in Pseudomonas putida. Scientific Reports, 7, Article ID 18055.
Open this publication in new window or tab >>The stringent response promotes biofilm dispersal in Pseudomonas putida
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 18055Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-141910 (URN)10.1038/s41598-017-18518-0 (DOI)000418644100014 ()29273811 (PubMedID)
Note

Originally published in thesis in manuscript form.

Available from: 2017-11-15 Created: 2017-11-15 Last updated: 2018-06-09Bibliographically approved
Shingler, V. (2016). Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals. In: Fernando Rojo (Ed.), Aerobic utililization of hydrocarbons, oils and Lipids. Handbook of hydrocarbon and lipid microbiology: (pp. 1-13). Cham: Springer
Open this publication in new window or tab >>Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals
2016 (English)In: 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.

Place, publisher, year, edition, pages
Cham: Springer, 2016
Series
Handbook of hydrocarbon and lipid microbiology
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-156938 (URN)10.1007/978-3-319-39782-5_34-1 (DOI)978-3-319-39782-5 (ISBN)
Funder
Swedish Research Council
Available from: 2019-03-03 Created: 2019-03-03 Last updated: 2019-04-17Bibliographically approved
del Peso-Santos, T. & Shingler, V. (2016). Inter-sigmulon communication through topological promoter coupling. Nucleic Acids Research, 44(20), 9638-9649
Open this publication in new window or tab >>Inter-sigmulon communication through topological promoter coupling
2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 20, p. 9638-9649Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-128510 (URN)10.1093/nar/gkw639 (DOI)000393817800017 ()27422872 (PubMedID)
Available from: 2016-12-06 Created: 2016-12-06 Last updated: 2018-06-09Bibliographically approved
Moreno, R., Hernandez-Arranz, S., La Rosa, R., Yuste, L., Madhushani, A., Shingler, V. & Rojo, F. (2015). The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs. Environmental Microbiology, 17(1), 105-118
Open this publication in new window or tab >>The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs
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2015 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 1, p. 105-118Article in journal (Refereed) Published
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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-100780 (URN)10.1111/1462-2920.12499 (DOI)000349152800010 ()24803210 (PubMedID)
Available from: 2015-04-01 Created: 2015-03-09 Last updated: 2018-06-07Bibliographically approved
Projects
Mechanisms of bacterial adaptation to nutritional stress [2008-03557_VR]; Umeå UniversityHierarchical regulatory circuits and signal integration in bacteria [2011-04791_VR]; Umeå UniversityA mechano-transcriptional activator of Vibrio cholerae type VI secretion as a novel anti-virulence drug target [2016-02047_VR]; Umeå University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-7349-1678

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