| 1. |
- Beljantseva, Jelena, et al.
(författare)
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Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:14, s. 3726-3731
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Jimmy, Steffi, 1988-
(författare)
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Novel RelA-SpoT Homolog toxin-antitoxin systems that inhibit bacterial growth through production of toxic alarmone ppApp
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The stress alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively known as (p)ppGpp, are the molecular mediators of the bacterial stringent response — a bacterial stress response implicated in virulence, antibiotic tolerance and biofilm formation. At high concentrations, (p)ppGpp halts bacterial growth, inhibits protein synthesis and rewires transcription and metabolism. In Escherichia coli (E. coli ) the (p)ppGpp levels are controlled by two large multi-domain proteins RelA and SpoT, the namesakes of Rel-SpoT Homolog (RSH) protein family. During amino acid starvation, RelA directly senses the acylation status of the A-site tRNA on the ribosome. In the presence of uncharged tRNA, RelA is activated to synthesize (p)ppGpp using ATP and either GDP or GTP as substrates; SpoT opposes the activity of RelA by hydrolyzing the alarmone. The RSH family also includes single domain, monofunctional enzymes: Small Alarmone Synthetases (SASs), which can synthesize (p)ppGpp, and Small Alarmone Hydrolases (SAHs), which can hydrolyze (p)ppGpp. Acting together with ‘long’ RSHs such as RelA and SpoT, these enzymes control the intracellular alarmone levels. Using conservation of genomic neighborhoods analysis of RSH sequences, we have identified several families of SAS factors encoded in conserved bicistronic architectures that are similar to the so-called toxin-antitoxin operons. We experimentally validated five of these SASs as being the toxins (toxSASs) which are neutralized by the products of the six neighboring antitoxin genes. The SAS enzyme from Cellulomonas marina (C. marina ) FaRel inhibits the growth of E. coli cells by synthesizing alarmones ppGpp and ppApp, which in turn leads to the depletion of cellular ATP and GTP. These toxic effects can be countered by the C. marina SAH antitoxin through degradation of ppGpp and ppApp alarmones.Since (p)ppGpp plays such a crucial role in bacterial virulence and antibiotic tolerance, the (p)ppGpp-mediated signaling has emerged as a target for developing new antibacterials. ppGpp-mimetics are a promising strategy for direct inhibition of RSH enzymes. We tested a targeted chemical library of ppGpp analogs in enzymatic assays with purified E. coli RelA activated by the ribosome. Although the screen has yielded several potent inhibitors, none of them were effective in live bacterial cells. Despite their limited utility as antibacterials, these compounds are useful tools for future structural and biochemical work. We took an alternative approach and developed a High Throughput Screening (HTS) assay which utilized amino acid auxotroph Bacillus subtilis lacking (p)ppGpp. We have performed an HTS screen with a diverse compound library and identified a set of compounds sharing a common 4-(6-(phenoxyl) alkyl)-3,5-dimethyl-1H-pyrazole core as possible stringent response inhibitors. Our follow-up characterization of these compounds as well as reported potential inhibitors — the ppGpp analog Relacin and cationic peptide 1018 — revealed that neither compound is sufficiently specific to warrant further development.
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| 3. |
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| 4. |
- Park, Kwang-Hyun, et al.
(författare)
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Tetrameric architecture of an active phenol-bound form of the AAA(+) transcriptional regulator DmpR
- 2020
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.
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| 5. |
- Shingler, Vicky
(författare)
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Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals
- 2016
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Ingår i: Aerobic utililization of hydrocarbons, oils and Lipids. Handbook of hydrocarbon and lipid microbiology. - Cham : Springer. - 9783319397825 ; , s. 1-13
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Bokkapitel (refereegranskat)abstract
- 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.
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| 6. |
- Shingler, Vicky
(författare)
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Experimental evolution of novel regulatory activities in response to hydrocarbons and related chemicals
- 2019
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Ingår i: Aerobic utililization of hydrocarbons, oils and Lipids. Handbook of hydrocarbon and lipid microbiology. - Cham : Springer. - 9783319504179 - 9783319504186 ; , s. 737-749
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Bokkapitel (refereegranskat)abstract
- 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.
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| 7. |
- Österberg, Sofia
(författare)
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Metabolism-dependent taxis and control of motility in Pseudomonas putida
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria living in soil and aquatic habitats rapidly adapt to changes in physico-chemical parameters that influence their energy status and thus their ability to proliferate and survive. One immediate survival strategy is to relocate to more metabolically optimal environments. To aid their movement through gradients (a process called taxis), many bacteria use whip like flagella organelles. Soil-dwelling Pseudomonas putida possesses a polar bundle of flagella that propel the bacterium forward in directed swimming motility. P. putida strains are generally fast growing, have a broad metabolic capacity, and are resistant to many harmful substances – qualities that make them interesting for an array of industrial and biotechnological application. This thesis identifies some of the factors that are involved in controlling the flagella driven motility of P. putida.In the first part of the thesis, I present evidence that P. putida displays energy-taxis towards metabolisable substrates and that the surface located Aer2 receptor (named after its similarities to the Escherichia coli Aer receptor) is responsible for detecting the changes in energy-status and oxygen-gradients that underlie this response. Aer2 is expressed simultaneously with the flagella needed for taxis responses and its expression is ensured during nutrient scares conditions through the global transcriptional regulators ppGpp and DksA.In addition to Aer2, P. putida possesses two more Aer-like receptors (Aer1 and Aer3) that are differentially expressed. Like Aer2, Aer1 and Aer3 co-localize to one cell pole. Although the signals to which Aer1 and Aer3 respond are unknown, analysis of Aer1 uncovered a role in motility control for a protein encoded within the same operon. This protein, called PP2258, instigated the work described in the second part of my thesis on the involvement of the second messenger c-di-GMP in regulation of P. putida motility. Genetic dissection of the catalytic activities of PP2258 revealed that it has the unusual capacity to both synthesize and degrade c-di-GMP. Coupling of the c-di-GMP signal originating from PP2258 to motility control was traced to the c-di-GMP binding properties of the protein PP4397. In the last part of the thesis, I present possible mechanisms for how these different components might interact to create a signal transduction cascade – from the surface located Aer1 receptor to PP2258 and the c-di-GMP responsive PP4397, and from there to the flagella motors – to ultimately determine flagella performance and the motility status of P. putida.
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