DOCSLIB.ORG

Genes in Deinococcus Radiodurans S Jinhui Wang, Ye Tian, Zhengfu Zhou, Liwen Zhang, Wei Zhang, Min Lin, and Ming Chen*

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Genes in Deinococcus Radiodurans S Jinhui Wang, Ye Tian, Zhengfu Zhou, Liwen Zhang, Wei Zhang, Min Lin, and Ming Chen* J. Microbiol. Biotechnol. (2016), 26(12), 2106–2115 http://dx.doi.org/10.4014/jmb.1601.01017 Research Article Review jmb Identification and Functional Analysis of RelA/SpoT Homolog (RSH) Genes in Deinococcus radiodurans S Jinhui Wang, Ye Tian, Zhengfu Zhou, Liwen Zhang, Wei Zhang, Min Lin, and Ming Chen* Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China Received: January 11, 2016 Revised: July 20, 2016 To identify the global effects of (p)ppGpp in the gram-positive bacterium Deinococcus Accepted: August 10, 2016 radiodurans, which exhibits remarkable resistance to radiation and other stresses, RelA/SpoT homolog (RSHs) mutants were constructed by direct deletion mutagenesis. The results showed that RelA has both synthesis and hydrolysis domains of (p)ppGpp, whereas RelQ only First published online synthesizes (p)ppGpp in D. radiodurans. The growth assay for mutants and complementation August 24, 2016 analysis revealed that deletion of relA and relQ sensitized the cells to H2O2, heat shock, and *Corresponding author amino acid limitation. Comparative proteomic analysis revealed that the bifunctional RelA is Phone: +86-1082106106; involved in DNA repair, molecular chaperone functions, transcription, the tricarboxylic acid Fax: +86-1082106142; cycle, and metabolism, suggesting that relA maintains the cellular (p)ppGpp levels and plays a E-mail: [email protected] crucial role in oxidative resistance in D. radiodurans. The D. radiodurans relA and relQ genes are responsible for (p)ppGpp synthesis/hydrolysis and (p)ppGpp hydrolysis, respectively. (p)ppGpp integrates a general stress response with a targeted re-programming of gene S upplementary data for this regulation to allow bacteria to respond appropriately towards heat shock, oxidative stress, and paper are available on-line only at starvation. This is the first identification of RelA and RelQ involvement in response to http://jmb.or.kr. oxidative, heat shock, and starvation stresses in D. radiodurans, which further elucidates the pISSN 1017-7825, eISSN 1738-8872 remarkable resistance of this bacterium to stresses. Copyright© 2016 by The Korean Society for Microbiology Keywords: RelA, RelQ, (p)ppGpp, oxidative stress, Deinococcus radiodurans, RelA/SpoT homolog and Biotechnology Introduction synthetases (SAS), and small alarmone hydrolases (SAH). The long RSHs have (p)ppGpp synthetase, hydrolase, TGS, To adapt to environmental changes, bacteria utilize and ACT domains, whereas SASs and SAHs only contain guanosine tetraphosphate and guanosine pentaphosphate synthetase or hydrolase domain, respectively [3]. Gram- (collectively named (p)ppGpp) as signal molecules to negative bacteria such as Escherichia coli have two long regulate many physiological processes that are sensitive to RSHs, RelA and SpoT. RelA is considered to be a (p)ppGpp changing nutrient availability, such as growth [13, 47], synthetase. In response to amino acid starvation, RelA secondary metabolism [40], survival [18], persistence[29], senses uncharged tRNA stalled on the ribosome and then biofilm formation [11, 46], development [1], competence, synthesizes (p)ppGpp [50]. SpoT is a bifunctional enzyme and virulence [1, 15, 20, 38]. that is involved in both degradation and synthesis of Upon encountering a stress, (p)ppGpp is produced from (p)ppGpp and regulates (p)ppGpp levels in response to ATP and GTP (or GDP) and rapidly accumulates [45]. limitation of carbon sources, fatty acids, or iron [6, 43, 49, RelA/SpoT homolog (RSH) proteins, named for their 51]. Unlike E. coli, Vibrio cholerae has a novel RelA-SpoT- sequence similarity to the RelA and SpoT enzymes of independent (p)ppGpp synthetase gene [16]. Gram-positive E. coli, comprise a superfamily of enzymes that synthesize bacteria such as Bacillus subtilis have RelA, RelQ, and RelP. and/or hydrolyze the alarmone (p)ppGpp. The RSH The bifunctional enzyme RelA exists widely in gram- proteins can be divided into three major groups: long positive bacteria, whereas RelQ and RelP belong to SASs, RelA/SpoT homolog proteins (longRSH), small alarmone which only contain the synthesis domain [24, 26, 35, 36]. J. Microbiol. Biotechnol. RSH Genes in Deinococcus radiodurans 2107 (p)ppGpp-deficient mutants exhibit defects in growth D. radiodurans relA and relQ genes are responsible for regulation, decreases in viability under nutrient-limited (p)ppGpp synthesis/hydrolysis and (p)ppGpp hydrolysis, conditions, increases in susceptibility to oxidative stress, respectively. We show that relQ and relA responded to and defects in biofilm formation [46]. In Enterococcus several stresses, including oxidative stress, heat shock, and faecalis V583, the ΔrelA strain was highly sensitive to 45 mM amino acid limitation. Furthermore, comparative proteomic H2O2 and displayed reduced growth in a medium analysis showed that relA affected the expression of containing 1 M NaCl [52]. Mutants defective only in the multiple proteins involved in oxidation resistance. RSH synthase domain of Staphylococcus aureus did not show impaired bacterial growth under nutrient-rich Materials and Methods conditions; however, they were more sensitive to mupirocin, had lower viability when essential amino acids Bacterial Strains, Plasmids, and Growth Conditions were depleted from the medium, and were less virulent The bacterial strains and plasmids used in this study are listed than the wild-type [19]. in Table 1. The D. radiodurans strains were grown at 30°C in TGY Deinococcus radiodurans, a gram-positive bacterium, is broth (1% tryptone, 0.5% yeast extract, and 0.1% glucose) or on well known in its capacity to resist extreme ionizing TGY plates supplemented with agar (1.5%). Concentrations of 350 μg/ml spectinomycin or 8 μg/ml kanamycin were used for radiation, UV radiation, oxidative stress, and a variety of antibiotic selection of D. radiodurans mutant strains. The minimal DNA-damaging agents [10], allowing it to survive in medium was prepared as described by Venkateswaran et al. [48]: various extreme environments. In D. radiodurans, two fructose as the sole carbon source and L-histidine and L-cysteine as genes, DR_1838 (relA) and DR_1631 (relQ), are predicted to the amino acid supplements. The E. coli strains were grown at be associated with (p)ppGpp synthesis and hydrolysis. 37°C in LB broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) However, the role of these genes in response to stresses or on LB plates supplemented with agar (1.5%) or antibiotic as remains unclear. In this study, we demonstrated that the required. All chemicals were of molecular biology grade and Table 1. Strains and plasmids used in this study. Strain/plasmid Genotype/construction Source/reference D. radiodurans R1 Wid-type (ATCC1399) Lab stock ΔrelA As R1 but relA::spe This study ΔrelQ As R1 but relQ::kan This study ΔrelArelQ As R1 but relA::spe DR_1631::kan This study DR Z3-relA As R1 but Z3-rleA This study DR Z3-rel NTD As R1 but Z3-rel NTD This study ΔrelA Z3-relA As ΔrelA but Z3-relA This study ΔrelA Z3-rel NTD As ΔrelA but Z3-relA NTD This study E. coli Trans10 Host for plasmid subclone TransGen CF1648 Wild-type E. coli Xiao et al. [51] CF1652 As CF1648 but relA::kana relA::kan Xiao et al. [51] CF1693 As CF1648 but relA::kana spoT::cm Xiao et al. [51] CF1648 Z3 As CF1648 with pRADZ3 This study CF1648 Z3-relA As CF1648 with Z3-relA This study CF1693 Z3 As CF1693 with pRADZ3 This study CR1693 Z3-relA As CF1693 with Z3-relA This study Plasmid pRADZ3 Shuttle plasmid between E. coli and D. radiodurans R1 Ap-R in E. coli Cm-R in D. radiodurans R1 Lab stock pBlueScript SK Cloning vector (Ap-R) Lab stock pRAZA pRADZ3 derivative carrying relA promoter This study pRAZQ pRADZ3 derivative carrying relQ promoter This study December 2016 ⎪ Vol. 26⎪ No. 12 2108 Wang et al. purchased from New England BioLabs, Takara Bio Inc., Promega, requirements of the ΔrelArelQ strain, the cells were grown in Sigma Chemicals Co., and General Electric Company. medium supplemented with only 17 amino acids (except His and Cys), and the growth curve was monitored. Construction of D. radiodurans relA and relQ Mutant Strains The relA and relQ mutants were constructed by direct deletion Conserved Domain Database Search of RelA and RelQ Proteins mutagenesis [53]. The primers that were used in this study are The protein sequences were submitted for a NCBI Conserved listed in Table S1. The homologous fragments 674 bp downstream Domain Database search (http://www.ncbi.nlm.nih.gov/Structure/ and 584 bp upstream of DR_1838 were amplified with the primers cdd/wrpsb.cgi) to identify various domains present in the relA-1F and relA-1R and with relA-2F and relA-2R, respectively, proteins. The default settings were used. The databases used were and the spectinomycin cassette was amplified with aadAF and CDSEARCH/cdd ver. 3.13. aadAR. The homologous fragments and spectinomycin cassette were cloned into the pBlueScript SK(+) vector by restriction H2O2 and Heat Shock enzyme digestion. The region containing homologous fragments TGY liquid cultures were inoculated at an OD600 value of 0.1 and the spectinomycin cassette was digested with KpnI and SacI, using overnight cultures. When the OD600 value reached 0.8, an and then transformed into the wild-type D. radiodurans. The relQ aliquot (1 ml) of the cultures was supplemented with a final mutants were generated by replacing the target gene with a concentration of 0, 20, 40, or 60 mM H2O2 for 0.5 h. The heat shock kanamycin cassette via a fusion PCR. The regions that were assay was performed by cultivating bacteria at OD600 =1.0, and located 339 bp upstream and 389 bp downstream of DR_1631 then shifting them to 48°C for 2, 3, or 4 h. were amplified with the primers relQ-1F and relQ-1R and with relQ-2F and relQ-2R, respectively, from the D. radiodurans RNA Preparation and qRT-PCR genome.
Load more

Recommended publications
  • Adaptive Responses by Transcriptional Regulators to Small Molecules in Prokaryotes
    Adaptive Responses by Transcriptional Regulators to small molecules in Prokaryotes Structural studies of two bacterial one-component signal transduction systems DntR and HpNikR Cyril Dian Stockholm University Doctoral thesis © Cyril Dian, Stockholm 2007 ISBN 978-91-7155-500-7 Department of Biochemistry and Biophysics The Arrhenius Laboratories for Natural Sciences Stockholm University SE-106 91 Stockholm Sweden All previously published papers are reprinted With permission from the publishers Intellecta Docusys, Stockholm 2007 Abstract Prokaryotes are continually exposed to environmental changes in their physiological conditions. In order to survive such unstable conditions, or to compete with others species for the same environmental niche, prokaryotes must monitor signals about both their extracellular environment and intracellular physiological status and provide rapid and appropriate responses to variations in their surroundings. This adaptive response to environmental signals is triggered mainly by transcriptional regulators via two components, the one- and two-component signal transduction systems. These scan intra- and extracellular small-molecule mixtures and modulate gene expression to provide the appropriate physiological response to the prevailing conditions. Most prokaryotic one component regulators are simple transcription factors comprising of a small-molecule binding domain (SMBD) and a DNA binding domain (DBD). Although the effects of transcription factors on the transcription machinery are well understood, the exact location
    [Show full text]
  • Molecular Insights Into DNA Interference by CRISPR-Associated Nuclease-Helicase Cas3
    Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3 Bei Gonga,1, Minsang Shinb,1, Jiali Suna, Che-Hun Junga,b, Edward L. Boltc, John van der Oostd, and Jeong-Sun Kima,b,2 aInterdisciplinary Graduate Program in Molecular Medicine, Chonnam National University, Gwangju 501-746, Korea; bDepartment of Chemistry, Chonnam National University, Gwangju 500-757, Korea; cSchool of Life Sciences, University of Nottingham Medical School, Nottingham NG72UH, United Kingdom; and dLaboratory of Microbiology, Wageningen University, 6703 HB, Wageningen, The Netherlands Edited by Wei Yang, National Institutes of Health, Bethesda, MD, and approved September 26, 2014 (received for review June 10, 2014) Mobile genetic elements in bacteria are neutralized by a system 21), Cascade complex by itself is not a nuclease for degradation based on clustered regularly interspaced short palindromic repeats of invader DNA (13). In Escherichia coli K-12 (type IE), Cascade (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas recognizes and binds crRNA to complimentary sequence in systems use a “Cascade” ribonucleoprotein complex to guide RNA target DNA, generating an RNA mediated displacement loop specifically to complementary sequence in invader double-stranded (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid DNA (dsDNA), a process called “interference.” After target recogni- within double-stranded (ds) target DNA (13). Formation of the tion by Cascade, formation of an R-loop triggers recruitment of R-loop structure induces conformational change in Cascade subunits, triggering recruitment of Cas3 protein that is a nucle- a Cas3 nuclease-helicase, completing the interference process by – destroying the invader dsDNA.
    [Show full text]
  • Regulation of Stringent Factor by Branched-Chain Amino Acids
    Regulation of stringent factor by branched-chain amino acids Mingxu Fanga and Carl E. Bauera,1 aMolecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405 Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved May 9, 2018 (received for review February 21, 2018) When faced with amino acid starvation, prokaryotic cells induce a Under normal growth conditions, the synthetase activity of Rel is stringent response that modulates their physiology. The stringent thought to be self-inhibited; however, during times of amino acid response is manifested by production of signaling molecules starvation, Rel interacts with stalled ribosomes, which activates guanosine 5′-diphosphate,3′-diphosphate (ppGpp) and guanosine synthetase activity to produce (p)ppGpp. The regulation of hy- 5′-triphosphate,3′-diphosphate (pppGpp) that are also called drolase activity is less understood but may involve one or more alarmones. In many species, alarmone levels are regulated by a downstream domains called the TGS and ACT domains. The TGS multidomain bifunctional alarmone synthetase/hydrolase called domain of SpoT has been shown to interact with an acyl carrier Rel. In this enzyme, there is an ACT domain at the carboxyl region protein, so it is presumed to sense the status of fatty acid metab- that has an unknown function; however, similar ACT domains are olism in E. coli (4). The function of the ACT domain is not as clear; present in other enzymes that have roles in controlling amino acid however, recent cryo-EM structures of E. coli RelA show that this metabolism. In many cases, these other ACT domains have been domain is involved in binding deacyl-tRNA as well as the ribosome shown to allosterically regulate enzyme activity through the bind- (5–7).
    [Show full text]
  • The Hns Gene of Escherichia Coli Is Transcriptionally Down-Regulated by (P)Ppgpp
    microorganisms Article The hns Gene of Escherichia coli Is Transcriptionally Down-Regulated by (p)ppGpp Anna Brandi, Mara Giangrossi, Attilio Fabbretti and Maurizio Falconi * School of Biosciences and Veterinary Medicine, University of Camerino, 62032 Camerino, Italy; [email protected] (A.B.); [email protected] (M.G.); [email protected] (A.F.) * Correspondence: [email protected]; Tel.: +39-0737-403274 Received: 25 August 2020; Accepted: 8 October 2020; Published: 10 October 2020 Abstract: Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the E. coli hns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex.
    [Show full text]
  • Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link Between GLUT1 and the Cytoskeleton Robert C
    Molecular Biology of the Cell Vol. 10, 819–832, April 1999 Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton Robert C. Bunn, Mari Anne Jensen, and Brent C. Reed* The Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, Shreveport, Louisiana 71130-3932 Submitted October 27, 1998; Accepted January 19, 1999 Monitoring Editor: Guido Guidotti Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, a-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.
    [Show full text]
  • Développement D'hybrides Aptamère-Peptide
    UNIVERSITÉ DU QUÉBEC INSTITUT NATIONAL DE LA RECHERCHE SCIENTIFIQUE INSTITUT ARMAND-FRAPPIER DÉVELOPPEMENT D’HYBRIDES APTAMÈRE-PEPTIDE ANTIMICROBIEN UN MODÈLE POUR CIBLER DES BACTÉRIES PATHOGÈNES Par Amal Thamri Mémoire présenté pour l’obtention du grade de Maître en sciences (M.Sc.) en microbiologie appliquée Jury d’évaluation Examinateur externe: Dr. Roger.C Lévesque, Université de Laval Examinateur interne: Dr. Frédéric Veyrier, INRS-IAF Directeur de recherche: Dr. Jonathan Perreault, INRS-IAF Co-directrice de recherche: Dre. Annie Castonguay, INRS-IAF Remerciements Cette maîtrise a été réalisée dans le cadre d’une coopération entre INRS et le ministère d’enseignement supérieur tunisien. Les recherches objets de ce mémoire ont été réalisées dans différents laboratoires de l’institut Armand-Frappier: Laboratoires des professeurs: Jonathan Perreault, Annie Castonguay, Eric Déziel, David Chatenet. Je tiens à remercier toutes les personnes qui ont participé de près ou de loin au bon déroulement et à l’avancement des travaux de cette maîtrise. Je remercie spécialement mon directeur de recherche Jonathan Perreault pour avoir cru en mes capacités d’intégrer sa jeune équipe et travailler sur un projet novateur. Je remercie très sincèrement ma co-directrice Annie Castonguay pour ses encouragements tout au long de ces deux années. Je les remercie également d'avoir bien assuré la direction et l'encadrement de mes travaux de recherche. Merci au professeur Eric Déziel pour sa compréhension et ses conseils précieux. J’adresse aussi mes remerciements au professeur David Chatenet pour sa disponibilité, ses directions bénéfiques et ses remarques pertinentes. J’adresse mes sentiments de respect et de gratitude à Myriam Letourneau et Marie- Christine Groleau pour leur générosité et leur aide à mettre en place et améliorer plusieurs expériences.
    [Show full text]
  • Regulation of Stringent Factor by Branched-Chain Amino Acids
    Regulation of stringent factor by branched-chain amino acids Mingxu Fanga and Carl E. Bauera,1 aMolecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405 Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved May 9, 2018 (received for review February 21, 2018) When faced with amino acid starvation, prokaryotic cells induce a Under normal growth conditions, the synthetase activity of Rel is stringent response that modulates their physiology. The stringent thought to be self-inhibited; however, during times of amino acid response is manifested by production of signaling molecules starvation, Rel interacts with stalled ribosomes, which activates guanosine 5′-diphosphate,3′-diphosphate (ppGpp) and guanosine synthetase activity to produce (p)ppGpp. The regulation of hy- 5′-triphosphate,3′-diphosphate (pppGpp) that are also called drolase activity is less understood but may involve one or more alarmones. In many species, alarmone levels are regulated by a downstream domains called the TGS and ACT domains. The TGS multidomain bifunctional alarmone synthetase/hydrolase called domain of SpoT has been shown to interact with an acyl carrier Rel. In this enzyme, there is an ACT domain at the carboxyl region protein, so it is presumed to sense the status of fatty acid metab- that has an unknown function; however, similar ACT domains are olism in E. coli (4). The function of the ACT domain is not as clear; present in other enzymes that have roles in controlling amino acid however, recent cryo-EM structures of E. coli RelA show that this metabolism. In many cases, these other ACT domains have been domain is involved in binding deacyl-tRNA as well as the ribosome shown to allosterically regulate enzyme activity through the bind- (5–7).
    [Show full text]
  • The Link Between Purine Metabolism and Production of Antibiotics in Streptomyces
    antibiotics Review The Link between Purine Metabolism and Production of Antibiotics in Streptomyces Smitha Sivapragasam and Anne Grove * Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA; [email protected] * Correspondence: [email protected] Received: 10 May 2019; Accepted: 3 June 2019; Published: 6 June 2019 Abstract: Stress and starvation causes bacterial cells to activate the stringent response. This results in down-regulation of energy-requiring processes related to growth, as well as an upregulation of genes associated with survival and stress responses. Guanosine tetra- and pentaphosphates (collectively referred to as (p)ppGpp) are critical for this process. In Gram-positive bacteria, a main function of (p)ppGpp is to limit cellular levels of GTP, one consequence of which is reduced transcription of genes that require GTP as the initiating nucleotide, such as rRNA genes. In Streptomycetes, the stringent response is also linked to complex morphological differentiation and to production of secondary metabolites, including antibiotics. These processes are also influenced by the second messenger c-di-GMP. Since GTP is a substrate for both (p)ppGpp and c-di-GMP, a finely tuned regulation of cellular GTP levels is required to ensure adequate synthesis of these guanosine derivatives. Here, we discuss mechanisms that operate to control guanosine metabolism and how they impinge on the production of antibiotics in Streptomyces species. Keywords: c-di-GMP; guanosine and (p)ppGpp; purine salvage; secondary metabolism; Streptomycetes; stringent response 1. Introduction Bacteria experience constant challenges, either in the environment or when infecting a host. They utilize various mechanisms to survive such stresses, which may include changes in temperature, pH, or oxygen content as well as limited access to carbon or nitrogen sources.
    [Show full text]
  • Quantification of Guanosine Triphosphate and Tetraphosphate In
    Quantification of guanosine triphosphate and tetraphosphate in plants and algae using stable isotope-labelled internal standards Julia Bartoli, Sylvie Citerne, Gregory Mouille, Emmanuelle Bouveret, Ben Field To cite this version: Julia Bartoli, Sylvie Citerne, Gregory Mouille, Emmanuelle Bouveret, Ben Field. Quantification of guanosine triphosphate and tetraphosphate in plants and algae using stable isotope-labelled internal standards. Talanta, Elsevier, 2020, 219, pp.121261. 10.1016/j.talanta.2020.121261. cea-02961710 HAL Id: cea-02961710 https://hal-cea.archives-ouvertes.fr/cea-02961710 Submitted on 19 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Quantification of guanosine triphosphate and tetraphosphate in plants and algae using stable isotope- labelled internal standards. Julia Bartoli1, Sylvie Citerne2, Gregory Mouille2, Emmanuelle Bouveret3 and Ben Field4* 1Aix Marseille Univ CNRS, LISM, UMR 7255, IMM FR 3479, 31 Chemin Joseph Aiguier, 13009, Marseille, France 2 Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay,
    [Show full text]
  • Guanosine Pentaphosphate Phosphohydrolase of Escherichia Coli Is a Long-Chain Exopolyphosphatase J
    Proc. Natl. Acad. Sci. USA Vol. 90, pp. 7029-7033, August 1993 Biochemistry Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase J. D. KEASLING*, LEROY BERTSCHt, AND ARTHUR KORNBERGtI *Department of Chemical Engineering, University of California, Berkeley, CA 94720-9989; and tDepartment of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307 Contributed by Arthur Kornberg, April 14, 1993 ABSTRACT An exopolyphosphatase [exopoly(P)ase; EC MATERIALS AND METHODS 3.6.1.11] activity has recently been purified to homogeneity from a mutant strain of Escherichia coi which lacks the Reagents and Proteins. Sources were as follows: ATP, principal exopoly(P)ase. The second exopoly(P)ase has now ADP, nonradiolabeled nucleotides, poly(P)s, bovine serum been identified as guanosine pentaphosphate phosphohydro- albumin, and ovalbumin from Sigma; [y-32P]ATP at 6000 lase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the Ci/mmol (1 Ci = 37 GBq) and [y-32P]GTP at 6000 Ci/mmol sequences of five btptic digestion fragments of the purified from ICN; Q-Sepharose fast flow, catalase, aldolase, Super- protein are found in the translated gppA gene, (u) the size ofthe ose-12 fast protein liquid chromatography (FPLC) column, protein (100 kDa) agrees with published values for GPP, and and Chromatofocusing column and reagents from Pharmacia (iu) the ratio of exopoly(P)ase activity to GPP activity remains LKB; DEAE-Fractogel, Pll phosphocellulose, and DE52 constant throughout a 300-fold purification in the last steps of DEAE-cellulose from Whatman; protein standards for SDS/ the procedure.
    [Show full text]
  • Diana Manuela Pinto Barros Classification and Structure-Based Inference of Transcriptional Regulatory Proteins
    Universidade do Minho Departamento de Informatica´ Diana Manuela Pinto Barros Classification and Structure-Based Inference of Transcriptional Regulatory Proteins June, 2016 Universidade do Minho Departamento de Informatica´ Diana Manuela Pinto Barros Classification and Structure-Based Inference of Transcriptional Regulatory Proteins Tese de Mestrado Mestrado em Bioinformatica´ Trabalho efetuado sobre a orientac¸ao˜ de Sonia´ Carneiro Analia´ Lourenc¸o June, 2016 Acknowledgements I’d like to thank my advisors, Sonia´ Carneiro, for all of the essential input, guidance and time she has conceded this work, otherwise impossible to develop and Analia´ Lourenc¸o, for her valuable time and advices throughout this journey. I want to give the biggest thanks to my family. To my parents, Fernando and Inesˆ Barros for always believing in me and for giving me opportunities that weren’t given to them. Thank you for not giving up on me and for making me the person I am today. To my brothers, Sergio´ e Pedro. You are examples of perseverance and success that I can only hope to achieve one day. To my grandparents, Maria da Conceic¸ao˜ Silva e Antonio´ Ferreira Pinto for being the type of people I want to become some day and for being an example that I could always look up to. To my aunts: Em´ılia Pinto and Luz Pinto, my extra mothers, for being light in times of need and providing me the hope and strength when I had none left. I wouldn’t have made it this far without you. I want to thank all my friends that have accompanied me throughout this adventure.
    [Show full text]
  • The C-Di-AMP Receptor Darb Controls (P)Ppgpp Synthesis in Bacillus Subtilis
    ARTICLE https://doi.org/10.1038/s41467-021-21306-0 OPEN A meet-up of two second messengers: the c-di-AMP receptor DarB controls (p)ppGpp synthesis in Bacillus subtilis Larissa Krüger1, Christina Herzberg1, Dennis Wicke1, Heike Bähre2, Jana L. Heidemann3, Achim Dickmanns3, ✉ Kerstin Schmitt4, Ralf Ficner 3 & Jörg Stülke 1 1234567890():,; Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium con- centrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB–Rel interaction results in the Rel-dependent accumu- lation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.
    [Show full text]