DOCSLIB.ORG

Diphosphate (Ppgpp) in Plants

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Diphosphate (Ppgpp) in Plants Identification of the bacterial alarmone guanosine 5؅-diphosphate 3؅-diphosphate (ppGpp) in plants Kosaku Takahashi, Koji Kasai, and Kozo Ochi* National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan Edited by Frederick M. Ausubel, Harvard Medical School, Boston, MA, and approved January 30, 2004 (received for review December 22, 2003) Stringent control mediated by the bacterial alarmone guanosine Tris-acetate-phosphate medium at 25°C under continuous white .(5؅-diphosphate 3؅-diphosphate (ppGpp) is a key regulatory process fluorescent light with rotary shaking (8 governing bacterial gene expression. By devising a system to measure ppGpp in plants, we have been able to identify ppGpp in Abiotic Treatments. Plants were wounded by cutting the shoots a the chloroplasts of plant cells. Levels of ppGpp increased markedly small interval with a knife. Treatment with salt, acid, alkali, or when plants were subjected to such biotic and abiotic stresses as heavy metal entailed transplantation to an aqueous solution of wounding, heat shock, high salinity, acidity, heavy metal, drought, NaCl (250 mM), HCl (pH 3.0), NaOH (pH 10.0), or CuSO4 (1 and UV irradiation. Abrupt changes from light to dark also caused mM), respectively. For heat and cold treatment, temperature a substantial elevation in ppGpp levels. In vitro, chloroplast RNA was shifted from 25 to 40 or 10°C. Plants were dehydrated by polymerase activity was inhibited in the presence of ppGpp, removing them from the soil and allowing them to dry at 25°C Ͻ demonstrating the existence of a bacteria-type stringent response with humidity of 20%. For UV irradiation, plants were ex- in plants. Elevation of ppGpp levels was elicited also by treatment posed to UV light (predominantly 254 nm) from a distance of 15 with plant hormones jasmonic acid, abscisic acid, and ethylene, but cm by using a germicidal lamp (15 W, National, Osaka). To treat these effects were blocked completely by another plant hormone, them with plant hormones, plants were floated on an aqueous Ϯ indole-3-acetic acid. On the basis of these findings, we propose solution containing 0.3 mM ( )-jasmonic acid (Sigma–Aldrich), ethephon (an ethylene generator, Wako Pure Chemical), or that ppGpp plays a critical role in systemic plant signaling in Ϯ response to environmental stresses, contributing to the adaptation ( )-abscisic acid (Sigma–Aldrich). Indole-3-acetic acid (Sigma– of plants to environmental changes. Aldrich) was added at a concentration of 0.1 mM. Plants were subjected to stress or hormone treatments for Ϸ1 or 2 h unless indicated otherwise. he ability of organisms to survive under a wide range of Tadverse environmental conditions depends on diverse mo- ppGpp and Guanosine 5؅-Triphosphate 3؅-Diphosphate Analysis. To lecular mechanisms that adjust patterns of gene expression to the analyze levels of ppGpp and guanosine 5Ј-triphosphate 3Ј- changing environment. In bacteria, one of the most important diphosphate (pppGpp) in plants, shoots (20 g) from each plant processes regulating gene expression is ‘‘stringent control,’’ to be tested were frozen immediately in liquid nitrogen, crushed, which enables cells to adapt to nutrient-limiting conditions (1). and extracted with 100 ml of 2 M HCOOH for1hat4°C. After The effector molecule of stringent control, called an alarmone, removing cell debris by centrifugation (10,000 ϫ g, 10 min) and is a hyperphosphorylated guanosine nucleotide, guanosine 5Ј- filtration, water was added to the extract to give a final volume diphosphate 3Ј-diphosphate (ppGpp), which binds to the core of 200 ml. This solution then was extracted further with 200 ml RNA polymerase, eventually leading to activation or repression of water-saturated n-BuOH. For C. reinhardtii, 3.1 g of cells were of gene expression. Ribosome-dependent ppGpp synthesis is collected by centrifugation (6,000 ϫ g, 10 min), soaked in 20 ml catalyzed by the relA gene product ppGpp synthetase (also called of 2 M HCOOH, and sonicated for 5 min. After removing the stringent factor), which is expressed in response to the binding cell debris by centrifugation (8,000 ϫ g, 10 min), the supernatant of uncharged tRNA to the ribosomal A site (1). Likewise, plants was extracted with 20 ml of water-saturated n-BuOH. The water have a complex signal transduction network activated in re- layer was applied immediately to an epichlorohydrin triethanol- Ϫ sponse to such stressful conditions as pathogenic infection, amine (ECTEOLA) cellulose column (10 ml, HCOO form, wounding, heat shock, drought, and high salinity (2, 3). Plant Wako Pure Chemical), previously equilibrated with 1 M ͞ hormones such as ethylene, jasmonic acid, and abscisic acid HCOOH, and eluted with 100 ml of 0.3 M HCOOH 0.3 M HCOONH4 followed by 30 ml of 2 M HCOONH4. The second occupy critical positions in this signal transduction network ϩ (4–7), although details on their molecular mechanisms remain effluent was charged onto Dowex 50W (50 ml, H form, unknown. Despite the apparent significance of ppGpp in bac- Sigma–Aldrich), previously equilibrated with water, washed with terial gene expression, its importance in plant biology has been 50 ml of water, and then lyophilized. The resultant residue first largely overlooked. By devising a system to measure small was dissolved in 4 ml of water and then extracted several times with an excess of n-BuOH to remove the water. The resultant amounts of ppGpp precisely, however, we have been able to Ϸ ␮ ␮ demonstrate unambiguously that ppGpp is produced in the concentrated solution ( 300 l) was mixed with 40 l of MeOH chloroplasts of plant cells in response to stressful conditions. (to make the solution clear) and water to give a final volume of 400 ␮l. All procedures described above were carried out in a cold Materials and Methods room (4°C). To assay ppGpp, a portion (200 ␮l) of the extract solution was Plant Seedlings and Green Algae. Pea, wheat, spinach, and rice subjected to HPLC (L-7000, Hitachi, Tokyo) by using a Partisil seeds were grown on moist vermiculite at 25°C under white fluorescent light (12 h of light per day) for 3 weeks. In addition, Arabidopsis thaliana was grown for 6 weeks under the same This paper was submitted directly (Track II) to the PNAS office. conditions. Tobacco was grown on half-strength Murashige and Abbreviations: ppGpp, guanosine 5Ј-diphosphate 3Ј-diphosphate; pppGpp, guanosine Skoog medium containing 3% sucrose and 0.3% Gelrite (Wako 5Ј-triphosphate 3Ј-diphosphate. Pure Chemical, Osaka) in a growth chamber at 25°C for 4 weeks. *To whom correspondence should be addressed at: National Food Research Institute, ϩ Chlamydomonas reinhardtii TW3 strain (thi10 cw15 mt ), which 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. E-mail: [email protected]. harbors the cell-wall deficiency mutation cw15, was cultured in © 2004 by The National Academy of Sciences of the USA 4320–4324 ͉ PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308555101 Downloaded by guest on September 30, 2021 SAX-10 column (4.6 ϫ 250 mm, Whatman). The nucleotides were eluted at a flow rate of 1 ml͞min by using a gradient made up of low (7 mM KH2PO4, adjusted to pH 4.0 with H3PO4) and high (0.5 M KH2PO4 plus 0.5 M Na2SO4, adjusted to pH 5.4 with KOH) ionic strength buffers. The proportion of the high ionic strength buffer was increased for 20 min from 0% to 100% and then maintained for 25 min at 100%. When assaying pppGpp, we used the same high ionic strength buffer but adjusted the pH to 4.0; pppGpp was eluted at 44 min. The recovery of ppGpp and pppGpp throughout the process was 20%, as estimated from results using standard ppGpp and pppGpp (purity Ͼ98%), which were prepared enzymatically in our laboratory by using Strep- tomyces morookaensis. Analysis of ppGpp in Chloroplasts. Intact chloroplasts were isolated from untreated and wounded pea plants (150 g) in a cold room (4°C) by using the method of Perry et al. (9), after which the chloroplasts (300 mg) were soaked in 10 ml of 2 M HCOOH and then sonicated for 5 min. This solution then was extracted first with 10 ml of phenol saturated with water and then with 10 ml of chloroform. The water layer was freeze-dried, and the residue was then dissolved in 4 ml of water and concentrated by using an excess of n-BuOH. The resultant concentrate was used for ppGpp analysis. The recovery of ppGpp in this process was 80%, as estimated by using standard ppGpp. RNA Polymerase Assay. Intact chloroplasts were prepared from pea plants (9). Purification of the DNA–protein complex for the RNA polymerase assay was done according to the method of Briat et al. (10). The DNA–protein complex (Ϸ5–10 ␮g) was suspended in 20 ␮l of buffer containing 50 mM Tris⅐HCl (pH ͞ ͞ ͞ 7.6) 10 mM (NH4)2SO4 40 mM 2-mercaptoethanol 4mM EDTA͞25% glycerol͞0.1% Triton X-100 and then incubated at 30°Cfor1hinthepresence of 30 ␮l of buffer containing 50 mM ⅐ ͞ ͞ ͞ ͞ Tris HCl (pH 7.9) 10 mM MgCl2 0.2 mM ATP 0.2 mM GTP 0.2 mM CTP͞0.2 mM [3H]UTP [2 ␮Ci͞100 ␮l(1Ciϭ 37 GBq)]͞50 mM KCl. After running the reaction for the indicated times, the reaction was stopped by the addition of 30 ␮lof1% sodium dodecylsulfate solution containing 50 mM sodium py- rophosphate. An aliquot (25 ␮l) was deposited on a DE81 DEAE cellulose filter (Whatman), and nucleotide triphosphates not incorporated into RNA were removed by washing the filter six times with 5% Na2HPO4, twice in water, and finally once in 99% ethanol.
Load more

Recommended publications
  • Cyclic Di-AMP Signaling in Listeria Monocytogenes by Aaron Thomas
    Cyclic di-AMP signaling in Listeria monocytogenes By Aaron Thomas Whiteley A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Infectious Diseases and Immunity in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Daniel A. Portnoy, Chair Professor Sarah A. Stanley Professor Russell E. Vance Professor Kathleen R. Ryan Summer 2016 Abstract Cyclic di-AMP signaling in Listeria monocytogenes by Aaron Thomas Whiteley Doctor of Philosophy in Infectious Diseases and Immunity University of California, Berkeley Professor Daniel A. Portnoy, Chair The Gram positive facultative intracellular pathogen Listeria monocytogenes is both ubiquitous in the environment and is a facultative intracellular pathogen. A high degree of adaptability to different growth niches is one reason for the success of this organism. In this dissertation, two facets of L. monocytogenes, growth and gene expression have been investigated. The first portion examines the function and necessity of the nucleotide second messenger c-di-AMP, and the second portion of this dissertation examines the signal transduction network required for virulence gene regulation. Through previous genetic screens and biochemical analysis it was found that the nucleotide second messenger cyclic di-adenosine monophosphate (c-di-AMP) is secreted by the bacterium during intracellular and extracellular growth. Depletion of c-di- AMP levels in L. monocytogenes and related bacteria results in sensitivity to cell wall acting antibiotics such as cefuroxime, decreased growth rate, and decreased virulence. We devised a variety of bacterial genetic screens to identify the function of this molecule in bacterial physiology. The sole di-adenylate cyclase (encoded by dacA) responsible for catalyzing synthesis of c-di-AMP in L.
    [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 Alarmone (P)Ppgpp Is Part of the Heat Shock Response of Bacillus
    bioRxiv preprint doi: https://doi.org/10.1101/688689; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 The alarmone (p)ppGpp is part of the heat shock response of Bacillus 2 subtilis 3 4 Heinrich Schäfer1,2, Bertrand Beckert3, Wieland Steinchen4, Aaron Nuss5, Michael 5 Beckstette5, Ingo Hantke1, Petra Sudzinová6, Libor Krásný6, Volkhard Kaever7, Petra 6 Dersch5,8, Gert Bange4*, Daniel Wilson3* & Kürşad Turgay1,2* 7 8 Author affiliations: 9 1 Institute of Microbiology, Leibniz Universität Hannover, Herrenhäuser Str. 2, D-30419, 10 Hannover, Germany. 11 2 Max Planck Unit for the Science of Pathogens, Charitéplatz 1, 10117 Berlin, Germany 12 3 Institute for Biochemistry and Molecular Biology, Martin-Luther-King Platz 6, University of 13 Hamburg, 20146 Hamburg, Germany. 14 4 Philipps-University Marburg, Center for Synthetic Microbiology (SYNMIKRO) and 15 Department of Chemistry, Hans-Meerwein-Strasse, 35043 Marburg, Germany. 16 5 Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, 17 Braunschweig, Germany 18 6 Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20, Prague, 19 Czech Republic. 20 7 Hannover Medical School, Research Core Unit Metabolomics, Carl-Neuberg-Strasse 1, 21 30625, Hannover, Germany. 22 8 Institute of Infectiology, Von-Esmarch-Straße 56, University of Münster, Münster, Germany 23 24 Short title: The interplay of heat shock and stringent response 25 1 bioRxiv preprint doi: https://doi.org/10.1101/688689; this version posted July 1, 2019.
    [Show full text]
  • (P)Ppgpp Synthesis by the Ca2+-Dependent Rela-Spot Homolog
    bioRxiv preprint doi: https://doi.org/10.1101/767004; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Plastidial (p)ppGpp synthesis by the Ca2+-dependent RelA-SpoT homolog regulates the adaptation of chloroplast gene expression to darkness in Arabidopsis Sumire Ono1,4, Sae Suzuki1,4, Doshun Ito1 Shota Tagawa2, Takashi Shiina2, and Shinji Masuda3,5 1School of Life Science & Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan 2Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan 3Center for Biological Resources & Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan 4These authors contributed equally to the study 5Corresponding author: E-mail, [email protected] Abbreviations: CRSH, Ca2+-activated RSH; flg22, flagellin 22; (p)ppGpp, 5'-di(tri)phosphate 3'-diphosphate; PAMPs; pathogen-associated molecular patterns; RSH, RelA-SpoT homolog; qRT-PCR, quantitative real-time PCR 1 bioRxiv preprint doi: https://doi.org/10.1101/767004; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract In bacteria, the hyper-phosphorylated nucleotides, guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp), function as secondary messengers in the regulation of various metabolic processes of the cell, including transcription, translation, and enzymatic activities, especially under nutrient deficiency. The activity carried out by these nucleotide messengers is known as the stringent response.
    [Show full text]
  • Tetracycline Modulates the Valine Induced Stringent Response and Decreases Expressed Rpos in Escherichia Coli
    Journal of Experimental Microbiology and Immunology (JEMI) Vol. 15: 117 – 124 Copyright © April 2011, M&I UBC Tetracycline Modulates the Valine Induced Stringent Response and Decreases Expressed RpoS in Escherichia coli Natasha Castillon, Krysta Coyle, June Lai, and Cindy Yang Department of Microbiology & Immunology, University of British Columbia The stringent response in Escherichia coli is a physiological response to stress characterized by accumulation of the alarmone (p)ppGpp and the down-regulation of various processes involved in cellular growth and protein synthesis. It has been previously shown that activation of the stringent response by amino acid starvation is able to confer protection against various classes of antibiotics, including tetracycline and ampicillin. However, no observations have been made on partial activation of stringency. This investigation attempted to repress the level of bacterial stringent response in E. coli CP78 through the use of tetracycline. Tetracycline is an antibiotic which has been shown to restrict the cellular accumulation of (p)ppGpp in CP78 Escherichia coli. The effectiveness of the starvation by valine was monitored by measuring turbidity. The modulation of stringent response was monitored by the level of RpoS, since RpoS production is known to be enhanced by activated stringent response. As measured by turbidity, we successfully established an amino acid starvation condition with valine treatment as seen in the inhibited growth of CP78 cells as compared to untreated cells. The valine inhibition of the growth of the bacteria appeared to be reduced by the addition of tetracycline. However, cultures treated with high concentrations of tetracycline seemed to become more turbid than the untreated control even though they had similar levels of protein.
    [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]
  • (P)Ppgpp in Plants and Algae Ben Field
    Green magic: regulation of the chloroplast stress response by (p)ppGpp in plants and algae Ben Field To cite this version: Ben Field. Green magic: regulation of the chloroplast stress response by (p)ppGpp in plants and algae. Journal of Experimental Botany, Oxford University Press (OUP), 2018, 69 (11), pp.2797-2807. 10.1093/jxb/erx485. hal-01855838 HAL Id: hal-01855838 https://hal-amu.archives-ouvertes.fr/hal-01855838 Submitted on 8 Aug 2018 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. Green magic: regulation of the chloroplast stress response by (p)ppGpp in plants and algae Ben Field Aix Marseille Univ, CEA, CNRS, France Correspondence: [email protected] Abstract The hyperphosphorylated nucleotides guanosine pentaphosphate and tetraphosphate [together referred to as (p)ppGpp, or ‘magic spot’] orchestrate a signalling cascade in bacteria that controls growth under optimal conditions and in response to environmental stress. (p)ppGpp is also found in the chloroplasts of plants and algae where it has also been shown to accumulate in response to abiotic stress. Recent studies suggest that (p)ppGpp is a potent inhibitor of chloroplast gene expression in vivo, and is a significant regulator of chloroplast function that can influence both the growth and the development of plants.
    [Show full text]
  • Regulation of Factor Competition by the Alarmone Ppgpp
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of ␴ factor competition by the alarmone ppGpp Miki Jishage,1,3,4 Kristian Kvint,1,4 Victoria Shingler,2 and Thomas Nyström1,5 1Department of Cell and Molecular Biology-Microbiology, Göteborg University, 405 30 Go¨teberg, Sweden; 2Department of Cell and Molecular Biology, Umeå University, 901 87 Umeå, Sweden Many regulons controlled by alternative ␴ factors, including ␴S and ␴32, are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of ␴S-dependent promoters because underproduction of ␴70, specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-␴70) restored expression from ␴S-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 ␴70 to compete with ␴32 for core binding and the mutant ␴70 proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type ␴70. Similarly, an in vivo competition assay showed that the ability of both ␴32 and ␴S to compete with ␴70 is diminished in cells lacking ppGpp. Consistently, the fraction of ␴S and ␴32 bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of ␴ factors in accordance with cellular demands during physiological stress. [Key Words: Stringent response; ␴ factor competition; RpoD; RpoS; RpoH; Rsd] Received February 14, 2002; revised version accepted April 2, 2002.
    [Show full text]
  • The Nucleotide Pgpp Acts As a Third Alarmone in Bacillus, with Functions Distinct from Those of (P)Ppgpp
    ARTICLE https://doi.org/10.1038/s41467-020-19166-1 OPEN The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp Jin Yang 1, Brent W. Anderson 1, Asan Turdiev2, Husan Turdiev2, David M. Stevenson1, ✉ ✉ Daniel Amador-Noguez 1, Vincent T. Lee 2 & Jue D. Wang 1 1234567890():,; The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly refer- red to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′- diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp. 1 Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. 2 Department of Cell Biology and Molecular Genetics, University of ✉ Maryland, College Park, MD, USA.
    [Show full text]
  • Discovery of Small Alarmone Synthetases and Their Inhibitors As
    bioRxiv preprint doi: https://doi.org/10.1101/575399; this version posted March 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Title: Discovery of small alarmone synthetases and their inhibitors as toxin-antitoxin loci Authors: Steffi Jimmy1,2, Chayan Kumar Saha1, Constantine Stavropoulos1, Abel Garcia-Pino3, Vasili Hauryliuk1,2,4,*, Gemma C. Atkinson1,* 1Department of Molecular Biology, Umeå University, Building 6K, 6L University Hospital Area, 901 87 Umeå, Sweden 2Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Building 6K and 6L, University Hospital Area, SE-901 87 Umeå, Sweden 3Cellular and Molecular Microbiology, Faculté des Sciences, Université Libre de Bruxelles, 12 rue des Professeurs Jeener et Brachet, 6041 Gosselies, Belgium 4University of Tartu, Institute of Technology, 50411 Tartu, Estonia * denotes the corresponding authors Contact details of corresponding authors: Vasili Hauryliuk: [email protected], +46 907 850 807 Gemma C. Atkinson: [email protected], +46 706070315 Summary Under stressful conditions, bacterial RelA-SpoT Homologue (RSH) enzymes synthesise the alarmone (p)ppGpp, a nucleotide messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and at high concentrations inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single domain Small Alarmone Synthetases (SAS) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain, and regulatory C-terminal domains of the long RSHs such as Rel, RelA and SpoT.
    [Show full text]
  • Viewed in (104)]
    The Role in Translation of Editing and Multi-Synthetase Complex Formation by Aminoacyl-tRNA Synthetases Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Medha Vijay Raina, M.Sc. Ohio State Biochemistry Graduate Program The Ohio State University 2014 Dissertation Committee: Dr. Michael Ibba, Advisor Dr. Juan Alfonzo Dr. Irina Artsimovitch Dr. Kurt Fredrick Dr. Karin Musier-Forsyth Copyright by Medha Vijay Raina 2014 ABSTRACT Aminoacyl-tRNA synthetases (aaRSs) catalyze the first step of translation, aminoacylation. These enzymes attach amino acids (aa) to their cognate tRNAs to form aminoacyl-tRNA (aa-tRNA), an important substrate in protein synthesis, which is delivered to the ribosome as a ternary complex with translation elongation factor 1A (EF1A) and GTP. All aaRSs have an aminoacylation domain, which is the active site that recognizes the specific amino acid, ATP, and the 3′ end of the bound tRNA to catalyze the aminoacylation reaction. Apart from the aminoacylation domain, some aaRSs have evolved additional domains that are involved in interacting with other proteins, recognizing and binding the tRNA anticodon, and editing misacylated tRNA thereby expanding their role in and beyond translation. One such function of the aaRS is to form a variety of complexes with each other and with other factors by interacting via additional N or C terminal extensions. For example, several archaeal and eukaryotic aaRSs are known to associate with EF1A or other aaRSs forming higher order complexes, although the role of these multi-synthetase complexes (MSC) in translation remains largely unknown.
    [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]