The Alarmone (P)Ppgpp Is Part of the Heat Shock Response of Bacillus
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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. -
Assessing the Role of Spot and Rela in Capsular Polysaccharide
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 15: 52 – 58 Copyright © April 2011, M&I UBC Assessing the Role of SpoT and RelA in Capsular Polysaccharide Synthesis after Treatment with Sub-lethal Concentrations of Kanamycin to Confer Decreased Antibiotic Sensitivity in Escherichia coli Wesley Chenne, Louisa Ng, and Mary Rose Pambid Department of Microbiology & Immunology, University of British Columbia Resistance to various antibiotics has been associated with increased capsular polysaccharide production through activation of RelA and SpoT, regulators of the stringent response, through their respective synthesis and hydrolysis of guanosine tetraphosphate in Escherichia coli. In order to further characterize the role of SpoT and RelA in capsular polysaccharide production and in conferring antibiotic resistance, growth patterns and induced capsular polysaccharide levels of various strains were determined following sub- lethal treatment with kanamycin and further treatment at inhibitory levels of kanamycin. Contrary to previous reports that capsular polysaccharide confers antibiotic resistance, it was found that spoT mutants produced the least capsular polysaccharide but had higher levels of resistance in comparison to relA mutants, which produced the most polysaccharide but exhibited lower resistance. As expected, both these mutants exhibited lower resistance to kanamycin as a result of pre-treatment than the wild-type strain. Thus, we propose that bacterial survival and resistance development upon antibiotic administration -
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. -
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). -
(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. -
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. -
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. -
N. Meningitidis
Stringent response regulation and its impact on ex vivo survival in the commensal pathogen Neisseria meningitidis Regulation der stringenten Kontrolle und ihre Auswirkungen auf das ex vivo Überleben des kommensalen Erregers Neisseria meningitidis Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Laura Violetta Hagmann (geb. Kischkies) Frankfurt am Main Würzburg, 2016 Eingereicht am: …………………………… Mitglieder der Promotionskommission: Vorsitzender: …………………………………………………….. Gutachter: PD Dr. rer. nat. Dr. med. Christoph U. Schoen Gutachter: PD Dr. rer. nat. Knut Ohlsen Tag des Promotionskolloquiums: …………………………... Doktorurkunde ausgehändigt am: …………………………... EIDESSTATTLICHE ERKLÄRUNG Hiermit versichere ich, dass ich die vorliegende Dissertation selbstständig angefertigt und nur die angegebenen Quellen und Hilfsmittel verwendet habe. Ich versichere zudem, dass diese Arbeit in dieser oder ähnlicher Form in keinem anderen Prüfungsverfahren vorgelegen hat. Bis auf den Titel der Diplom-Biologin habe ich bislang keinen anderen akademischen Grad erworben oder zu erwerben versucht. Würzburg, den 13.10.2016 ……………………………………. Laura Violetta Hagmann Table of Content 1 Summary .......................................................................................................... 1 2 Zusammenfassung .......................................................................................... 2 3 Introduction..................................................................................................... -
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. -
Ribosomal RNA
Ribosomal RNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal DNA (rDNA) and then bound to ribosomal proteins to form small and large ribosome subunits. rRNA is the physical and mechanical factor of the ribosome that forces transfer RNA (tRNA) and messenger RNA (mRNA) to process and translate the latter into proteins.[1] Ribosomal RNA Three-dimensional views of the ribosome, showing rRNA in dark blue (small subunit) is the predominant form of RNA found in most cells; it makes and dark red (large subunit). Lighter colors up about 80% of cellular RNA despite never being translated represent ribosomal proteins. into proteins itself. Ribosomes are composed of approximately 60% rRNA and 40% ribosomal proteins by mass. Contents Structure Assembly Function Subunits and associated ribosomal RNA In prokaryotes In eukaryotes Biosynthesis In eukaryotes Eukaryotic regulation In prokaryotes Prokaryotic regulation Degradation In eukaryotes In prokaryotes Sequence conservation and stability Significance Human genes See also References External links Structure Although the primary structure of rRNA sequences can vary across organisms, base-pairing within these sequences commonly forms stem-loop configurations. The length and position of these rRNA stem-loops allow them to create three-dimensional rRNA structures that are similar across species.[2] Because of these configurations, rRNA can form tight and specific interactions with ribosomal proteins to form ribosomal subunits. These ribosomal proteins contain basic residues (as opposed to acidic residues) and aromatic residues (i.e. -
(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. -
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.