DOCSLIB.ORG

Kasugamycin-Resistance in Escherichia Coli (Ribosomal Proteins/Ribosomal RNA/Reconstitution/Phosphocellulose Chromatography) ROBERT A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Kasugamycin-Resistance in Escherichia Coli (Ribosomal Proteins/Ribosomal RNA/Reconstitution/Phosphocellulose Chromatography) ROBERT A Proc. Nat. Acad. Sci. USA Vol. 70, No. 1, pp. 71-75, January 1973 Alteration of Ribosomal Protein S4 by Mutation Linked to Kasugamycin-Resistance in Escherichia coli (ribosomal proteins/ribosomal RNA/reconstitution/phosphocellulose chromatography) ROBERT A. ZIMMERMANN*, YOSHIKO IKEYAt, AND P. FREDERICK SPARLINGt§ * D~partement de Biologie Mol6culaire, Universit6 de Gen6ve, 30, Quai de l'Ecole-de-Medecine, CH-1211, Geneva 4, Switzerland; and t Departments of Medicine and Bacteriology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514 Communicated by Cyrus Levinthal, October 20, 1972 ABSTRACT An alteration in the chromatographic We report experiments that confirm many of these findings. mobility of 30S ribosomal protein S4 from two kasugamy- In contrast to Helser et al. (7, 8), however, we have found that cin-resistant (ksgA) strains of E. coli was observed. The locus determining this alteration frequently cotrans- two ksgA mutants possess an altered 30S subunit protein as duced with ksgA but could be separated from it by trans- well as undermethylated 16S RNA. Both of the mutants were duction and conjugation. Since the structural gene for obtained by nitrosoguanidine mutagenesis and are probably protein S4 is thought to lie some 30 min from ksgA on the double mutants. The altered protein has been identified as E. coli chromosome, the product of the newly-identified or structural gene may modify protein S4. We propose to designate this S4 (9) p4a (10). Several reports have described gene ramB. changes in protein S4 (11-14) resulting from mutations in the Reconstitution of 30S subunits from RNA and protein cluster of ribosomal protein genes near strA (12-15). The gene components of ksgA ramB and ksgA+ ramB+ strains dem- for alteration of protein S4 that we describe is shown to be onstrated that ribosomal resistance to kasugamycin was close to, but separable from, ksgA, and is therefore at a con- due to altered 16S RNA and not to altered protein S4. The altered 16S RNA was undermethylated, and a methyl- siderable distance fromn strA on the E. coli chromosome. The ating enzyme that acts on 16S RNA from ksgA strains implications of this finding are discussed. was present in ksgA+ but not in ksgA strains. MATERIALS AND METHODS Resistance to the aminoglycoside antibiotic, kasugamycin Bacterial Strains. Bacterial strains are listed in Table 1. results from alteration of the 30S ribosomal subunit in The kasugamycin-resistant strains in which altered protein Escherichia coli (1). The gene for ribosomal kasugamycin S4 was found were selected on tryptone-yeast extract plates resistance (ksgA) is closely linked to leu and maps near containing 1 mg of kasugamycin/ml after nitrosoguanidine 0.5 min on the E. coli chromosome (1-3). By contrast, loci mutagenesis (16) to the extent of 10% survival of exposed determining ribosomal resistance to the aminoglycosides, cells. Spontaneous kasugamycin-resistant mutants were streptomycin and spectinomycin, have been placed in the selected on the same plates containing 250 lsg of kasugamycin cluster of 30S and 50S ribosomal protein genes near strA at per ml. Kasugamycin was a gift of Bristol Laboratories. 64 min (see ref. 4), and in both cases, antibiotic sensitivity or Strains were designated ksgA if the locus for kasugamycin resistance is mediated by specific ribosomal proteins (5, 6). resistance cotransduced 90% with pdxA, and if cell-free The unusual map position of the ksgA locus suggested a novel extracts were resistant to kasugamycin (2). Strains were mechanism for kasugamycin resistance (1). designated ksgB if they contained a locus for kasugamycin In a recent report, Helser, Davies, and Dahlberg demon- resistance that was located between 30-35 min on the E. strated that resistance to kasugamycin is attributable to an coli chromosome, and if cell-free extracts were sensitive to alteration of the 16S RNA component of the 30S subunit (7). kasugamycin (2). Transductions were performed with phage Specifically, the 16S RNA from resistant mutants lacks Plkc by standard methods (17). methylation of two adjacent adenine residues that are both dimethylated in the RNA of sensitive strains. The same Chromatography of Ribosomal Proteins. Growth of cells, authors later found that ksgA+, but not ksgA, bacteria con- preparation of ribosomal proteins, and chromatographic tain an enzyme that dimethylates the relevant adenine resi- analysis were performed as described by Zimmerman et al. dues of "resistant" RNA and that 30S particles reconstituted (11) with two exceptions: cells were labeled with either from such RNA after in vitro methylation are sensitive to [3H]- or ['4C]amino-acid mixtures (New England Nuclear the drug (8). Moreover, the enzyme acted only on under- Corp.) and high-capacity phosphocellulose (Mann, 1.1 methylated 16S RNA from kasugamycin-resistant mutants meq/g) was used for chromatography. and not on mature 16S RNA from sensitive strains, 23S RNA, Preparation of Ribosomal Components for Reconstitution. or protein. It was concluded that the product of the ksgA gene Cells were grown to mid log-phase in tryptone-yeast extract is a methylating enzyme that modifies 16S RNA at some broth and harvested after pouring on ice. Ribosomes were time during maturation of the 30S subunit. prepared as described (18), except that no DNase was used. Ribosomes were not washed in high-salt (0.5 M NH4Cl) buffers. 30S and 50S subunits were prepared by suspension of § To whom reprint requests should be sent. pelleted 70S ribosomes in standard buffer [10 mM Tris HCI 71 Downloaded by guest on September 29, 2021 72 Biochemistry: Zimmerman et al. Proc. Nat. Acad. Sci. USA 70 (1978) TABLE 1. Strains of E. coli K12 used TABLE 2. Reconstitution of SOS ribosomes from total protein and 16S RNA Strain Source Genotype Reconstituted 30S JC411 W. K. Mass* F-; argG met leu his strA ribosomes Incorporation cpm (% JC12 W. K. Mass* HFr; purC met control) FS131 Nitrosoguanidine As JC12 but ramBI ksgAl9 16S Total ug of kasugamycin per ml mutagenesis RNA protein 0 50 100 FS157 Nitrosoguanidine As JC12 but ramB2 ksgA23 ksgB1 mutagenesie S S 607 367 (61) 289 (48) FS215 Recombinant from As JC411 but his+ ramB2 ksgA23 S R 503 251 (50) 179 (35) FS157 x JC411 R S 324 281 (87) 220 (68) FS216 Recombinant from As JC411 but his+ ramB2 ksgBl R R 357 288 (81) 290 (73) FS157 x JC411 FS232 Transductant of As JC411 but leu+ ramBI ksgAl9 30S ribosomal subunits were reconstituted as described in JC411 from Methods and assayed for activity in poly(U,G)-dependent FS131 [P4C]valine incorporation in vitro. Reaction mixture (100 pl FS233 Transductant of As JC411 but leu+ ramB2 ksgA23 total) contained 0.7-0.8 A26 units each of reconstituted 30S par- JC411 from ticles and 50S subunits, 10ul of S-100 fraction, 10 pg of poly(U,G) FS157 (1:1, Miles Laboratories), 8 mM Mg acetate, 0.01 mM [14C]- Q13 W. Gilbert via met tyr pnp-13 valine (50 pCi/pmol, New England Nuclear Corp.), and other M. Nomurat components as described (18). S indicates component derived from FS240 Spontaneous As Q13 but ksgA3O strain JC411 (ksgA+ ramB+); R indicates component derived from mutant strain FS232 (ksgAI9 ramBi). FS241 Nitrosoguanidine As Q13 but leu mutagenesis FS242 Transductant of As FS241 but leu+ ksgA23 gation on a 5-20% linear sucrose gradient in TAS buffer for FS241 from 9.5 hr at 21,000 rpm in an SW27 rotor. Ribosomes and sub- FS157 units were stored at -70° in standard buffer containing 10 mM MgC12. * Dept. of Microbiology, New York University School of Med- The 16S RNA and total 30S ribosomal protein fractions icine, New York, N.Y. were prepared by phenol extraction and ureag-LiCl treatment, t Institute for Enzyme Research, Univ. of Wisconsin, Madison, respectively (20). Reconstitution of 30S subunits from Wisc. 16S RNA and total 30S proteins was performed according to Traub and Nomura (20). The RNA precipitate resulting from (pH 7.6)-30 mM NH4C1-6 mM 2-mercaptoethanol] con- treatment of 30S subunits with urea-LiCl was also used and taining 0.3 mMI MgCl2, followed by dialysis against TAS was as active as that prepared by phenol extraction. buffer [20 mM Tris HCl (pH 7.6)-100 mM NH4Cl-1 mM Fingerprint Analysis. [14C]Methyl-labeled oligonucleotides MgCl2-0.5 mM EDTA-4 mM 2-mercaptoethanol] for 3-4 hr from 16S RNA were prepared and fingerprinted (21). at 0° (19). Subunits were separated on discontinuous 0.2-1.0 M sucrose gradients (19) by centrifugation in an SW27 rotor for 20 Methykation. 23S core particles from 30S subunits (22) were hr at 27,000 rpm. 30S and 50S subunits were precipitated with methylated by enzymes prepared from 8-100 fractions and 0.6 volume of ethanol (19), and further purified by centrifu- from the 1.0 M NH4Cl supernatant of washed 70S ribosomes (8); S-adenosyl-['H ]methionine was purchased from New England Nuclear Corp. RESULTS .1. :.: i i 1500 1. AI Altered Ribosomal Protein S4 in Certain Kasugamycin- Resistant Strains. In our early efforts to identify the ribosomal I I00o .I looo l component altered in kasugamycin-resistant strains of :11 E. coli, 'H- and "C-labeled 30S subunit proteins from JC411 0 0 j.., I. (ksgA+) and FS215 (a ksgA23 recombinant derived by con- 500 jugation of JC411 with FS157) were compared by cochroma- tography on phosphocellulose columns. Fig. 1 demonstrates that one 30S protein from the mutant eluted at a significantly o -O 50 100 150 200 250 300 lower salt concentration than its counterpart from the FRACTION NUMBER wild-type strain, whereas the elution positions of all other 30S FIG. 1. Elution profile of 30S total proteins from 3H-labeled proteins from the two strains coincided.
Load more

Recommended publications
  • Kasugamycin Crops Identification of Petitioned Substance
    United States Department of Agriculture Agricultural Marketing Service | National Organic Program Document Cover Sheet https://www.ams.usda.gov/rules-regulations/organic/national-list/petitioned Document Type: ☐ National List Petition or Petition Update A petition is a request to amend the USDA National Organic Program’s National List of Allowed and Prohibited Substances (National List). Any person may submit a petition to have a substance evaluated by the National Organic Standards Board (7 CFR 205.607(a)). Guidelines for submitting a petition are available in the NOP Handbook as NOP 3011, National List Petition Guidelines. Petitions are posted for the public on the NOP website for Petitioned Substances. ☒ Technical Report A technical report is developed in response to a petition to amend the National List. Reports are also developed to assist in the review of substances that are already on the National List. Technical reports are completed by third-party contractors and are available to the public on the NOP website for Petitioned Substances. Contractor names and dates completed are available in the report. Kasugamycin Crops Identification of Petitioned Substance 1 Chemical Names: Other Name: 2 Kasugamycin Kasugamycin hydrochloride hydrate 3 2-amino-2-[(2R,3S,5S,6R)-5-amino-2-methyl-6- Kasugamycin monohydrochloride 4 [(2R,3S,5S,6S)-2,3,4,5,6- 5 pentahydroxycyclohexyl]oxyoxan-3- Trade Names: 6 yl]iminoacetic acid (IUPAC name) Kasumin 2L, Kasumin 4L 7 8 3-O-[2-amino-4-[(carboxyiminomethyl)amino]- CAS Numbers: 9 2,3,4,6-tetradeoxy-a-D-arabino-hexopyranosyl]- Kasugamycin (6980-18-3) 10 D-chiro-inositol Kasugamycin monohydrochloride (19408-46-9) 11 12 Other Codes: 13 Kasugamycin Pub Chem CID 65174 14 15 16 Summary of Petitioned Use 17 18 The National Organic Program (NOP) was petitioned to add kasugamycin as an allowed synthetic to the 19 synthetic substances National List at 7 CFR §205.601.
    [Show full text]
  • Ribosomal RNA Degradation Induced by the Bacterial RNA Polymerase Inhibitor Rifampicin
    Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin. Lina Hamouche1, Leonora Poljak1, and Agamemnon J. Carpousis1,2† 1LMGM, Université de Toulouse, CNRS, UPS, CBI, Toulouse, France 2TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France Running title: Rifampicin-induced rRNA degradation †Corresponding author: [email protected] 1 Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin- induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete thus permitting the re-initiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect.
    [Show full text]
  • Kasugamycin Is a Novel Chitinase 1 Inhibitor with Strong Antifibrotic Effects on Pulmonary Fibrosis
    bioRxiv preprint doi: https://doi.org/10.1101/2021.02.25.432796; this version posted February 26, 2021. 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. Kasugamycin is a novel chitinase 1 inhibitor with strong antifibrotic effects on pulmonary fibrosis Jae-Hyun Lee1,*, Chang-Min Lee2,*, Mun-Ock Kim3, Jin Wook Park2, Suchita Kamle2, Bedia Akosman2, Erica L. Herzog4, Jack A. Elias2,5,#, and Chun Geun Lee2,# 1Division of Allergy and Immunology, DePartment of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea 2Molecular Microbiology and Immunology Brown University 185 Meeting St. Providence, RI. 02912, USA 3Natural Medicine Research Center, KRIBB Cheongju-si, Chungcheongbuk-do RePublic of Korea, 28116 4Yale School of Medicine Section of Pulmonary, Critical Care, and SleeP Medicine DePartment of Internal Medicine 300 Cedar Street (TAC S441) New Haven, CT 06520 4Division of Medicine and Biological Sciences, Brown University, Warren AlPert School of Medicine, Box G-A1, 97 Waterman Street, Providence, RI 02912 *Equal contribution to the work #Corresponding authors: Jack A. Elias Box G-A1, 97 Waterman Street, Providence, RI 02912 jack_elias @brown.edu, 401-863-3336 Chun Geun Lee (to whom requests for rePrints) Box G-L, 185 Meeting St, Providence, RI 02912 [email protected], 401-863-5932 Key Words: Kasugamycin, TGFBRAP1, Pulmonary fibrosis, Chitinase 1, TGF-beta Running Title: Kasugamycin and Pulmonary Fibrosis Total Word Count: 3434 Subject code: 9.24 Interstitial Lung Disease bioRxiv preprint doi: https://doi.org/10.1101/2021.02.25.432796; this version posted February 26, 2021.
    [Show full text]
  • Emerging Insights Into the Moonlighting Functions And
    www.symbiosisonline.org Symbiosis www.symbiosisonlinepublishing.com Opinion SOJ Genetic Science Open Access Emerging Insights into the Moonlighting Functions and Evolutionary Origins of Mitochondrial RNA Methyltransferases Sam Manna and Ashley Harman* Department of Microbiology, La Trobe University, Melbourne, Victoria, Australia Received: 26 November, 2014; Accepted: 20 January, 2015; Published: April 17, 2015 *Corresponding author: Ashley Harman, Department of Microbiology, La Trobe University,Melbourne, Victoria, Australia, Tel: +613-9479-2657; Fax: +613-9479-1222; E-mail: [email protected] Keywords: Mitochondria; RNA Methyltransferase; Pentatricopeptide repeat; Mitochondrial transcription factor homolog into these mutants restores methylation and sensitivity RNA methyltransferases mediate the addition of methyl to kasugamycin [4]. Furthermore, the methyltransferase activity mitochondrialof mtTFB has small been subunit implicated rRNA and in increasedaminoglycoside-induced aminoglycoside for the structure and function of RNA. While methylation is deafness, a disease involving hypermethylation of mutated reportedgroups to inribonucleotides. mitochondrial Such RNAs, modifications the enzymes have that implications catalyze subunitsensitivity rRNA [3]. structure. This sensitivity is caused by an increased binding in sequencing technologies, genomic analysis is beginning to affinity for aminoglycosides to the mutated mitochondrial small these reactions often remain elusive; however with advances of mitochondria, little is known about the origin and function of uncover their identities. Due to the complex evolutionary history is capableMetazoans of both possess functions, two there mtTFB is a markedhomologs difference (TFB1M in theirand TFB2M) as a result of gene duplication. While each human mtTFB methyltransferases. This article explores two emerging families of mitochondrial RNA methyltransferases. We discuss the evidence primary activity. TFB1M is primarily a rRNA methyltransferase whereas TFB2M is largely a transcriptional activator [2,3].
    [Show full text]
  • Discovery of Small-Molecule Inhibitors of Multidrug-Resistance Plasmid Maintenance Using a High-Throughput Screening Approach
    Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach Katelyn E. Zulaufa,b and James E. Kirbya,b,1 aDepartment of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215; and bHarvard Medical School, Boston, MA 02215 Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved October 12, 2020 (received for review March 30, 2020) Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug- DNA, cause DNA strand breakage, affect supercoiling, inhibit resistant pathogens for which new treatments are desperately conjugation, or cause plasmid-dependent fitness defects (5). needed. Carbapenemases and other types of antibiotic resistance Almost all, where investigated, acted nonspecifically and were genes are carried almost exclusively on large, low-copy-number highly toxic to the host bacterial cell, for example chemothera- plasmids (pCRE). Accordingly, small molecules that efficiently evict peutic intercalators. Of interest, however, DeNap et al. (6) and pCRE plasmids should restore much-needed treatment options. We Thomas et al. (7) identified several positively charged amino- therefore designed a high-throughput screen to identify such com- glycosides that affected the RNA-regulated plasmid replication pounds. A synthetic plasmid was constructed containing the plas- machinery of the IncB incompatibility group plasmid, pMU2403. mid replication machinery from a representative Escherichia coli These findings supported the possibility of identifying anti- CRE isolate as well as a fluorescent reporter gene to easily monitor plasmid agents with greater mechanistic specificity. plasmid maintenance. The synthetic plasmid was then introduced As pCRE plasmids encode carbapenemase genes, we hypoth- into an E. coli K12 tolC host.
    [Show full text]
  • Antibiotic Blocks Mrna Path on the Ribosome
    NEWS AND VIEWS Antibiotic blocks mRNA path on the ribosome Alexander Mankin Many translation initiation inhibitors block transfer RNA binding or placement in the ribosome. Structures of kasugamycin bound to the bacterial ribosome now indicate that it instead blocks proper mRNA placement. In 1965, a compound was isolated from a EPA mRNA EPA EPA Streptomyces strain found near the Kasuga a SD SD SD shrine in Nara, Japan. The drug could pre- http://www.nature.com/nsmb Small vent growth of a fungus causing rice blast subunit Subsequent steps of disease and later was found to inhibit bacte- translation fMet-tRNA Large rial growth. It was called kasugamycin. subunit Like many (actually, almost half) of all the known natural antibiotics, kasugamycin inhibits proliferation of bacteria by tamper- Kasugamycin ing with their ability to make new proteins, EPA EPA the ribosome being the major target of such b SD SD x drugs. In essence, the ribosome is an RNA Initiation is blocked machine, and most antibiotics that inhibit Nature Publishing Group Group Nature Publishing translation bind ribosomal RNA and affect 6 rRNA interactions with the ligands of the 200 ribosome: aminoacyl- and peptidyl-tRNAs, EPA EPA © translation factors or the nascent peptide. c A great variety of drugs that target the ribo- some inhibit elongation of translation. In Subsequent contrast, only a small fraction of the known steps of translation protein-synthesis inhibitors affect transla- tion initiation. In many cases, the mechanism of action of such drugs is obscure. During translation initiation, the small ribosomal sub- unit locates a specific segment in mRNA known EPA EPA as the translation-initiation region (Fig.
    [Show full text]
  • Abctransporter Genes, Kasklm,Responsible for Self-Resistance of a Kasugamycin Producer Strain
    VOL. 53 NO. 4, APR. 2000 THE JOURNAL OF ANTIBIOTICS pp.373 - 384 ABCTransporter Genes, kasKLM,Responsible for Self-resistance of a Kasugamycin Producer Strain Souichi Ikeno*, Yasuhiro Yamane, Yoko Ohishi, Naoko Kinoshita1*, Masa Hamada1", Kayoko S. Tsuchiya and Makoto Hori Showa College of Pharmaceutical Sciences, 3-3 1 65 Higashi Tamagawagakuen^ Machida-shi, Tokyo 194-8543, Japan institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021 , Japan (Received for publication November 19, 1999) Wepreviously reported that a 7.6-kb DNAfragment from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSMacetyltransferase gene (kac33S) and some other genes possibly involved in KSMbiosynthesis. As an extension of that study, a 10-kb Sacl-Kpnl DNAfragment, located 5~ 15-kb upstream of kac?3%, was cloned and a 4.2-kb Sacl- EcoRl fragment there from was sequenced, revealing one incomplete (designated ORFJ) and three complete open reading frames (designated kasK, kasL and kasM). The coding frames of kasK, L and Moverlap one another with terminator/initiator ATGAsequence. RT-PCRanalysis of a DNAregion including kasKLMindicated the presence of one transcript that is long enough to span the three genes. The kasK gene potentially encodes an ATP-binding protein of the ATP- binding cassette (ABC) transporter super family. Homology search for the deduced KasK protein shows similarity to other ABCtransporters involved in self-resistance of a mithramycin and possibly doxorubicin producer strain. The kasL and kasMgenes encode different integral membraneproteins, both having six putative transmembrane helices. Anexpression plasmid for kasKLM(pTV^KLM)was constructed and these genes were expressed in E.
    [Show full text]
  • Curr Drug Targets
    Current Drug Targets - Infectious Disorders, 2002, 2, 169-186 169 Antibiotics Targeting Ribosomes: Crystallographic Studies Tamar Auerbach1,2, Anat Bashan1, Joerg Harms3, Frank Schluenzen3, Raz Zarivach1, Heike Bartels3, Ilana Agmon1, Maggie Kessler1, Marta Pioletti4, François Franceschi4 and Ada Yonath1,3,* 1Dept. of Structural Biology, Weizmann Institute, 76100 Rehovot, Israel; 2FB Biologie, Chemie, Pharmazie, Frei Univ. Berlin, Takustr. 3,14195 Berlin, Germany; 3Max-Planck-Res. Unit for Ribosomal Structure, Notkestr. 85, 22603 Hamburg, Germany and 4Max-Planck- Institut for Moleculare Genetics, Ihnestr. 73, 14195 Berlin, Germany Abstract: Resistance to antibiotics is a major problem in modern therapeutics. Ribosomes, the cellular organelle catalyzing the translation of the genetic code into proteins, are targets for several clinically relevant antibiotics. The ribosomes from eubac- teria are excellent pathogen models. High resolution structures of the large and small ribosomal subunits were used as references that allowed unambiguous localization of almost a dozen antibiotic drugs, most of which are clinically relevant. Analyses of these structures showed a great diversity in the antibiotics’ modes of action, such as interference with substrate binding, hindrance of the mobility required for the biosynthetic process and the blockage of tunnel which provides the path of exit for nascent proteins. All antibiotics studied by us were found to bind primarily to ribosomal RNA and, except for one allosteric effect, their binding did not cause major conformational changes. Antibiotics of the small ribosomal subunit may hinder tRNA binding, decoding, translocation, and the initiation of the entire biosynthetic process. The large subunit agents may target the GTPase center, interfere with peptide bond formation, or block the entrance of the nascent protein exit tunnel.
    [Show full text]
  • Kasugamycin Human Health Risk Assessment DP Barcode D433630
    Kasugamycin Human Health Risk Assessment DP Barcode D433630 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, DC 20460 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION MEMORANDUM DATE: 27 Septem her 2017 SUBJECT: Kasugamycin. Human Health Risk Assessment forthe Proposed Section 3 Registration of New Uses of the Antibiotic Fungicide on Cherry Subgroup 12-12A and Walnuts. DP Barcode: PC Code: 230001 D433630 Registration Number: Decision Number: 513997 66330-404 Regulatory Action: Petition Number: 6E8450 Section 3 Registration (Tolerance Petition) Risk Assessment Type: Single Chemical Aggregate Case Number: 7045 TXR Number: NA CAS Number: 6980-18-3 (19408-46-9, HCI salt) MRID Number: NA 40CF�{\1,_80.6�4 � FROM: Linnea J. Hansen, Biologis��sor cJvv" William T. Drew, Chemist � � Gerad Thornton, Environmental Protection Specialist��--­ Registration Action Branch II Health Effects Division (HED), 7509P THROUGH: Christina Swartz, Chief Risk Assessment Branch II Health EffectsDivision (HED), 7509P To: Hope Johnson and Fatima Sow, PM Team 21 Fungicide Branch (FB) Registration Division (RD), 7505P and Tamica Cain Minor Use and Emergency Response Branch (MUERB)/RD, 7505P Page 1 of 45 Kasugamycin Human Health Risk Assessment DP Barcode D433630 Table of Contents 1.0 Executive Summary ............................................................................................................ 4 2.0 HED Recommendations...................................................................................................... 6 2.1 Data Deficiencies
    [Show full text]
  • Insights Into the Stress Response Triggered by Kasugamycin in Escherichia Coli
    antibiotics Article Insights into the Stress Response Triggered by Kasugamycin in Escherichia coli Christian Müller, Lena Sokol, Oliver Vesper, Martina Sauert and Isabella Moll * Max F. Perutz Laboratories, Center for Molecular Biology, Department of Microbiology, Immunobiology and Genetics, University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 9/4, A-1030 Vienna, Austria; [email protected] (C.M.); [email protected] (L.S.); [email protected] (O.V.); [email protected] (M.S.) * Correspondence: [email protected]; Tel.: +43-1-427-754-606 Academic Editor: Claudio O. Gualerzi Received: 1 April 2016; Accepted: 23 May 2016; Published: 1 June 2016 Abstract: The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12. Keywords: kasugamycin; translation initiation; leaderless mRNA; Escherichia coli 1. Introduction The bactericidal aminoglycoside antibiotic kasugamycin (Ksg) inhibits protein synthesis in eukaryotic and prokaryotic cells at an initial step [1] without affecting translational elongation [2].
    [Show full text]
  • Snapshot: Antibiotic Inhibition of Protein Synthesis I Daniel Sohmen,1 Joerg M
    SnapShot: Antibiotic Inhibition of Protein Synthesis I Daniel Sohmen,1 Joerg M. Harms,2 Frank Schlünzen,3 and Daniel N. Wilson1,4 1University of Munich, Germany; 2MPSD, University of Hamburg, Germany; 3DESY, Hamburg, Germany; 4CiPS-M, Munich, Germany Class Name Target Mechanism Aminoglycosides Apramycin, gentamycin, hygromycin B, Elongation (translocation) Most aminoglycoside antibiotics induce translational misreading by promoting kanamycin, neomycin, paromomycin, binding of near-cognate tRNAs, but the biological effect is probably due to inhibition tobramycin of the translocation reaction and promotion of back-translocation. Edeine Edeine A Initiation (fMet-tRNA Edeine prevents binding of the initiator tRNA to the 30S subunit in an mRNA- binding) dependent manner. GE81112 GE81112 Initiation (fMet-tRNA GE81112 inhibits binding of the initiator tRNA to the 30S subunit in an mRNA- binding) independent manner. Kasugamycin Kasugamycin Initiation (fMet-tRNA Kasugamycin inhibits translation initiation of canonical mRNAs by binding in the binding) path of the mRNA and preventing stable interaction of the initiator tRNA with the start codon. Pactamycin Pactamycin Initiation, elongation Early data suggest that pactamycin allows 30S but not 70S initiation complex (translocation) formation, whereas recently pactamycin was shown to inhibit translocation in a tRNA-mRNA-dependent manner. Spectinomycin Spectinomycin Elongation (translocation) Spectinomycin binds to the neck of the small subunit and prevents the relative movement of the head and body
    [Show full text]
  • Kasugamycin Potentiates Rifampicin and Limits Emergence of Resistance
    RESEARCH ARTICLE Kasugamycin potentiates rifampicin and limits emergence of resistance in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation Swarnava Chaudhuri1†, Liping Li2†, Matthew Zimmerman2, Yuemeng Chen1, Yu-Xiang Chen1, Melody N Toosky1,3, Michelle Gardner3, Miaomiao Pan1, Yang-Yang Li1, Qingwen Kawaji1, Jun-Hao Zhu1, Hong-Wei Su1, Amanda J Martinot4, Eric J Rubin3, Veronique Anne Dartois2*, Babak Javid1,3* 1Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China; 2Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, United States; 3Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, Boston, United States; 4Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States Abstract Most bacteria use an indirect pathway to generate aminoacylated glutamine and/or *For correspondence: asparagine tRNAs. Clinical isolates of Mycobacterium tuberculosis with increased rates of error in [email protected] gene translation (mistranslation) involving the indirect tRNA-aminoacylation pathway have (VAD); increased tolerance to the first-line antibiotic rifampicin. Here, we identify that the aminoglycoside [email protected] (BJ) kasugamycin can specifically decrease mistranslation due to the indirect tRNA pathway. †These authors contributed Kasugamycin but not the aminoglycoside streptomycin, can limit emergence of rifampicin equally to this work resistance in vitro and increases mycobacterial susceptibility to rifampicin both in vitro and in a Competing interests: The murine model of infection. Moreover, despite parenteral administration of kasugamycin being authors declare that no unable to achieve the in vitro minimum inhibitory concentration, kasugamycin alone was able to competing interests exist.
    [Show full text]