DOCSLIB.ORG

Identification of the Catalytic Motif of the Microbial Ribosome Inactivating Cytotoxin Colicin E3

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification of the Catalytic Motif of the Microbial Ribosome Inactivating Cytotoxin Colicin E3 This is a repository copy of Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/1051/ Article: WALKER, DANIEL, LANCASTER, LORNA, KLEANTHOUS, COLIN et al. (1 more author) (2004) Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3. Protein Science. pp. 1603-1611. ISSN 1469-896X https://doi.org/10.1110/PS.04658504 Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3 1,3 1,3 2 DANIEL WALKER, LORNA LANCASTER, RICHARD JAMES, AND COLIN KLEANTHOUS1 1Department of Biology, University of York, York YO10 5YW, UK 2Division of Microbiology and Infectious Diseases, School of Molecular Medical Sciences, University Hospital, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK (RECEIVED January 30, 2004; FINAL REVISION March 4, 2004; ACCEPTED March 4, 2004) Abstract Colicin E3 is a cytotoxic ribonuclease that specifically cleaves 16S rRNA at the ribosomal A-site to abolish protein synthesis in sensitive Escherichia coli cells. We have performed extensive mutagenesis of the 96-residue colicin E3 cytotoxic domain (E3 rRNase), assayed mutant colicins for in vivo cytotoxicity, and tested the corresponding E3 rRNase domains for their ability to inactivate ribosome function in vitro. From 21 alanine mutants, we identified five positions where mutation resulted in a colicin with no measurable cytotoxicity (Y52, D55, H58, E62, and Y64) and four positions (R40, R42, E60, and R90) where mutation caused a significant reduction in cytotoxicity. Mutations that were found to have large in vivo and in vitro effects were tested for structural integrity through circular dichroism and fluorescence spectroscopy using purified rRNase domains. Our data indicate that H58 and E62 likely act as the acid–base pair during catalysis with other residues likely involved in transition state stabilization. Both the Y52 and Y64 mutants were found to be highly destabilized and this is the likely origin of the loss of their cytotoxicity. The identification of important active site residues and sequence alignments of known rRNase homologs has allowed us to identify other proteins containing the putative rRNase active site motif. Proteins that contained this active site motif included three hemagglutinin-type adhesins and we speculate that these have evolved to deliver a cytotoxic rRNase into eukaryotic cells during pathogenesis. Keywords: colicin E3; ribonuclease; ribosome; active site; mutagenesis Colicins are plasmid-encoded multidomain protein antibi- to aid in competition for limited nutrient resources, but has otics that are produced by and are active against Escherichia also been implicated in the maintenance of biodiversity coli and closely related bacteria. Production of the toxin is (Kerr et al. 2002) and in bacterial adaptation (Walker et al. induced under conditions that cause damage to the bacterial 2004). Most colicins characterized to date possess either a chromosome and forms part of the bacterial SOS response pore-forming activity that causes depolarization of the cy- (Walker 1996). Accordingly, colicin production is believed toplasmic membrane or a lethal nuclease activity that spe- cifically targets rRNA, tRNA, or chromosomal DNA. The DNase-type colicins (E2, E7, E8, and E9) kill cells through Reprint requests to: Colin Kleanthous, Department of Biology, Univer- sity of York, York YO10 5YW, UK; e-mail: [email protected], fax: +44 nonspecific, magnesium-dependent cleavage of chromo- (0)1904 328825. somal DNA (Pommer et al. 2001; Walker et al. 2002). 3These authors contributed equally to this work. These enzymes belong to the HNH family of endonucleases Abbreviations: E3 rRNase, the ribonuclease domain of colicin E3; Im3, immunity protein of colicin E3; E9 DNase, the endonuclease domain of that includes the caspase-activated DNases, responsible for colicin E9; Im9, immunity protein of colicin E9; Col−, a noncytotoxic the degradation of chromatin in eukaryotic apoptosis colicin phenotype; KPi, potassium phosphate; CD, circular dichroism; (Walker et al. 2002; Scholz et al. 2003). The RNase-type ␭max, wavelength of maximum fluorescence intensity. Article published online ahead of print. Article and publication date are at colicins D and E5 cleave specific, albeit different, tRNA http://www.proteinscience.org/cgi/doi/10.1110/ps.04658504. species (Ogawa et al. 1999; Tomita et al. 2000), and colicin Protein Science (2004), 13:000–000. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society 1 Walker et al. E3 (and by homology colicins E4 and E6 and cloacin DF13) Here we extend this work to assign unambiguously active specifically cleaves at the A-site of the 16S rRNA, between site residues through extensive site-directed mutagenesis bases A1493 and G1494. This inactivates the ribosome and and in vivo cytotoxicity assays by using purified mutant arrests protein synthesis, inducing a cold shock-like tran- colicins. Mutant rRNase domains were tested in in vitro scriptional response prior to cell death (Bowman et al. 1971; ribosome inactivation assays and their structural integrity Walker et al. 2004). and stability assessed through spectroscopic methods. Our Cleavage of 16S rRNA by colicin E3 has been reported to findings support the idea that the rRNase activity of colicin occur only in the context of the intact 70S ribosome (Boon E3 is mediated in a similar way to that of barnase and other 1972), although other studies indicate that at high concen- members of the T1 ribonuclease family, with H58 and E62 trations of the toxin, both isolated 30S subunits (Ohno- of E3 rRNase acting as the acid–base pair. We identify other Iwashita and Imahori 1977) and even naked 16S rRNA residues, the mutations of which abolish or significantly (Ohno and Imahori 1978) are cleaved, although in the latter reduce cytotoxicity without significant perturbation of the case with a lack of specificity. Colicin E3 is cytotoxic only rRNase structure, giving a fuller picture of the arrangement against E. coli and closely related bacteria because more of the E3 rRNase active site. In addition, we identified a distantly related bacteria lack a suitable receptor and/or further class of mutations that led to a loss of cytotoxicity translocation system for uptake of the colicin. However, in through destabilization of the rRNase structure. Defining vitro colicin E3 is capable of inactivating ribosomes from the E3 rRNase active site has allowed us to identify a mini- distantly related bacterial species (Sidikaro and Nomura mal catalytic motif used to identify other E3-like rRNase 1973). Further to this, colicin E3 has been shown to inac- domains involved in biological processes such as plant– tivate chloroplast ribosomes from Euglena gracilis (Steege microbe interactions. et al. 1982) and has a cytostatic activity against leukemic cells (Smarda et al. 2001). Like other cytotoxic microbial Results ribonucleases, colicin E3 is of potential interest as a novel chemotherapeutic agent (for review, see Makarov and Ilin- Identification of Col− mutants skya 2003). The nuclease-type colicins are coexpressed and released Mutant colicin E3 proteins were purified in complex with in complex with a specific immunity protein that binds with the immunity protein Im3 and tested for cytotoxicity against high affinity to the cytotoxic domain and abolishes its en- colicin-sensitive E. coli cells in an agar plate spot test assay zymatic activity. The equilibrium dissociation constant (Kd) (Fig. 1). In this assay, serial fivefold dilutions of the toxin for both the colicin E3-Im3 and colicin E9-Im9 complexes are spotted onto a growing lawn of E. coli cells and cyto- has been measured as 10−14 M in 200 mM salt (Wallis et al. toxicity indicated by a clear or hazy zone in the cell lawn. 1995; Walker et al. 2003). However, the affinity of the For this in vivo activity assay, the intact colicin E3-Im3 complex formed between the isolated rRNase domain and complex was used because it displays a substantially greater Im3 is two orders of magnitude weaker (10−12 M) and this cytotoxic activity than the uncomplexed colicin (Walker et reflects additional weak interactions between Im3 and the al. 2003). We initially targeted residues identified by Carr et translocation domain of the toxin as revealed by the struc- al. (2000) as likely involved in catalysis or substrate binding ture of the colicin E3-Im3 complex (Soelaiman et al. 2001). through a structural similarity of the putative E3 rRNase Structures for the E3 rRNase domain Im3 complex and the active site with the active site of barnase. In total, 21 mutant full-length colicin E3-Im3 complex have been solved at 2.4 colicin complexes were purified and tested for cytotoxicity Å and 3.0 Å, respectively (Carr et al. 2000; Soelaiman et al. by this method. 2001). The location of the active site of colicin E3 was Spot tests showed that the mutant colicins Y52A, D55A, originally predicted through the similar structural arrange- H58A, E62A, and Y64A are devoid of cytotoxic activity ment of H58 and E62 of E3 rRNase with the catalytic resi- and the activity of the R40A, R42A, E60A, and R90A mu- dues of the nonspecific ribonuclease barnase (Carr et al.
Load more

Recommended publications
  • Colicin E2 Is a DNA Endonuclease
    Proc. Nati. Acad. Sci. USA Vol. 73, No. 11, pp. 3989-S993'November 1976 Biochemistry Colicin E2 is a DNA endonuclease (colicin-E2-immunity protein/colicin E3/colicin-E3-immunity protein) KLAUS SCHALLER AND MASAYASU NOMURA University of Wisconsin, Institute for Enzyme Research, 1710 University Avenue, Madison, Wisc. 53706 Communicated by Henry Lardy, September 10, 1976 ABSTRACT Colicin E2 purified by conventional methods immunity protein (20). These authors demonstrated that the contains a tightly bound low-molecular-weight protein, as has immunity protein could be separated from the E3 protein by been found with purified colicin E3 [Jakes, N. & Zinder, N. D. preparative electrophoresis in sodium dodecyl sulfate/poly- (1974) Proc. Nat. Acad. Sci. USA 71, 3380-33841. Such E2 preparations do not cause DNA cleavage in vitro. After sepa- acrylamide gels and that E3 protein (called "ES*") free of ration from the low-molecular-weight protein, colicin E2 re- immunity protein was much more active than the "complexed" tained the original in vivo killing activity, and in addition ES preparation in ribosome inactivation in vitro (20). They also showed a high activity in vitro in cleaving various DNA mole- noted the presence of a small-molecular-weight protein in cules, such as a ColE1 hybrid plasmid and DNAs from Esche- highly purified E2 preparations (cited in ref. 20). From the richia coli, X phage, 4X174 phage, and simian virus 40. The low-molecular-weight protein ("E2-immunity protein") specif- analogy of the colicin E3-immunity protein complex, they ically prevented this in vitro DNA cleavage reaction, i.e., had inferred that this small-molecular-weight protein is probably an "immunity function." The results demonstrate that colicin the E2-immunity protein.
    [Show full text]
  • Broad and Efficient Control of Major Foodborne Pathogenic Strains Of
    Broad and efficient control of major foodborne PNAS PLUS pathogenic strains of Escherichia coli by mixtures of plant-produced colicins Steve Schulza,1, Anett Stephana,1, Simone Hahna,1, Luisa Bortesia,2, Franziska Jarczowskib, Ulrike Bettmannb, Anne-Katrin Paschkeb, Daniel Tuséc, Chad H. Stahld, Anatoli Giritcha,3, and Yuri Glebaa aNomad Bioscience GmbH, Biozentrum Halle, D-06120 Halle (Saale), Germany; bIcon Genetics GmbH, Biozentrum Halle, D-06120 Halle (Saale), Germany; cDT/Consulting Group, Sacramento, CA 95818; and dDepartment of Animal and Avian Sciences, University of Maryland, College Park, MD 20742 Edited by Charles J. Arntzen, Arizona State University, Tempe, AZ, and approved August 7, 2015 (received for review July 7, 2015) Enterohemorrhagic Escherichia coli (EHEC) is one of the leading involve heating or organic acids, which can adversely modify the causes of bacterial enteric infections worldwide, causing ∼100,000 taste and quality of the products. Currently approved bacteriophage illnesses, 3,000 hospitalizations, and 90 deaths annually in the United mixtures enable narrow and specific control of O157:H7 but not of States alone. These illnesses have been linked to consumption of other pathogenic strains (4). Use of traditional antibiotics for the contaminated animal products and vegetables. Currently, other than treatment of food is not appropriate and should be considered thermal inactivation, there are no effective methods to eliminate unacceptable, particularly due to the increase of antibiotic re- pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial sistance seen among E. coli strains found in food (5). Among proteins, produced by E. coli strains that kill or inhibit the growth of nonantibiotic antibacterials, several colicins have been shown to be other E.
    [Show full text]
  • Colicin E1 Fragments Potentiate Antibiotics by Plugging Tolc
    bioRxiv preprint doi: https://doi.org/10.1101/692251; this version posted July 4, 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. 1 Colicin E1 Fragments Potentiate Antibiotics by 2 Plugging TolC 3 4 S. Jimmy Budiardjoa, Jacqueline J. Deayb, Anna L. Calkinsc, Virangika K. Wimalasenab, 5 Daniel Montezano b, Julie S. Biteenc and Joanna S.G. Sluskya,b,1 6 7 aCenter for Computational Biology, The University of Kansas, 2030 Becker Dr., 8 Lawrence, KS 66045-7534 bDepartment of Molecular Biosciences, The University of 9 Kansas, 1200 Sunnyside Ave. Lawrence KS 66045 cDepartment of Chemistry, 10 University of Michigan, Ann Arbor MI 48109-1055 11 12 13 14 15 1To whom correspondence may be addressed. E-mail: [email protected] 785-864-6519 16 1 bioRxiv preprint doi: https://doi.org/10.1101/692251; this version posted July 4, 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. 17 Abstract 18 The double membrane architecture of Gram-negative bacteria forms a barrier 19 that is effectively impermeable to extracellular threats. Accordingly, researchers have 20 shown increasing interest in developing antibiotics that target the accessible, surface- 21 exposed proteins embedded in the outer membrane. TolC forms the outer membrane 22 channel of an antibiotic efflux pump in Escherichia coli. Drawing from prior observations 23 that colicin E1, a toxin produced by and lethal to E.
    [Show full text]
  • Isolation of Catenated and Replicating DNA Molecules of Colicin Factor El from Minicells (E
    Proc. Nat. Acad. Sci. USA Vol. 68, No. 11, pp. 2839-2842, November 1971 Isolation of Catenated and Replicating DNA Molecules of Colicin Factor El from Minicells (E. coli/sucrose gradients/ethidium bromide/CsCl/electron microscopy) JOSEPH INSELBURG AND MOTOHIRO FUKE* Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755; and Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115 Communicated by Luigi Gorini, September 14, 1971 ABSTRACT Various catenated and replicating mole- at 37°C) of minicells previously incubated in spheroplast cules of colicin El isolated from minicells have been medium was introduced into the DNA extraction procedure identified in regions of neutral sucrose density gradients or cesium chloride-ethidium bromide density gradients. before the addition of Sarkosyl NL30 and subsequent phenol These colicin molecules have been found in those regions extraction (2, 11). Neutral sucrose density gradient sedimen- of the gradient where pulse-labeled DNA accumulates. tation analysis (2) and cesium chloride-ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridium bromide) den- Adler et al. (1) isolated a mutant of Escherichia coli, P678-54, sity-gradient analysis of DNA (2, 12) and electron that produced significant numbers of small, DNA-less progeny microscope techniques (7, 13, 14) were also described in ("minicells") under normal growth conditions. Subsequent detail. Buffer solutions used are Tris-saline (TS) 0.05 M work has shown that if plasmid or episomal DNAs are carried Tris-0.05 M NaCl (pH 8.0); Tris-EDTA-Saline (TES), by that mutant, they can segregate into (2-6) and replicate which is TS + 5 mM EDTA; and saline-sodium citrate in (2, 6, 7) the minicells.
    [Show full text]
  • Structural and Functional Alterations of a Colicin-Resistant Mutant of Ompf
    Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10675-10679, October 1994 Biophysics Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli (bacteroi sentlty/coe N/porin channel/x-ray analysis) DENIS JEANTEUR*, TILMAN SCHIRMERt, DIDIER FOUREL0, VALERIE SIMONETt, GABRIELE RUMMEL§, CHRISTINE WIDMER§, JURG P. ROSENBUSCH§, FRANC PATTUS*¶, AND JEAN-MARIE PAGtSfII *European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012 Heidelberg, Germany; Departments of tStructural Biology and *Microbiology, Biozentrum, University of Basel, CH4056, Basel, Switzerland; and *Unit6 Propre de Recherche 9027, Centre de Biochimie et de Biologie Moleculaire, Centre National de la Recherche Scientifique, 31 Chemin Joseph-Aiguier, B.P. 71, Marseille Cedex 20, France Communicated by Eugene P. Kennedy, July 7, 1994 ABSTRACT A strain of Escherichia coli, selected on the residues that protrude from the barrel wall near the threefold basis of Its resistance to colicin N, reveals distinct structural molecular axis, faces two acidic side chains located on L3. and functional alterations in unspecific OmpF porin. A single This establishes a strong electrostatic field parallel to the mutation [Gly-119 -- Asp (G119D)] was identified in the membrane plane (11). Of the four colicin N-resistant point internal loop L3 that contributes critically to the formation of mutations that have recently been isolated and characterized the constriction inside the lumen of the pore. X-ray structure in OmpF porin (7), three are located on the external loops, analysis to a resolution of 3.0 A reveals a locally altered presumably impairing binding ofthe toxin. The fourth is aGly peptide backbone, with the side chain of residue Asp-119 Asp substitution at position 119 (G119D), located in loop protruding into the channe, causing the area of the constric- L3, far from the external surface of the molecule.
    [Show full text]
  • Reconstitution of Membrane Proteins Into Model Membranes: Seeking Better Ways to Retain Protein Activities
    Int. J. Mol. Sci. 2013, 14, 1589-1607; doi:10.3390/ijms14011589 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities Hsin-Hui Shen 1,2,*, Trevor Lithgow 1 and Lisandra L. Martin 2 1 Department of Biochemistry and Molecular Biology, Monash University, Melbourne 3800, Australia; E-Mail: [email protected] 2 School of Chemistry, Monash University, Clayton, VIC 3800, Australia; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +61-3-9545-8159. Received: 20 December 2012; in revised form: 9 January 2013 / Accepted: 10 January 2013 / Published: 14 January 2013 Abstract: The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity.
    [Show full text]
  • Colicin M for the Control of Ehec Colicin M Zur Kontrolle Von Ehec Colicine M Pour Le Contrôle De Ehec
    (19) *EP003097783B1* (11) EP 3 097 783 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A01N 63/02 (2006.01) A01P 1/00 (2006.01) 13.11.2019 Bulletin 2019/46 (21) Application number: 15181133.8 (22) Date of filing: 14.08.2015 (54) COLICIN M FOR THE CONTROL OF EHEC COLICIN M ZUR KONTROLLE VON EHEC COLICINE M POUR LE CONTRÔLE DE EHEC (84) Designated Contracting States: • L.SARADA NANDIWADA ET AL: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB "Characterization of an E2-type colicin and its GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO application to treat alfalfa seeds to reduce PL PT RO RS SE SI SK SM TR Escherichia coli O157:H7", INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, vol. 93, no. (30) Priority: 26.05.2015 US 201562166379 P 3, 1 June 2004 (2004-06-01), pages 267-279, XP055226209, NL ISSN: 0168-1605, DOI: (43) Date of publication of application: 10.1016/j.ijfoodmicro.2003.11.009 30.11.2016 Bulletin 2016/48 • H. TOSHIMA ET AL: "Enhancement of Shiga Toxin Production in Enterohemorrhagic (73) Proprietor: Nomad Bioscience GmbH Escherichia coli Serotype O157:H7 by DNase 80333 München (DE) Colicins", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 73, no. 23, 1 December 2007 (72) Inventors: (2007-12-01), pages 7582-7588, XP055226172, US • GIRITCH, Anatoli ISSN: 0099-2240, DOI: 10.1128/AEM.01326-07 06108 Halle (DE) • DATABASE MEDLINE [Online] US NATIONAL • HAHN, Simone LIBRARY OF MEDICINE (NLM), BETHESDA, MD, 06217 Merseburg (DE) US; 2011, ETCHEVERRÍA A I ET AL: "Reduction • SCHULZ, Steve of Adherence of E.
    [Show full text]
  • Channel Formation by Antiapoptotic Protein Bcl-2
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 5113–5118, May 1997 Cell Biology Channel formation by antiapoptotic protein Bcl-2 SHARON L. SCHENDEL*†,ZHIHUA XIE*†,MYRTA OBLATT MONTAL†‡,SHIGEMI MATSUYAMA*, MAURICIO MONTAL‡§, AND JOHN C. REED*§ *Program on Apoptosis and Cell Death Research, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037; and ‡Department of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0366 Communicated by Carlo M. Croce, Thomas Jefferson University, Philadelphia, PA, March 4, 1997 (received for review January 15, 1997) ABSTRACT Bcl-2 is the prototypical member of a large Bcl-2 can regulate Ca21 fluxes and protein translocation across family of apoptosis-regulating proteins, consisting of blockers membranes (10–14), has prompted speculations that Bcl-2 family and promoters of cell death. The three-dimensional structure of proteins may be involved in some aspect of either ion or protein a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the transport (1). Recently, the three-dimensional structure of Bcl- pore-forming domains of diphtheria toxin and the bacterial XL, an antiapoptotic homolog of Bcl-2, has been solved, revealing colicins, prompting exploration of whether Bcl-2 is capable of striking similarity to the pore-forming domains of diphtheria forming pores in lipid membranes. Using chloride efflux from toxin (DT) and the bacterial colicins (15). KCl-loaded unilamellar lipid vesicles as an assay, purified re- Bcl-XL, DT, and the colicins A and E1 all contain a pair of combinant Bcl-2 protein exhibited pore-forming activity with central hydrophobic a-helices, arranged in a hairpin structure properties similar to those of the bacterial toxins, diphtheria that is surrounded by 5–8 amphipathic a-helices.
    [Show full text]
  • Molecular Mechanisms of Colicin Evolution’
    Molecular Mechanisms of Colicin Evolution’ Margaret A. Riley Department of Biology, Yale University This review explores features of the origin and evolution of colicins in Escherichia coli. First, the evolutionary relationships of 16 colicin and colicin-related proteins are inferred from amino acid and DNA sequence comparisons. These comparisons are employed to detail the evolutionary mechanisms involved in the origin and diversification of colicin clusters. Such mechanisms include movement of colicin plasmids between strains of E. coli and subsequent plasmid cointegration, trans- position- and recombination-mediated transfer of colicin and related sequences, and rapid diversification of colicin and immunity proteins through the action of positive selection. The wealth of information contained in colicin sequence com- parisons makes this an ideal system with which to explore molecular mechanisms of evolutionary change. Introduction Colicins are toxic proteins produced by and active against Escherichia coli and related bacteria. Nineteen colicins have been described in E. coli, distinguished by the absence of cross-immunity between the producing strains (Fredericq 1957; Nomura 1967; Pugsley 1984, 1985). Although colicins differ in their precise mode of killing, they share ( 1) a similar genetic structure, including the usual presence of three colicin- related genes- a colicin, a lysis, and an immunity gene, termed a “colicin cluster”; (2) the lethality of colicin release from the cell; (3) the specific protection afforded by the immunity protein; and (4) carriage on plasmids (Hardy 1975; Pugsley 1984; Luria and Suit 1987). The biochemistry and molecular biology of colicins have been studied in great detail (reviewed in Konisky 1982; Luria and Suit 1987).
    [Show full text]
  • Analysis of the Genome and Outer Membrane Proteome
    pathogens Article How Bacteria Change after Exposure to Silver Nanoformulations: Analysis of the Genome and Outer Membrane Proteome Anna K˛edziora 1,* , Mateusz Speruda 1, Maciej Wernecki 1 , Bartłomiej Dudek 1, Katarzyna Kapczynska 2 , Eva Krzyzewska˙ 2 , Jacek Rybka 2 and Gabriela Bugla-Płosko ´nska 1,* 1 Department of Microbiology, Faculty of Biological Sciences, University of Wroclaw, 51-148 Wroclaw, Poland; [email protected] (M.S.); [email protected] (M.W.); [email protected] (B.D.) 2 Department of Immunology of Infectious Diseases, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland; [email protected] (K.K.); [email protected] (E.K.); [email protected] (J.R.) * Correspondence: [email protected] (A.K.); [email protected] (G.B.-P.); Tel.: +487-1375-6323 (A.K.) Abstract: Objective: the main purpose of this work was to compare the genetic and phenotypic changes of E. coli treated with silver nanoformulations (E. coli BW25113 wt, E. coli BW25113 AgR, E. coli J53, E. coli ATCC 11229 wt, E. coli ATCC 11229 var. S2 and E. coli ATCC 11229 var. S7). Silver, as the metal with promising antibacterial properties, is currently widely used in medicine and the biomedical industry, in both ionic and nanoparticles forms. Silver nanoformulations are usually Citation: K˛edziora,A.; Speruda, M.; considered as one type of antibacterial agent, but their physical and chemical properties determine Wernecki, M.; Dudek, B.; Kapczynska, the way of interactions with the bacterial cell, the mode of action, and the bacterial cell response K.; Krzyzewska,˙ E.; Rybka, J.; Bugla-Płosko´nska,G.
    [Show full text]
  • Inhibition of a Ribosome-Inactivating Ribonuclease: the Crystal Structure Of
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Research Article 949 Inhibition of a ribosome-inactivating ribonuclease: the crystal structure of the cytotoxic domain of colicin E3 in complex with its immunity protein Stephen Carr1, Daniel Walker2, Richard James2, Colin Kleanthous2 and Andrew M Hemmings1,2* Background: The cytotoxicity of most ribonuclease E colicins towards Addresses: Colicin Research Group, 1School of Escherichia coli arises from their ability to specifically cleave between bases Chemical Sciences, University of East Anglia, 2 1493 and 1494 of 16S ribosomal RNA. This activity is carried by Norwich NR4 7TJ, UK and School of Biological Sciences, University of East Anglia, Norwich the C-terminal domain of the colicin, an activity which if left unneutralised NR4 7TJ, UK. would lead to destruction of the producing cell. To combat this the host E. coli cell produces an inhibitor protein, the immunity protein, which forms *Corresponding author. a complex with the ribonuclease domain effectively suppressing its activity. E-mail: [email protected] Key words: crystal structure, immunity protein, Results: We have solved the crystal structure of the cytotoxic domain of the ribonuclease colicin, ribosome inactivation ribonuclease colicin E3 in complex with its immunity protein, Im3. The structure of the ribonuclease domain, the first of its class, reveals a highly twisted central Received: 18 April 2000 Revisions requested: 21 June 2000 β-sheet elaborated with a short N-terminal helix, the residues of which form a Revisions received: 11 July 2000 well-packed interface with the immunity protein.
    [Show full text]
  • The Role of the Bcl-2 Family in the Regulation of Outer Mitochondrial Membrane Permeability
    Cell Death and Differentiation (2000) 7, 1182 ± 1191 ã 2000 Macmillan Publishers Ltd All rights reserved 1350-9047/00 $15.00 www.nature.com/cdd Review The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability 1 ,1 MH Harris and CB Thompson* Introduction 1 Abramson Family Cancer Research Institute, University of Pennsylvania, Mitochondria became the subject of intensive scientific Philadelphia, PA 19104, USA investigation in the mid-twentieth century when their role as * Corresponding author: CB Thompson, Abramson Family Cancer Research cellular energy producers was discovered. This culminated in Institute, 450 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160, USA. the discovery and characterization of oxidative phosphoryla- E-mail: [email protected] tion as the mechanism by which most cellular ATP is Received 29.6.00; accepted 14.9.00 produced. Free exchange of substrate and ATP/ADP Edited by G Kroemer between mitochondria and the cytosol was proposed to provide the homeostatic mechanism by which glycolysis and oxidative phosphorylation are coupled to maintain the Abstract intracellular ATP/ADP ratio.1,2 In the last few years, there has been a resurgence of interest in mitochondria following Mitochondria are well known as sites of electron transport and the discovery that mitochondria play a crucial role in the generators of cellular ATP. Mitochondria also appear to be regulation of programmed cell death, or apoptosis.3±9 A sites of cell survival regulation. In the process of programmed number of molecules involved in the execution of apoptosis cell death, mediators of apoptosis can be released from normally reside in mitochondria, safely sequestered from their mitochondria through disruptions in the outer mitochondrial targets and co-factors.10 ± 16 Following an apoptotic stimulus, membrane; these mediators then participate in the activation these proteins can be released from mitochondria and initiate of caspases and of DNA degradation.
    [Show full text]