skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

Abstract

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.

Authors:
ORCiD logo [1];  [2];  [3];  [4];  [5];  [3];  [6];  [7];  [3];  [6]
  1. Barcelona Institute of Science and Technology, Barcelona (Spain); Molecular Biology Institute of Barcelona, Barcelona (Spain); International Institute of Molecular and Cell Biology in Warsaw, Warsaw (Poland)
  2. Barcelona Institute of Science and Technology, Barcelona (Spain); Molecular Biology Institute of Barcelona, Barcelona (Spain); CELLS-ALBA Synchrotron Light Source, Cerdanyola del Valles (Spain)
  3. Consejo Superior de Investigaciones Cientificas, Madrid (Spain)
  4. Barcelona Institute of Science and Technology, Barcelona (Spain); Molecular Biology Institute of Barcelona, Barcelona (Spain); SLAC National Accelerator Lab., Menlo Park, CA (United States)
  5. Barcelona Institute of Science and Technology, Barcelona (Spain); Barcelona Institute of Science and Technology, Barcelona (Spain)
  6. Barcelona Institute of Science and Technology, Barcelona (Spain); Molecular Biology Institute of Barcelona, Barcelona (Spain)
  7. Barcelona Institute of Science and Technology, Barcelona (Spain); Barcelona Institute of Science and Technology, Barcelona (Spain); Univ. of Barcelona, Barcelona (Spain)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1390289
Grant/Contract Number:
AC02-76SF00515; SGR2009-1309; 2014-SGR134; 2014-SGR1530; GA No. 260644; ERC_SimDNA; BFU2008-02372/BMC; CSD-2006-23; BFU2011-22588; BIO2013-49148-C2-2-R; BFU2014-53550-P; BIO2015-69085-REDC; BIO2015-64802; CSD-2008/00013
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 114; Journal Issue: 32; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; histidine relaxase; antibiotic resistance; horizontal gene transfer; X-ray structure; Staphylococcus aureus

Citation Formats

Pluta, Radoslaw, Boer, D. Roeland, Lorenzo-Diaz, Fabian, Russi, Silvia, Gomez, Hansel, Fernandez-Lopez, Cris, Perez-Luque, Rosa, Orozco, Modesto, Espinosa, Manuel, and Coll, Miquel. Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance. United States: N. p., 2017. Web. doi:10.1073/pnas.1702971114.
Pluta, Radoslaw, Boer, D. Roeland, Lorenzo-Diaz, Fabian, Russi, Silvia, Gomez, Hansel, Fernandez-Lopez, Cris, Perez-Luque, Rosa, Orozco, Modesto, Espinosa, Manuel, & Coll, Miquel. Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance. United States. doi:10.1073/pnas.1702971114.
Pluta, Radoslaw, Boer, D. Roeland, Lorenzo-Diaz, Fabian, Russi, Silvia, Gomez, Hansel, Fernandez-Lopez, Cris, Perez-Luque, Rosa, Orozco, Modesto, Espinosa, Manuel, and Coll, Miquel. 2017. "Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance". United States. doi:10.1073/pnas.1702971114.
@article{osti_1390289,
title = {Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance},
author = {Pluta, Radoslaw and Boer, D. Roeland and Lorenzo-Diaz, Fabian and Russi, Silvia and Gomez, Hansel and Fernandez-Lopez, Cris and Perez-Luque, Rosa and Orozco, Modesto and Espinosa, Manuel and Coll, Miquel},
abstractNote = {Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOBV family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOBV relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOBV histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.},
doi = {10.1073/pnas.1702971114},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 32,
volume = 114,
place = {United States},
year = 2017,
month = 7
}

Journal Article:
Free Publicly Available Full Text
This content will become publicly available on July 24, 2018
Publisher's Version of Record

Save / Share:
  • Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 {angstrom} resolution, respectively, andmore » results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m{sup 7}G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.« less
  • MexR functions as the primary regulator of the mexAB-oprM multidrug efflux expression in Pseudomonas aeruginosa. It has been shown that MexR senses oxidative stress by interprotomer disulphide bond formation between redox-active cysteines. This oxidation induces MexR to dissociate from the promoter DNA, thus activating the transcriptional expression of efflux pump genes. In this study, we present the crystal structure of MexR in its oxidized form at a resolution of 2.1 {angstrom}. This crystal structure reveals the mechanism by which oxidative signal allosterically derepresses the MexR-controlled transcription activation.
  • No abstract prepared.
  • No abstract prepared.
  • Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI*) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of themore » A138T protein to the GAATTC site has S more favorable and H less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI* sites that structurally resemble the specific wild-type complex with GAATTC.« less