Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Antimicrobial Agents and Chemotherapy
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Mechanisms of Resistance

Transcriptional and Translational Control of the mlr Operon, Which Confers Resistance to Seven Classes of Protein Synthesis Inhibitors

Lisa K. Smith, Alexander S. Mankin
Lisa K. Smith
Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 S. Ashland Ave., Chicago, Illinois 60607
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alexander S. Mankin
Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 S. Ashland Ave., Chicago, Illinois 60607
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: shura@uic.edu
DOI: 10.1128/AAC.01583-07
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    Organization of the monocistronic operon containing cfr from plasmid pSCFS1 (A) and organization of the mlr operon containing the erm(B) and the cfr genes in the chromosome of MRSA isolate CM05 (B). In pSCFS1, cfr is preceded by two overlapping ORFs (shown as ORF 1 and ORF 2) and a putative promoter. In CM05, the insertion of Tn917, which carries erm(B) and its regulatory elements, disrupts ORF 1 and the putative cfr promoter. An additional 35-nucleotide sequence present in pSCFS1 immediately after ORF 2 (indicated by brackets) is absent from CM05. (C) The scheme of the RT-PCR experiment for the detection of a transcript spanning the erm(B)-cfr intergenic spacer is shown. cDNA was synthesized from primer c2, which is complementary to the 5′ proximal region of the cfr gene, and was subsequently used as a template for PCR amplification with primers e2 and c3.

  • FIG. 2.
    • Open in new tab
    • Download powerpoint
    FIG. 2.

    Maps of plasmids pMS2 and pLXM1 used in this study.

  • FIG. 3.
    • Open in new tab
    • Download powerpoint
    FIG. 3.

    Primer extension analysis of the erm(B) and cfr transcription start sites. Total RNA isolated from S. aureus RN4220 cells transformed with plasmid pMS2 was used as the template. Primers e1 and c1 were used in the experiment and were complementary to the 5′ proximal region of the erm(B) ORF (lane e1) or the 5′ proximal region of cfr ORF (lane c1). Extension products carried on pMS2 DNA were separated on a 6% denaturing polyacrylamide gel along with sequencing reactions with primer e1 (lanes G, A, T, and C). The band in lane e1 that indicates the transcription start site from the Perm promoter corresponds to a 106-nucleotide-long cDNA product. 32P-labeled 1,443-bp and 495-bp DNA markers (lane M) indicate the approximate sizes of the putative cDNA products extending from primer c1 and terminating at the erm(B) transcription start site or a putative site within the erm(B)-cfr spacer, respectively.

  • FIG. 4.
    • Open in new tab
    • Download powerpoint
    FIG. 4.

    Effect of Perm promoter deletion on expression of erm(B) and cfr genes. (A) Deletions engineered in plasmid pMS2, yielding plasmids pErmBΔ1, pErmBΔ2, and pErmBΔ3. (B) Disk diffusion assay. The plates carried lawns of S. aureus RN4220 cells transformed with plasmid pMS2, pLI50, or pErmBΔ1. Antibiotic disks contained 20 μg erythromycin (disks E) or 30 μg florfenicol (disks F). (C) Primer extension analysis of modification of A2058 and A2503 in 23S rRNA in cells transformed with plasmid pMS2, pLI50, or pErmBΔ1.

  • FIG. 5.
    • Open in new tab
    • Download powerpoint
    FIG. 5.

    Mapping of the translation start site of the cfr gene. (A) The 5′ terminal nucleotide sequence showing two putative initiator codons of the cfr gene and the mutations engineered in plasmids pMS2X1 and pMS2X2 plasmids; (B) antibiotic sensitivities of S. aureus RN4220 cells transformed with pMS2, empty vector pLI50, or plasmids pMS2X1 and pMS2X2; (C) primer extension analysis of the extent of Cfr-dependent modification of A2503 in S. aureus cells transformed with pMS2, pMS2X1, pMS2X2, or the empty vector.

  • FIG. 6.
    • Open in new tab
    • Download powerpoint
    FIG. 6.

    Differences in nucleotide sequences of the leader peptide ORF in transposon Tn917 [inducible erm(B)] (32) and the mlr operon [noninducible erm(B)]. The nucleotide sequence of the Tn917 erm(B) leader ORF is shown, and the nucleotide changes observed in mlr are indicated. The corresponding changes in the amino acid sequence of the encoded leader peptide are shown under the nucleotide sequence.

  • FIG. 7.
    • Open in new tab
    • Download powerpoint
    FIG. 7.

    (A) Relative positions of A2058, A2503, and tylosin in the nascent peptide exit tunnel of the ribosome. The segment of 23S rRNA from positions 2058 to 2061 is shown as sticks (beige). The adenine bases of the residues 2058 and 2503 are highlighted in red. Tylosin is shown in orange, with the desosamine-mycarose side chain highlighted in olive. The position of the tRNA CCA 3′ end (cyan) and the attached amino acid (blue) in the P site of the peptidyltransferase center is shown as a landmark. The figure was prepared with the PyMol program (6) on the basis of the structures of antibiotic complexes of the Haloarcula marismortui large ribosomal subunits (PDB accession numbers 1K9M and 1YI2) (10, 37). (B) The structure shown in panel A was rotated by ca. 90 degrees clockwise around the y axis to illustrate the proximity of the desosamine-mycarose side chain of tylosin to A2503. The shortest distances (in Å) between the drug and the A2503 base are indicated.

Tables

  • Figures
  • TABLE 1.

    Primers used in this study

    PrimerSequence
    atgx1AAATTGATTCTTAACTAGAGCAAATTGTGAAAGGCAGAAAGAAATGAATTTTAATAATAAAACAAAGTATG
    atgx2CTTAACTAGAGCAAATTGTGAAAGGATGAAAGAACAGAATTTTAATAATAAAACAAAGTATGGTAAAATAC
    c1TTCCTGTATTTTACCATACTTTG
    c2ACATGATATAACTTCCCTG
    c3CTATAATCAGGCTCATTATTACTT
    e1GTCTGTTTCAAAACAGTAGATG
    e2GATTGTTGAAGAAGGATTCTAC
    e3GTAATTAAGAAGGAGGGATTCG
    e4GTTATCTATTATTTAACGGGAGG
    pdel2GCGCAAGCTTAGGTATAGGGCACCTCTAATA
    pdel3GCGCAAGCTTGTATGTTTTGACTTTCGGCAC
    permBdel1GGGCAAGCTTCGTCATGTTGGTATTCCAAAT
    RevpMS2GGCTAATAGGGAATACATTACCA
    SAL2060GTAAAGCTCCACGGGGTC
    SAL2507CCAGGATGCGATGAGCCG
    tRNADirATCCGGCCCCCGCAACC
  • TABLE 2.

    Antibiotic sensitivity profiles of clinical MRSA isolate CM05 and S. aureus laboratory strain RN4220 transformed with mlr operon-expressing plasmid pMS2 or empty vector pLI50

    AntibioticMIC (μg/ml)
    RN4220(pLI50)RN4220(pMS2)CM05
    ChloramphenicolNDaND64
    Clindamycin<1.0>1,024>1,024
    Erythromycin0.5>1,024>1,024
    Florfenicol4128ND
    Linezolid248
    Quinupristin-dalfopristin<0.050.52
    Tiamulin1128512
    Tylosin2>1,024ND
    • ↵ a ND, not determined.

  • TABLE 3.

    Sensitivity of S. aureus strain RN4220 transformed with various plasmids to erythromycin and florfenicol

    StrainMIC (μg/ml)
    ErythromycinFlorfenicol
    RN4220(pMS2)>1,024128
    RN4220(pLI50)a0.54
    RN4220(pErmBΔ1)b0.54
    RN4220(pMS2X1)c>1,024128
    RN4220(pMS2X2)d>1,0244
    • ↵ a Empty vector.

    • ↵ b pMS2 with deletion of erm(B) promoter.

    • ↵ c pMS2 with first AUG of cfr mutagenized to CAG.

    • ↵ d pMS2 with second AUG of cfr mutagenized to CAG.

  • TABLE 4.

    Extent of modification of A2058 and A2503 in clinical MRSA isolate CM05 and S. aureus RN4220 transformed with pMS2 or empty vector pLI50 before and after incubation with erythromycin or florfenicol

    StrainSite% Modification after treatment witha:
    No treatmentErythromycinFlorfenicol
    RN4220(pLI50)A20581NDbND
    A25030NDND
    RN4220(pMS2)A2058122320
    A250381116
    CM05A2058343532
    A2503252720
    • ↵ a The percentages shown were deduced from primer extension data and are the averages of at least two experiments.

    • ↵ b ND, not determined.

  • TABLE 5.

    Sensitivity of S. aureus RN4220 transformed with plasmids harboring erm(B) and/or cfr to clindamycin, quinupristin-dalfopristin, tylosin, josamycin, and spiramycin

    Plasmid (phenotype)aMIC (μg/ml)b
    CLIQ-DTYLJOSSPI
    pMS2 [erm(B)+cfr+]>1,0240.396,000>2,000>4,000
    pLI50 [erm(B)−cfr−]<1<0.1118
    pLXM1 [erm(B)−cfr+]1,0240.39264128
    pMS2X2 [erm(B)+cfr−]>1,024<0.12,000>2,000>4,000
    • ↵ a erm(B)+, erm(B) positive; cfr+, cfr positive; erm(B)−, erm(B) negative; cfr−, cfr negative.

    • ↵ b CLI clindamycin; Q-D, quinupristin-dalfopristin; TYL, tylosin; JOS, josamycin; SPI, spiramycin.

PreviousNext
Back to top
Download PDF
Citation Tools
Transcriptional and Translational Control of the mlr Operon, Which Confers Resistance to Seven Classes of Protein Synthesis Inhibitors
Lisa K. Smith, Alexander S. Mankin
Antimicrobial Agents and Chemotherapy Apr 2008, 52 (5) 1703-1712; DOI: 10.1128/AAC.01583-07

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Antimicrobial Agents and Chemotherapy article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Transcriptional and Translational Control of the mlr Operon, Which Confers Resistance to Seven Classes of Protein Synthesis Inhibitors
(Your Name) has forwarded a page to you from Antimicrobial Agents and Chemotherapy
(Your Name) thought you would be interested in this article in Antimicrobial Agents and Chemotherapy.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Transcriptional and Translational Control of the mlr Operon, Which Confers Resistance to Seven Classes of Protein Synthesis Inhibitors
Lisa K. Smith, Alexander S. Mankin
Antimicrobial Agents and Chemotherapy Apr 2008, 52 (5) 1703-1712; DOI: 10.1128/AAC.01583-07
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Operon
Protein Biosynthesis
Protein Synthesis Inhibitors
Transcription, Genetic

Related Articles

Cited By...

About

  • About AAC
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • AAC Podcast
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AACJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0066-4804; Online ISSN: 1098-6596