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

Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

Mark Veleba, Paul G. Higgins, Gerardo Gonzalez, Harald Seifert, Thamarai Schneiders
Mark Veleba
aCentre for Infection and Immunity, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul G. Higgins
bInstitute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gerardo Gonzalez
cLaboratorio de Investigación en Agentes Antibacterianos, Departamento de Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Harald Seifert
bInstitute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Thamarai Schneiders
aCentre for Infection and Immunity, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AAC.00456-12
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Fig 1
    • Open in new tab
    • Download powerpoint
    Fig 1

    (A) Comparison of genomic organizations of rarA (KPN_02968) locus in Klebsiella pneumoniae strain MGH 78578 to those of other Enterobacteriaceae harboring the locus. (B) Predicted phylogenetic tree of MarA-type protein homologues in Klebsiella pneumoniae determined using PhyML 3.0 (14). The amino acid sequences for MarA (KPN_01624), Rob (KPN_04851), RarA (KPN_02968), RamA (KPN_00556), SoxS (KPN_04462), and KPN_01709 were used in Muscle alignment software to generate the phylogenetic tree. All amino acid sequences were obtained from the genome sequence of MGH 78578 (NC_009648). Of note, different branch lengths denote relative amino acid identities.

  • Fig 2
    • Open in new tab
    • Download powerpoint
    Fig 2

    Nucleotide changes within the intergenic region between KPN_02968 (rarA) and KPN_02969 (oqxA). Changes observed within the intergenic region between rarA and oqxA in the different clinical strains tested are shown. The numbering scheme is based on the first nucleotide before the ATG of rarA as position 1. The transcription start site of rarA, determined by 5′ RACE analysis, is labeled TSS. The Shine-Dalgarno sequence is underlined; putative −10 and −35 promoter regions determined through Softberry analysis are shown boxed and labeled accordingly.

  • Fig 3
    • Open in new tab
    • Download powerpoint
    Fig 3

    Fold change in expression levels of rarA and oqxA among clinical isolates compared to K. pneumoniae Ecl8. All QPCR experiments were performed as outlined in Materials and Methods. For strains Ecl8Mdr1 [represented by EcL8(R)], Kp342, TS152, TS165, TS202, GC9, GC12, GC19, and GC21, fold change values were generated after normalizing to 16S levels for each strain and then using oqxA and rarA levels derived from the sensitive K. pneumoniae Ecl8 strain. To assess whether rarA could activate the expression of the efflux pump oqxAB, transcription of oqxA was assessed in Ecl8/pACrarA-1 [EcL8(P)] and Ecl8 ΔrarA/pACrarA-2 [EcL8(Δ)]. In this case, the fold change was normalized as mentioned before but calibrated against either Ecl8/pACYC177 or Ecl8ΔrarA/pACYC184.

Tables

  • Figures
  • Table 1

    List of strains used in experiments

    StrainRelevant genotype and phenotypeaReference or source
    E. coli strains
        AG100E. coli wt strain11
        AG100/pACrarA-1AG100 + pACrarA-1 (wt rarA cloned into pACYC177 (BamHI, ScaI), KanrThis work
        AG100/pACYC177AG100 + pACYC177 (Kanr, Ampr)This work
        AG100/pACrarA-2AG100 + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), CmrThis work
        AG100/pACYC184AG100 + pACYC184 (Cmr, Tetr)This work
        AG100AacrAB efflux pump-deleted strain28
        AG100A/pACrarA-2AG100A + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), CmrThis work
        AG100A/pACYC184AG100A + pACYC184 (Cmr, Tetr)This work
        MG1655E. coli K-12 laboratory isolateIn-house strain collection
        MG1655/pACrarA-1MG1655 + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), KanrThis work
        MG1655/pACYC177MG1655 + pACYC177 (Kanr, Ampr)This work
        MG1655 ΔmarAmarA-deleted strainS. B. Levy, L. McMurry
        MG1655 ΔmarA/pACrarA-1MG1655 ΔmarA + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), KanrThis work
        MG1655 ΔmarA/pACYC177MG1655 ΔmarA + pACYC177 (Kanr, Ampr)This work
        AG100 ΔsoxS Δrob ΔmarAsoxS-, rob-, marA-deleted strainS. B. Levy, L. McMurry
        AG100 ΔsoxS Δrob ΔmarA/pACrarA-2AG100 ΔsoxS Δrob ΔmarA + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), CmrThis work
        AG100 ΔsoxS Δrob ΔmarA/pACYC184AG100 ΔsoxS Δrob ΔmarA + pACYC184 (Cmr, Tetr)This work
    K. pneumoniae strains
        MGH 78578ATCC 70072119
        Ecl8K. pneumoniae wt strain8
        Ecl8/pACrarA-1Ecl8 + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), KanrThis work
        Ecl8/pACYC177Ecl8 + pACYC177 (Kanr, Ampr)This work
        Ecl8Mdr1Spontaneous MDR mutant of Ecl810
        Ecl8 ΔramAramA-deleted strain derived from Ecl827
        Ecl8 ΔramA/pACrarA-1Ecl8 ΔramA + pACrarA-1 (wt rarA cloned into pACYC177 [BamHI, ScaI]), KanrThis work
        Ecl8 ΔramA/pACYC177Ecl8 ΔramA + pACYC177 (Kanr, Ampr)This work
        Kp342K. pneumoniae isolated from maize9
        Ecl8 ΔacrABacrAB-deleted strain derived from Ecl8, KanrThis work
        Ecl8 ΔacrAB/pACrarA-2Ecl8 ΔacrAB + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), CmrThis work
        Ecl8 ΔacrAB/pACYC184Ecl8 ΔacrAB + pACYC184 (Cmr, Tetr)This work
        Ecl8 ΔrarArarA (KPN_02968)-deleted strain derived from Ecl8, KanrThis work
        Ecl8 ΔrarA/pACrarA-2Ecl8 ΔrarA + pACrarA-2 (wt rarA cloned into pACYC184 [BamHI, HindIII]), CmrThis work
        Ecl8 ΔrarA/pACYC184Ecl8 ΔrarA + pACYC184 (Cmr, Tetr)This work
        TS152, TS165K. pneumoniae clinical isolates (Turkey)27
        TS180, TS202K. pneumoniae clinical isolates (Chile)27
        GC9, GC12, GC19, GC21K. pneumoniae clinical isolate (Germany)This work
        GC9, GC12, GC19, GC21/pACoqxRGC isolates + pACoqxR (wt oqxR cloned into pACYC177 [BamHI, ScaI]), KanrThis work
        GC9, GC12, GC19, GC21/pACYC177GC isolates + pACYC177 (Kanr, Ampr)This work
    • ↵a wt, wild type; Kan, kanamycin; Amp, ampicillin; Cm, chloramphenicol; Tet, tetracycline.

  • Table 2

    List of primers used in experiments

    PrimerSequence (5′–3′)a
    Cloning
        02968FBCGGGATCCCACTATCGCGGCGATTGTA
        02968RSAAAAGTACTTCATGCGGATCGCTGACG
        02968HRCCCAAGCTTTCATGCGGATCGCTGACG
        02968BFCGGGATCCTTGCACTTTATGTGCGGT
        OqxRbamHIFCGGGATCCTTTACTTCGCAGGCTAAC
        OqxRsca1RAGTACTTCATTTTCTGGTGACGAAAA
    Deletion
        Ci-02968CCGTTCCAAGCGGCCGCAAGAGCGCATCTCGTCAGCGATCCGCATG
        Co-02968AAAAAGTCGACTCAGTCCTGACCCGGCCCATG
        Ni-02968CGCTCTTGCGGCCGCTTGGAACGGTCGCGGCCG GTTAAAAGCATC
        No-02968AAAAACTGCAGGTATGGACTAATTTATCGGTGC
        02968delchkFTCAGCCAGATGGCAACCG
        02968delchkRACCGCTTCAATGCGACCG
        Ci-acrBCGCTCTTGCGGCCGCTTGGAACGGGGCGCGCCTCTCCTGGTT
        Co-acrBAAAAACTCGAGCCACTTTTGCAAATCCGTAGAG
        Ni-acrACGCTCTTGCGGCCGCTTGGAACGGGTGTCCAATTTCAAAATGTTC
        No-acrAAAAAACTGCAGGCGTAGCGTCGGG CAGAATTG
        AcrABdelchkFGAGCGAATGTGGCAATGTGC
        AcrABdelchkRCACGGTCATTTCACCGACG
    Sequencing
        02968FBCGGGATCCCACTATCGCGGCGATTGTA
        02968RSAAAAGTACTTCATGCGGATCGCTGACG
        02698intFGACAGATCCTGAATGAT
        02698intRTGAATGTTTCCCCAGGTT
        OqxRFGTCACCAGAAAATGATTAATGCGC
        OqxRRGCCTTTGCCCGTGAAATCAG
    Q-RTPCR
        Kp02968FTGGATCGACAACCATCTTGA
        Kp02968RAAGGACTGCTGGGAGTCAAA
        Kp_16SQ_FGTTACCCGCAGAAGAAGCAC
        Kp_16SQ_RCTACGCATTTCACCGCTACA
        02969FAAGGTGCTGGTGAAGTCGAT
        02969RGGAGACGAGGTTGGTATGGA
        Kp_ramAQ_FAGCCTGGGGCGCTATATT
        Kp_ramAQ_RGTGGTTCTCTTTGCGGTAGG
        Kp_marAFTCGAGGATAACCTGGAATCG
        Kp_marARACAAATGGGCTCATTGCTC
        oqxRqpcrFTAACGAAGCCTGCTCTGCTT
        oqxRqpcrRAATGGTTCCGCTAACTCGTG
    5′ RACE
        GSP1ATGTCTGCGCAACAG
        GSP2GCTCGTCGGGCAACGGTGTC
        GSP3CCAGACGGCTATCAAGATGGTTG
    • ↵a Underlined sequences denote restriction enzyme cut sites.

  • Table 3

    Susceptibility profiles of E. coli strains transformed with pACrarA and vector-only controla

    StrainMIC (μg/ml)
    CHLOQXTETTIGNORCIP
    AG1004164< 0.5000.1250.031
    AG100/pACrarA-116328210.062
    AG100/pACYC1774164< 0.5000.1250.031
    AG100/pACrarA-2*32*20.2500.062
    AG100/pACYC184*16*< 0.50.1250.031
    AG100A*8*0.2500.0080.004
    AG100A/pACrarA-2*8*0.2500.0080.004
    AG100A/pACYC184*8*0.2500.0080.004
    AG100 ΔsoxS Δrob ΔmarA*16*0.5000.0320.031
    AG100 ΔsoxS Δrob ΔmarA/pACrarA-2*32*40.2500.062
    AG100 ΔsoxS Δrob ΔmarA/pACYC184*16*0.5000.0310.031
    MG1655416440.5000.062
    MG1655/pACrarA-116328810.125
    MG1655/pACYC177416440.5000.062
    MG1655 ΔmarA48440.2500.031
    MG1655 ΔmarA/pACrarA-1321616810.125
    MG1655 ΔmarA/pACYC17748440.2500.031
    • ↵a A pACYC177 or pACYC184 backbone was used to clone the rarA open reading frame. Depending on the resistance cassettes already in situ, the appropriate recombinant constructs would be used. Asterisks denote that MIC testing for that particular antibiotic was not done due to presence of a conflicting antibiotic resistance cassette on the plasmid and/or construct. Entries in bold denote increased MICs over those seen with wild-type/parental strains. CHL, chloramphenicol, OQX, olaquindox, TET, tetracycline, TIG, tigecycline, NOR, norfloxacin, CIP, ciprofloxacin.

  • Table 4

    Susceptibility profiles of K. pneumoniae strains transformed with pACrarA and vector-only controla

    StrainMIC (μg/ml)
    CHLOQXTETTIGNORCIP
    Ecl8416420.2500.031
    Ecl8/pACrarA-1832840.5000.062
    Ecl8/pACYC177416420.2500.031
    Ecl8 ΔramA0.58110.2500.016
    Ecl8 ΔramA/pACrarA-1432240.5000.062
    Ecl8 ΔramA/pACYC1770.58110.2500.016
    Ecl8 ΔrarA*8*0.5000.0310.016
    Ecl8 ΔrarA/pACrarA-2*64*40.2500.062
    Ecl8 ΔrarA/pACYC184*8*0.5000.0310.016
    Ecl8 ΔacrAB*2*0.250.0310.016
    Ecl8 ΔacrAB/pACrarA-2*64*0.250.0310.016
    Ecl8 ΔacrAB/pACYC184*2*0.250.0310.016
    • ↵a A pACYC177 or pACYC184 backbone was used to clone the rarA open reading frame. Depending on the resistance cassettes already in situ, the appropriate recombinant constructs would be used. Asterisks denote that MIC testing for that particular antibiotic was not done due to presence of a conflicting antibiotic resistance cassette on the plasmid and/or construct. Entries in bold denote increased MICs over those seen with wild-type/parental strains. CHL, chloramphenicol; OQX, olaquindox; TET, tetracycline; TIG, tigecycline; NOR, norfloxacin; CIP, ciprofloxacin.

  • Table 5

    Sequence analysis of OqxR and rarA/oqxA expression levels of clinical K. pneumoniae isolatesa

    StrainOqxR change(s)Expression level of rarA calibrated against Ecl8Con/Comp log2 expression level(s)b
    rarAoqxA
    152Val130→Ala6.62—6.42/—
    165Arg25→His, Val130→Ala3.77—4.76/—
    202Ala133→Thr7.37—5.64/—
    GC9Phe6→Ser9.6017.97/17.538.68/−1.4
    GC12Gln11→Leu, Asp95→Glu, V113→Ile7.9121.77/23.62.89/0.64
    GC19Frameshift Δ, aa 73-778.2421.06/20.847.6/0.74
    GC21Gln11→Leu, Asp95→Glu, Val 113→Ile8.0923.34/21.753.72/0.01
    Kp342Asn38→Thr, Asp95→Glu, Val113→Ile, His159→Leu6.2121.76/22.031.77/−0.735
    Ecl8Mdr1Frameshift Δ, aa 88-9410.4617.88/17.449.06/0.28
    • ↵a Strains whose names are underlined were complemented with pACoqxR. Boldface font denotes amino acid changes present in several strains. Expression levels of rarA calibrated against sensitive K. pneumoniae strain Ecl8 are shown.

    • ↵b Con, control data representing either the wild-type or vector-only strains (pACYC177); Comp, complementation data representing increases or decreases of rarA or oqxA expression levels in the different strains after complementation with pACOqxR. Negative values indicate reduction of oqxA levels below the levels noted in the vector-only calibrators. —, strains not complemented due to high levels of innate kanamycin resistance. aa, amino acids.

  • Table 6

    Susceptibility profiles of K. pneumoniae strains after complementation with pACoqxRa

    StrainMICs (mg/liter) for strain transformed with pACoqxR/paCYC177
    CHLOQXTETTIGNORCIP
    GC964/64768/76816/164/48/88/8
    GC1224/24768/76816/168/88/812/12
    GC19256/256768/76832/324/44/41/1
    GC2124/24768/76816/168/88/812/12
    Kp34248/64384/51232/328/84/80.375/0.500
    Ecl8Mdr132/32384/5124/41/18/80.500/0.500
    • ↵a MICs show values for strains complemented with pACoqxR/strains complemented with pACYC177. Numbers in bold indicate MIC reductions seen with the complemented strain versus the vector-only control. CHL, chloramphenicol, OQX, olaquindox, TET, tetracycline, TIG, tigecycline, NOR, norfloxacin, CIP, ciprofloxacin.

PreviousNext
Back to top
Download PDF
Citation Tools
Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae
Mark Veleba, Paul G. Higgins, Gerardo Gonzalez, Harald Seifert, Thamarai Schneiders
Antimicrobial Agents and Chemotherapy Jul 2012, 56 (8) 4450-4458; DOI: 10.1128/AAC.00456-12

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.
Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae
(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
Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae
Mark Veleba, Paul G. Higgins, Gerardo Gonzalez, Harald Seifert, Thamarai Schneiders
Antimicrobial Agents and Chemotherapy Jul 2012, 56 (8) 4450-4458; DOI: 10.1128/AAC.00456-12
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

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