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Mechanisms of Resistance

Relative Strengths of the Class 1 Integron Promoter Hybrid 2 and the Combinations of Strong and Hybrid 1 with an Active P2 Promoter

Costas C. Papagiannitsis, Leonidas S. Tzouvelekis, Vivi Miriagou
Costas C. Papagiannitsis
1Laboratory of Bacteriology, Hellenic Pasteur Institute
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Leonidas S. Tzouvelekis
2Department of Microbiology, Medical School, University of Athens, Athens, Greece
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Vivi Miriagou
1Laboratory of Bacteriology, Hellenic Pasteur Institute
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  • For correspondence: miriagou@pasteur.gr
DOI: 10.1128/AAC.00912-08
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Tables

  • TABLE 1.

    Characteristics of class 1 integrons with the uncommon promoter sequence Pc hybrid 2 and the combinations of P2 with Pc hybrid 1 and Pc strong

    PromoterIntegron designationGene cassette(s) (5′-3′)SpeciesLocationGenBank accession no.Reference or source
    Pc hybrid 2In-t4 aadB, orfE, orf416, cmlA7 Klebsiella pneumoniae Plasmid pCC416 (IncA/C2) AJ704863 4
    aadB, int, cmlA1 K. pneumoniae Plasmid pKPN5 (IncF; 88.6 kb) CP000650 McClelland et al., unpublished
    aacA1, orfGa E. coli Plasmid pCMXR1 AB061794 6
    In7 bla VIM-2, aacA4 Pseudomonas aeruginosa Chromosome AM180753 8
    aacA7, blaVIM-2, dfrB5, aacC-A5 P. aeruginosa Unknown DQ522233 Shevchenko and Edelstein, unpublished
    In70 bla VIM-1, aacA4, aphA15, aadA1 Achromobacter xylosoxidans Plasmid pAX22 (30 kb) AJ278514 14
    Tn402-like oxa2, aacA4, aadA1Uncultured bacteriumPlasmid pTB11 (IncP; 68.9 kb) AJ744860 16
    aacA7, catB3, aadB, oxa2, orfD Enterobacter aerogenes Plasmid pBWH301 U13880 3
    aacA4 b Salmonella enterica serovar TyphimuriumPlasmid pST2 S45954 10
    Pc hybrid 1+P2 aadA2 E. coli Plasmid pMUR050 (IncN; 56.6 kb) AY522431 7
    Pc strong+P2In-h12 aacA7, blaVIM-12, aacA7 K. pneumoniae Plasmid p2873 (70 kb) DQ143913 12
    • ↵ a aacA1 and orfG occur in the same cassette.

    • ↵ b Only the first gene cassette has been characterized.

  • TABLE 2.

    Oligonucleotide primers

    Primer function and nameSequence (5′-3′)UsageGenBank accession no. (nt) or source
    Cloning and real-time PCR
        Int-FCGTTCCATACAGAAGCTGCloning of the In58-G1 integron EU598463 (1-18)
        3-CSAAGCAGACTTGACCTGACloning of the In58-G1 integron EU598463 (1625-1641)
        Ven-8PGTAATCTCTCTCCTGGGCTRT-PCR for blaGES-1 EU598463 (1343-1361)
        Ven-12CCCCAAGGAGAGATCGTRT-PCR for blaGES-1 EU598463 (744-762)
        Aph-FGGAAACGTCTTGCTCGAGGRT-PCR for aphA1 X06402 (1940-1958)
        Aph-RACTGAATCCGGTGAGAATGGRT-PCR for aphA1 X06402 (2480-2500)
    Site-directed mutagenesis
        P1st-FaAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGConstruction of the strong promoter from the weak promoterThis study
        P1st-RaCATTACAGTTTACGAACCGAACAGGCTTATGTCAACTGGGTTConstruction of the strong promoter from the weak promoterThis study
        P1h1-FbGCACGAACCCAGTGGACATAAGCCTGTTCGConstruction of hybrid 1 from the weak promoter (−35 box)This study
        P1h1-RbCGAACAGGCTTATGTCCACTGGGTTCGTGCConstruction of hybrid 1 from the strong promoter (−35 box)This study
        P1h2-FbGCACGAACCCAGTTGACATAAGCCTGTTCGConstruction of hybrid 2 from the weak promoter (−35 box)This study
        P1h2-RbCGAACAGGCTTATGTCAACTGGGTTCGTGCConstruction of hybrid 2 from the weak promoter (−35 box)This study
        P2ac-FcTGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGAConstruction of the P2 promoterThis study
        P2ac-RcTGCCCGAGGCATAGACTGTACCCCAAAAAAACAGTCAConstruction of the P2 promoterThis study
        PC-FdGGATGAAGGCACGAACCCAGAGCTTACGAACCGAACAGTGTCCAGTAATGCAAGTAGCGTATGCConstruction of a promoterless sequence upstream of the blaGES-1cassetteThis study
        PC-RdGCATACGCTACTTGCATTACTGGACACTGTTCGGTTCGTAAGCTCTGGGTTCGTGCCTTCATCCConstruction of a promoterless sequence upstream of the blaGES-1cassetteThis study
    • ↵ a Underlined bases correspond to the −35 and −10 hexamers of the Pc strong promoter.

    • ↵ b Underlined bases correspond to the −35 hexamer of the hybrid Pc promoters.

    • ↵ c Underlined bases correspond to the insertion of three G bases in the P2 promoter sequence.

    • ↵ d Bases in boldface type correspond to the reversed Pc weak promoter sequence. The −10 and −35 hexamers are underlined.

  • TABLE 3.

    Effect of integron promoter sequences on the relative amounts of blaGES-1 transcripts, β-lactamase activities, and β-lactam resistance levels in E. coli MC4100 strains carrying a plasmid-borne blaGES-1 gene cassette

    PromoterSequence at hexamerRelative amt of blaGES-1 transcriptbHydrolysis of nitrocefin (U)cEtest MIC (μg/ml) of β-lactama
    −35−10AmxPipCecCxmCazAtmFep
    Pc weakTGGACATAAGCT132>2562680.750.0470.032
    Pc strongTTGACATAAACT17.0838>25624256>256120.190.19
    Pc hybrid 1TGGACATAAACT1.9107>25648321.50.0470.064
    Pc hybrid 2TTGACATAAGCT4.6225>256122412840.0940.125
    Pc weak+P2TTGTTATACAGT4.7198>25612249640.0940.094
    Pc strong+P220.51,030>25624256>256160.250.19
    Pc hybrid 1+P26.7365>256162425660.1250.125
    ControldNot detectable<1411.520.0640.0320.032
    • ↵ a Amx, amoxicillin; Pip, piperacillin; Cec, cefaclor; Cxm, cefuroxime; Caz, ceftazidime; Atm, aztreonam; Fep, cefepime.

    • ↵ b Values are the means of three measurements differing by <25% and expressed as relative to that of the Pc weak promoter, set to 1.

    • ↵ c Values are the means of three measurements differing by <10%.

    • ↵ d Values are from E. coli MC4100 containing a promoterless blaGES-1-carrying integron sequence.

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Relative Strengths of the Class 1 Integron Promoter Hybrid 2 and the Combinations of Strong and Hybrid 1 with an Active P2 Promoter
Costas C. Papagiannitsis, Leonidas S. Tzouvelekis, Vivi Miriagou
Antimicrobial Agents and Chemotherapy Dec 2008, 53 (1) 277-280; DOI: 10.1128/AAC.00912-08

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Relative Strengths of the Class 1 Integron Promoter Hybrid 2 and the Combinations of Strong and Hybrid 1 with an Active P2 Promoter
Costas C. Papagiannitsis, Leonidas S. Tzouvelekis, Vivi Miriagou
Antimicrobial Agents and Chemotherapy Dec 2008, 53 (1) 277-280; DOI: 10.1128/AAC.00912-08
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  • Top
  • Article
    • ABSTRACT
    • Cloning of a GES-1-encoding integron and construction of the Pc-P2 sequence variants.
    • Characterization of blaGES-1 transcripts by Northern hybridization and real-time reverse transcription-PCR (RT-PCR).
    • β-Lactamase activities and resistance levels to β-lactams.
    • Conclusions.
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
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KEYWORDS

integrons
Promoter Regions, Genetic

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