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Molecular Pathogenesis

Identification and Characterization of a Novel ABC Iron Transport System, fit, in Escherichia coli

Zhiming Ouyang, Richard Isaacson
Zhiming Ouyang
Department of Veterinary and Biomedical Sciences, University of Minnesota, 1971 Commonwealth Ave., St. Paul, Minnesota 55108
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Richard Isaacson
Department of Veterinary and Biomedical Sciences, University of Minnesota, 1971 Commonwealth Ave., St. Paul, Minnesota 55108
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  • For correspondence: isaac015@umn.edu
DOI: 10.1128/IAI.00866-06
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  • FIG. 1.
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    FIG. 1.

    Organization and orientation of the E. coli fit system. The block arrow indicates the gene transcription direction. Intergenic regions are shown as boxes. P, promoter. Arrows indicate the locations of a putative fur box and the −10 and −35 boxes of fitA and fitB, respectively.

  • FIG. 2.
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    FIG. 2.

    Effect of streptonigrin on growth of E. coli AA93 derivatives. Growth of strain AA93 with plasmids in LB (A), α-MEM (B), or LB with 200 μM dipyridyl (C) and growth of strain AA93/feoB with plasmids in LB (D) were measured. Open symbols, strains carrying pfit1; filled symbols, strains carrying pCR; squares, growth in medium without streptonigrin; triangles, growth in medium with 1 μg/ml streptonigrin; diamonds, growth in medium with 5 μg/ml. All experiments were performed at least three times to assess reproducibility. The figure presents the results of one typical experiment.

  • FIG. 3.
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    FIG. 3.

    Measurement of the iron content of the E. coli AA93 derivatives by inductively coupled plasma atomic emission spectrometry. Cells were grown in α-MEM supplemented with 10 μM FeCl3. The data were calculated from three independent replicates. Error bars indicate standard deviations. The asterisk indicates statistical significance using Student's t test (P < 0.05).

  • FIG. 4.
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    FIG. 4.

    Expression of fit was induced by iron depletion. E. coli oy016 cells carrying pMP-fitB (A) or pMP-fitA (B) were first grown in LB medium for 3 h. Then the culture was split into two groups. Into one group, DIP was added at a final concentration of 200 μM, and into the other group no DIP was added. Cells were then allowed to grow, samples were taken at various time points, and β-galactosidase activities were measured as described elsewhere (21). Open bars, cells grown in LB; filled bars, cells grown in LB with DIP. Values are the means from three independent experiments. Error bars indicate standard deviations. Asterisks indicate statistical significance using Student's t test (P < 0.05).

  • FIG. 5.
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    FIG. 5.

    Expression of fit was repressed by iron. E. coli oy016 cells carrying pMP-fitB (A) and pMP-fitA (B) were grown in MM9 medium with various metal ions. A 100 μM concentration each of Mn2+, Ca2+, Fe2+, Cu2+, Ni2+, and Rb+, 50 μM each of Zn2+ and Co2+, and 10 μM Cd2+ were used in the experiment. Cells were harvested at mid-log phase, and β-galactosidase activities were measured as described previously (21). Error bars indicate standard deviations (n = 3). Asterisks indicate statistical significance using Student's t test (P < 0.05).

  • FIG. 6.
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    FIG. 6.

    Effects of pH and H2O2 on fit expression, based on β-galactosidase activities of oy016 cells carrying pMP-fitB (A and C) and pMP-fitA (B and D). (A and B) Cells were grown in LB at various pHs. (C and D) Cells were grown in LB, and when bacterial growth reached an A600 of 0.6, various amounts of H2O2 were added to the medium. After 10 min, cells were collected and β-galactosidase activities were measured as described elsewhere (21). Values are the means from three independent experiments. Error bars indicate standard deviations (n = 3). Asterisks indicate statistical significance using Student's t test (P < 0.05).

  • FIG. 7.
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    FIG. 7.

    Bacterial growth in iron-restricted medium. LB medium was made iron depleted by adding DIP to a final concentration of 200 μM. Bacteria were grown at 37°C with shaking, and the A600 was measured during growth. All tests were repeated three times, and the results of one typical experiment are presented here.

Tables

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  • TABLE 1.

    Bacterial strains and plasmids used in this study

    Strain or plasmidDescriptionSource or reference
    E. coli strains
        i484O25:H autoagluttinating; human isolate 18
        xy006i484, fitA::EZ-TN5 <Kan-2>This study
        xy005i484, fitB::EZ-TN5 <Kan-2>This study
        xy009i484, fepA::EZ-TN5 <DHFR>This study
        xy008i484, fepC::EZ-TN5 <DHFR>This study
        xy007i484, fhuA::EZ-TN5 <DHFR>This study
        oy016i484, lacZ::EZ-TN5 <DHFR>This study
        AA93F−araD139 ΔlacU169 rpsL150 relA1 deoC1 flbB5301 ptsF25 rbsR aroB fecB::Mud1 (Ap lac) 26
        oy097AA93; feoB::EZ-TN5 <DHFR>This study
        AB1515.199 purE42 proC14 leu-6 trpE38 thi-1 fhuA23 lacY1 (fepC::Tn5) 6
        123Field isolatePig
        124Field isolatePig
        252Field isolatePig
        EcoR33Field isolatePig
        263Field isolatePig
        431Field isolatePig
        987Field isolatePig
        1413Field isolatePig
        16Field isolateHuman
        17Field isolateHuman
        18Field isolateHuman
        EcoR40Field isolateHuman
        EcoR60Field isolateHuman
        EcoR62Field isolateHuman
        EcoR8Field isolateHuman
        EcoR26Field isolateHuman
        EcoR42Field isolateHuman
        EcoR45Field isolateHuman
        DH5α F − φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdS17 (rK− mK+) deoR thi-1 supE44 λ−gyrA96 relA1Invitrogen
    Plasmids
        pMP220Contains a promoterless lacZ gene, Tetr IncP 33
        pMP-fitA fit promoter cloned into pMP220 with direction of fitA transcriptionThis study
        pMP-fitB fit promoter cloned into pMP220 with direction of fitB transcriptionThis study
        pCRCloning plasmid pCR-XL-TOPOInvitrogen
        pCR63 lacZ cloned into pCR-XL-TOPOThis study
        pCR64 fitR cloned into pCR-XL-TOPOThis study
        pfit1Whole fit region cloned into pCR-XL-TOPOThis study
  • TABLE 2.

    Distribution of the E. coli fit system among E. coli clinical isolates

    E. coli strain source and typefit system
    Pig commensal strains
        123−
        124−
        252−
        EcoR33−
    Pig enterotoxigenic strains
        263−
        431−
        987−
        1413−
    Human extraintestinal pathogenic strains
        16+
        17−
        18+
        EcoR40−
        EcoR60+
        EcoR62+
    Human commensal strains
        EcoR8−
        EcoR26−
        EcoR42+
        EcoR45−
  • TABLE 3.

    Expression of β-galactosidase from E. coli oy016 derivatives grown in LB or MM9 medium

    β-Galactosidase activity (Miller units, mean ± SD)
    LBMM9-glucose
    oy0160.00.0 ± 0.3
    pMP2202.4 ± 0.30.75 ± 0.1
    pMP-fitA4.2 ± 0.311.7 ± 1.2
    pMP-fitB153 ± 12759 ± 82
  • TABLE 4.

    Growth promotion test of E. coli strainsa

    SubstrateGrowth of strain
    i484fitA-(xy006)fitB-(xy005)fhuA-(xy007)fepC-(xy008)fepA-(xy009)AA93/ pfit1AA93/ pCR
    Si484++++++++
    SAB1515++++−−++
    Heme+++NDNDND−−
    Heme-BSA+++NDNDND−−
    DHBA+++NDNDND−−
    Lactoferrin−−−NDNDND−−
    Transferrin−−−NDNDND−−
    Desferal+++NDNDND−−
    RA+++NDNDND++
    Ferrichrome+++−NDND++
    Iron citrate+++NDNDND−−
    FeSO4++++++++
    FeCl3++++++++
    dH2O−−−−−−−−
    • ↵ a Concentrations of iron compounds used are reported in Materials and Methods. Si484 and SAB1515 represent supernatants of E. coli i484 and AB1515.199 cells, respectively, grown in iron-restricted media. Heme-BSA was made by mixing 100 μM heme and 100 μM BSA at a 1:1 molar ratio. Iron citrate was made by mixing fresh FeSO4 with sodium citrate at a molar ratio of 1:1,000. +, positive; −, negative; ND, not determined; DHBA, dihydroxybenzoic acid; dH2O, distilled water.

Additional Files

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    • Supplemental file 1 - Table S1. Primers used for sequencing, PCR, and creation of mutations.
      MS Word document, 60K.
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Identification and Characterization of a Novel ABC Iron Transport System, fit, in Escherichia coli
Zhiming Ouyang, Richard Isaacson
Infection and Immunity Nov 2006, 74 (12) 6949-6956; DOI: 10.1128/IAI.00866-06

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Identification and Characterization of a Novel ABC Iron Transport System, fit, in Escherichia coli
Zhiming Ouyang, Richard Isaacson
Infection and Immunity Nov 2006, 74 (12) 6949-6956; DOI: 10.1128/IAI.00866-06
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KEYWORDS

ATP-Binding Cassette Transporters
Escherichia coli
iron

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