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Articles

Candida albicans Ferric Reductases Are Differentially Regulated in Response to Distinct Forms of Iron Limitation by the Rim101 and CBF Transcription Factors

Yong-Un Baek, Mingchun Li, Dana A. Davis
Yong-Un Baek
1Department of Microbiology, The University of Minnesota, Minneapolis, Minnesota 55455
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Mingchun Li
1Department of Microbiology, The University of Minnesota, Minneapolis, Minnesota 55455
2Laboratory of Molecular Microbiology and Technology, Department of Microbiology, Nankai University, Tianjin, China 300071
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Dana A. Davis
1Department of Microbiology, The University of Minnesota, Minneapolis, Minnesota 55455
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  • For correspondence: dadavis@umn.edu
DOI: 10.1128/EC.00108-08
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  • FIG. 1.
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    FIG. 1.

    Predicted FRP1 and FRE2 regulatory sites. Predicted Rim101 (lines) and Sfu1 (asterisk) regulatory sites within the FRP1 and FRE2 promoter regions are shown.

  • FIG. 2.
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    FIG. 2.

    EMSA of promoter regions containing putative Rim101 binding sites. Protein extracts from the wild-type (WT) (DAY286; lanes 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17) or rim101Δ/rim101Δ (DAY5; lanes 3, 6, 9, 12, 15, and 18) strain grown at pH 8 were incubated with radiolabeled DNA probes for endogenous (+) (lanes 1, 3, 4, 6, 7, and 9) or mutated (−) (lanes 2, 5, and 8) FRE2 (−876, −443, −22) oligomers or endogenous (+) (lanes 10, 12, 13, 15, 16, and 18) or mutated (−) (lanes 11, 14, and 17) FRP1 (−981, −626, −164) oligomers and analyzed by EMSA.

  • FIG. 3.
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    FIG. 3.

    FRE2 and FRP1 promoter lacZ fusions identify important Rim101 binding sites. β-Galactosidase assays were performed on PFRE2-lacZ and PFRP1-lacZ reporter strains grown at pH 4 or pH 8 or at pH 4 with 200 μM BIP. At least three independent transformants were used to determine average Miller units and standard deviation. n-fold induction was determined by comparison of the expression of a given construct in either pH-8- or BIP-grown cells to expression in pH-4-grown cells. rim101Δ/Δ, rim101Δ/rim101Δ.

  • FIG. 4.
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    FIG. 4.

    Deletion analysis of the FRP1 promoter. β-Galactosidase assays were performed with the PFRP1-lacZ and derivative reporter strains grown at pH 4 or pH 8 or at pH 4 with 200 μM BIP. At least three independent transformants were used to determine average Miller units and standard deviation. n-fold induction was determined by comparison of the expression a given construct in either pH-8- or BIP-grown cells to expression in pH-4-grown cells.

  • FIG. 5.
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    FIG. 5.

    EMSAs of the region predicted to contain the BIP-dependent site. Thirty-base-pair oligomers spanning −220/−190, −200/−170, −180/−150, −160/−130, −140/−110, and −120/−90 in relation to the FRP1 START codon were incubated with protein extracts from wild-type C. albicans (DAY286) grown at pH 4 (lanes 1, 4, 7, 10, 13, and 16), pH 4 with 200 μM BIP (lanes 2, 5, 8, 11, 14, and 17), or pH 8 (lanes 3, 6, 9, 12, 15, and 18). Free probe (open arrowhead), a nonspecific gel shift (closed arrowhead), and specific gel shifts (arrows A to C) are indicated.

  • FIG. 6.
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    FIG. 6.

    Competition assays between the −220/−190 and −160/−130 regions. Radiolabeled probes for the −160/−130 region (lanes 1 to 7) and −220/−190 region (lanes 8 to 14) were incubated with protein extracts from wild-type (DAY286) cells and −160/−130 region (lanes 2 to 4 and 12 to 14) or −220/−190 region (lanes 5 to 7 and 9 to 11) cold competitor. Competitor was added at 50-fold (lanes 2, 5, 9, and 12), 100-fold (lanes 3, 6, 10, and 13), or 500-fold (lanes 4, 7, 11, and 14) excess. Control reactions lacking competitor are included (lanes 1 and 8).

  • FIG. 7.
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    FIG. 7.

    The FRP1 promoter is bound by Hap5 via the CCAAT site. (A) EMSAs of the −160/−130 region containing the endogenous CCAAT site (lanes 1 to 4) or mutated TGCGC site (lanes 5 to 7). Protein extracts were obtained from wild-type (WT) (DAY286) cells grown in M199 (pH 4) (lanes 2 and 5), M199 (pH 4) plus 200 μM BIP (lanes 3 and 6), or M199 (pH 8) medium (lanes 4 and 7). (B) EMSAs of the −160/−130 region using protein extracts from wild-type (DAY286; lanes 2 to 4) or hap5Δ/hap5Δ (DAY1062; lanes 5 to 7) cells grown as for panel A. fp, free probe lane with no protein extract.

  • FIG. 8.
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    FIG. 8.

    Hap5 via the CCAAT site governs FRP1 promoter activity. β-Galactosidase assays were performed on wild-type (WT) cells containing the PFRP1-8-lacZ (data also shown in Fig. 4) and PFRP1-8*TGCGC-lacZ reporters and on hap5Δ/hap5Δ cells containing the PFRE2-lacZ and PFRP1-8-lacZ reporters grown at pH 4 or pH 8 or at pH 4 with 200 μM BIP. At least three independent transformants were used to determine average Miller units and standard deviation. Induction (n-fold) was determined by comparison of the expression of a given construct in either pH 8- or BIP-grown cells to expression in pH 4-grown cells.

  • FIG. 9.
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    FIG. 9.

    Hap43 is required for iron limitation-specific promoter binding. EMSAs of the −160/−130 probe incubated with protein extracts derived from wild-type (WT) (DAY286; lanes 1 to 3), hap5Δ/hap5Δ (DAY1062; lanes 4 to 6), hap41Δ/hap41Δ (DAY1083; lanes 7 to 9), or hap43Δ/hap43Δ (DAY1085; lanes 10 to 12) cells are shown. Protein extracts were obtained from cells grown in M199 (pH 4) (lanes 1, 4, 7, and 10), M199 (pH 4) plus 200 μM BIP (lanes 2, 5, 8, and 11), or M199 (pH 8) medium (lanes 3, 6, 9, and 12).

  • FIG. 10.
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    FIG. 10.

    Growth of the wild-type (WT) (DAY286), rim101Δ/rim101Δ (DAY5), hap5Δ/hap5Δ (DAY1062), ftr1Δ/ftr1Δ (DAY609), hap41Δ/hap41Δ (DAY1083), and hap43Δ/hap43Δ (DAY1085) strains on YPG plates with or without 150 μM BPS after 3 days of incubation at 37°C.

Tables

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  • TABLE 1.

    Strains used in this study

    NameGenotypeReference
    BWP17 ura3Δ::λimm434 his1::hisG arg4::hisG 58
    ura3Δ::λimm434 his1::hisG arg4::hisG
    DAY5 ura3Δ::λimm434 his1::hisG arg4::hisG rim101::URA3 58
    ura3Δ::λimm434 his1::hisG arg4::hisG rim101::ARG4
    DAY286 ura3Δ::λimm434 his1::hisG pARG4::URA3::arg4::hisG 12
    ura3Δ::λimm434 his1::hisG arg4::hisG
    DAY609 (CTA87.4) ura3Δ::λimm434 his1::hisG arg4::hisG ftr1::ARG4 31
    ura3Δ::λimm434 his1::hisG arg4::hisG ftr1::URA3
    DAY1061 ura3Δ::λimm434 his1::hisG arg4::hisG hap5::ARG4 This study
    ura3Δ::λimm434 his1::hisG arg4::hisG HAP5
    DAY1062 ura3Δ::λimm434 his1::hisG arg4::hisG hap5::ARG4 This study
    ura3Δ::λimm434 his1::hisG arg4::hisG hap5::URA3-dpl200
    DAY1082 ura3Δ::λimm434 his1::hisG arg4::hisG hap41::ARG4 This study
    ura3Δ::λimm434 his1::hisG arg4::hisG HAP41
    DAY1083 ura3Δ::λimm434 his1::hisG arg4::hisG hap41::ARG4 This study
    ura3Δ::λimm434 his1::hisG arg4::hisG hap41::URA3
    DAY1084 ura3Δ::λimm434 his1::hisG arg4::hisG hap43::ARG4 This study
    ura3Δ::λimm434 his1::hisG arg4::hisG HAP43
    DAY1085 ura3Δ::λimm434 his1::hisG arg4::hisG hap43::ARG4 This study
    ura3Δ::λimm434 his1::hisG arg4::hisG hap43::URA3
  • TABLE 2.

    Sequences of primers used in this study

    PrimerSequencea
    FRE2−22 5atgtacaattttcaacaCCAAGaagagtttcctaaa
    FRE2−22 3atttaggaaactcttCTTGGtgttgaaaattgtaca
    FRE2−22 mut5atgtacaattttcaacaGCCGGCagagtttcctaaa
    FRE2−22 mut3atttaggaaactctGCCGGCtgttgaaaattgtaca
    FRE2−443 5atttttataaattaatGCCAAGccatttttatttct
    FRE2−443 3aagaaataaaaatggCTTGGCattaatttataaaaa
    FRE2−443 mut5atttttataaattaatGCCGGCccatttttatttct
    FRE2−443 mut3aagaaataaaaatggGCCGGCattaatttataaaaa
    FRE2−876 5aagaactaattgaaaGCCAAGaaaagttatatggaa
    FRE2−876 3attccatataacttttCTTGGCtttcaattagttct
    FRE2−876 mut5aagaactaattgaaaGCCGGCaaaagttatatggaa
    FRE2−876 mut3attccatataacttttGCCGGCtttcaattagttct
    PFRP1−981 5aggtacatcatgaatCCAAGaattaccgatccgatt
    PFRP1−981 3aaatcggatcggtaattCTTGGattcatgatgtacc
    PFRP1−981 mut 5aggtacatcatgaatGCCGGCattaccgatccgatt
    PFRP1−981 mut 3aaatcggatcggtaatGCCGGCttcatgatgtacc
    PFRP1−636 5aaacacaacgaagtaGCCAAGaatctggcactagca
    PFRP1−636 3atgctagtgccagattCTTGGCtacttcgttgtgtt
    PFRP1−636 mut 5aaacacaacgaagtaGCCGGCaatctggcactagca
    PFRP1−636 mut 3atgctagtgccagattGCCGGCtacttcgttgtgtt
    PFRP1−164 5acgcagttttcggattCTTGGCatagaaacgataaa
    PFRP1−164 3aatttatcgtttctatGCCAAGaatccgaaaactgcg
    PFRP1−164 mut 5acgcagttttcggattGCCGGCatagaaacgataaa
    PFRP1−164 mut 3atttatcgtttctatGCCGGCaatccgaaaactgcg
    PFRE2−876 m1ataatagaactaattgaaaGCCGGCaaaagttatatggaattgct
    PFRE2−876 m2agcaattccatataacttttGCCGGCtttcaattagttctattat
    PFRE2−443 m1tcctttttttataaattaatGCCGGCccatttttatttcttacag
    PFRE2−443 m2ctgtaagaaataaaaatggGCCGGCattaatttataaaaaaagga
    PFRP1−981 m1cgggaattcgaatGCCGGCattaccgatccgattcctcct
    PFRP1−981 m2aggaggaatcggatcggtaatGCCGGCttcatgatgtacc
    PFRP1−636 m1aactgaacacaacgaagtaGCCGGCaatctggcactagcaacatc
    PFRP1−636 m2gatgttgctagtgccagattGCCGGCtacttcgttgtgttcagtt
    PFRP1−164 m1aatttcgcagttttcggattGCCGGCatagaaacgataaatccac
    PFRP1−164 m2gtggatttatcgtttctatGCCGGCaatccgaaaactgcgaaatt
    FRP1-1ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgatttgatgagcctgtagatgacg
    FRP1-2ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgattgcacttcctggagcagtaac
    FRP1-3ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgattaagcaggcatcatccaagta
    FRP1-4ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgattacattgaccaactaaaccag
    FRP1-5ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgatttttgcacagtctggtaataa
    FRP1-6ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgattcagcagatactaaaagaatc
    FRP1-7ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgattgttaaacccgaaagaaaagt
    FRP1-8ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgattgtaaaatttcgcagttttcg
    FRP1-9ggcgaattgggcccgacgtcgcatgctcccggccgccatggcggccgcgggaattcgatttgcatgaatgaaaagtatta
    LacZClaI+200ctacggaaataatcagccatgtcagc
    FRP1-8-1-5a′taaaaatgcttttgtgctgcgtaaaatttc
    FRP1-8-1-3a′gaaattttacgcagcacaaaagcattttta
    FRP1-8-2-5a′gtaaaatttcgcagttttcggattCTTGGC
    FRP1-8-2-3a′GCCAAGaatccgaaaactgcgaaattttac
    FRP1-8-3-5a′gattCTTGGCatagaaacgataaatccacc
    FRP1-8-3-3a′ggtggatttatcgtttctatGCCAAGaatc
    FRP1-8-4-5a′taaatccaccaattaaacgatggccgcatc
    FRP1-8-4-3a′gatgcggccatcgtttaattggtggattta
    FRP1-8-5-5a′tggccgcatcccttgtctgatgataataag
    FRP1-8-5-3a′cttattatcatcagacaagggatgcggcca
    FRP1-8-6-5a′tgataataaggtttattgaatgcatgaatg
    FRP1-8-6-3a′cattcatgcattcaataaaccttattatca
    FRP1-8-4-mut5a′taaatccaTGCGCtaaacgatggccgcatc
    FRP1-8-4-mut3a′gatgcggccatcgtttaGCGCAtggattta
    HAP5 5DRaacaaacattcaattaagacgaagaaatacagaaagaccccccaaaacaatactcaaacatttcccagtcacgacgtt
    HAP5 3DRtattattacaaaatcaaacactatttttaaaatgaacgaaaaaaaaaaaaaaatcctctagtggaattgtgagcggata
    HAP5 5detectaaaatgcgacaacggaaagg
    HAP5 3detectcaagtgaggaactgagaactg
    5′SnaBI CaHIS1gtagtggaagatattctttattgaaaaatagcttgtcaccggatcctggaggatgaggag
    3-detecttgtggaattgtgagcggataacaatttcac
    HAP41 5DRtacagtatatttattttttcccccatacattaattattctaatcttatgaaattaccatttttcccagtcacgacgtt
    HAP41 3DRcatctcatcacatcacatcacatcaaaaataaaatccgttaattttttactgttccttcagtggaattgtgagcggata
    HAP43 5DRcaaaaaaaaacaacagagtccaaaaaaatacataataattagaatttcaatttgaacaactttcccagtcacgacgtt
    HAP43 3DRgaaaagaaaagaaaaaaaaaactgaagtgtcggaaatacttcatactgtaagtcaaactagtggaattgtgagcggata
    HAP41 5detect newccactagttcactcaattac
    HAP41 3detect newccaaatcgagcaaaatcaaacc
    HAP43 5detecttgtcttccccccaatagact
    HAP43 3detecttacctttttgaatacattgt
    HAP41UPFWD2NDtgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgactgaaatttctataattttagcagaa
    HAP41UPREV2NDttttaaagaaagtagtaccagctcttttttttgtttccgtttataccatccaaatcccgcaatggtaatttcataagattagaat
    HAP41DOWNFWDaataattaaataagggtggtaattattactatttacaatcaaaggtggtccttctagatgtgaaggaacagtaaaaaattaacgg
    HAP41DOWNREVttacactttatgcttccggctcctatgttgtgtggaattgtgagcggataacaatttcacgtgcgtagtgttcggaagtgttgag
    HAP43UPFWDtgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacgctgacacaaacgggaacagaaata
    HAP43UPREV2NDttttaaagaaagtagtaccagctcttttttttgtttccgtttataccatccaaatcccgcgttgttcaaattgaaattctaatta
    HAP43DOWNFWDaataattaaataagggtggtaattattactatttacaatcaaaggtggtccttctagatgtagtttgacttacagtatgaagtat
    HAP43DOWNREVttacactttatgcttccggctcctatgttgtgtggaattgtgagcggataacaatttcactaaaggtattggtgctgccgcttat
    URA3FWD2NDgcgggatttggatggtataaacgg
    URA3REVcatctagaaggaccacctttgattg
    • ↵ a Uppercase letters denote the endogenous or mutated putative Rim101 binding site.

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Candida albicans Ferric Reductases Are Differentially Regulated in Response to Distinct Forms of Iron Limitation by the Rim101 and CBF Transcription Factors
Yong-Un Baek, Mingchun Li, Dana A. Davis
Eukaryotic Cell Jul 2008, 7 (7) 1168-1179; DOI: 10.1128/EC.00108-08

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Candida albicans Ferric Reductases Are Differentially Regulated in Response to Distinct Forms of Iron Limitation by the Rim101 and CBF Transcription Factors
Yong-Un Baek, Mingchun Li, Dana A. Davis
Eukaryotic Cell Jul 2008, 7 (7) 1168-1179; DOI: 10.1128/EC.00108-08
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KEYWORDS

CCAAT-Binding Factor
Candida albicans
FMN Reductase
Fungal Proteins
Gene Expression Regulation, Fungal
Iron

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