Hans-Knöll-Institut für Naturstoff-Forschung eV, Abteilung Mikrobielle Infektionsbiologie, Beutenbergstrasse 11,D-07745 Jena, Germany1
Author for correspondence: Raimund Eck. Tel: +49 3641 656852. Fax: +49 3641 656652. e-mail: reck{at}pmail.hki-jena.de
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ABSTRACT |
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Keywords: multicopper oxidase, Candida albicans, gene disruption, pathogenicity, iron uptake
Abbreviations: BPA, bathophenanthrolinedisulfonic acid
b The EMBL accession number for the sequence reported in this paper is Y09329.
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INTRODUCTION |
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Pathogens accumulate iron by different mechanisms. The most widespread mechanism involves the excretion of iron chelators (siderophores). Siderophore production occurs in various pathogenic fungi (Holzberg & Artis, 1983 ). One study reported that the dimorphic human pathogenic yeast Candida albicans can secrete siderophores of both the hydroxamate and the phenolate type (Ismail et al., 1985
). Another report only confirmed the production of the hydroxamate type (Sweet & Douglas, 1991
). However, it is not clear how C. albicans obtains Fe(II) from the ferric-siderophore complex or from other sources. Little is known about siderophore-independent mechanisms of iron uptake of C. albicans, although Morrissey et al. (1996
) determined a ferric reductase activity regulated similarly to the S. cerevisiae ferric reductase, which was induced in low-iron medium.
The well-characterized yeast Saccharomyces cerevisiae does not secrete its own siderophores. It uses foreign siderophores and a two-step reductive Fe(III) uptake system. The two steps are the reduction of Fe(III) to Fe(II) by the surface ferric reductases Fre1p and Fre2p (Dancis et al., 1994 ; Georgatsou & Alexandraki, 1994
), and the transport (and oxidation?) by a low- or high-affinity Fe(II) transport system (Dix et al., 1997
; Askwith et al., 1994
). The high-affinity system has a Km for iron of 0·15 µM (Eide et al., 1992
). The low-affinity system works at iron concentrations higher than 5 µM. The yeast Fet4p is responsible for the low-affinity iron-transport system. The yeast Fet3p is required for high-affinity iron transport and works downstream of the ferric reductases. It encodes a cell-surface ferroxidase with homology to the multicopper oxidase family (De Silva et al., 1995
). It is a type 1 membrane protein with only one potential transmembrane domain, in which the ferroxidase domain is located on the extracellular surface (De Silva at al., 1995
). The transcription of FET3 is regulated by the transcription factor Aft1p and the iron concentration. Transcripts for Fre1p, Fre2p and Fet3p are not detected in the absence of a functional AFT1 gene (Yamaguchi et al., 1995
). Cells grown in low-iron medium (110 µM) contain abundant FET3 mRNA, while cells grown in high-iron medium (1000 µM) show no detectable FET3 mRNA (Askwith et al., 1994
). The high-affinity iron uptake system in S. cerevisiae requires copper. Copper deficiency or mutations in genes involved in delivery of copper to Fet3p [CTR1, encoding the cellular copper uptake transporter (Dancis et al., 1994
); CCC2, encoding the intracellular copper transporter (Yuan et al., 1995
)] abrogate iron uptake as a secondary effect. The human multicopper oxidase ceruloplasmin exhibits similarity to the yeast Fet3p oxidase. Ceruloplasmin plays an important role in iron uptake by human cells.
The amino acid sequence of Fet3p shows no homology to the family of permeases and it has only a single transmembrane domain. Therefore, it is difficult to understand how Fet3p could mediate the iron transport. A candidate for the transport protein is Ftr1p. This protein contains six transmembrane domains and shows homology to Fth1p, a permease of unknown function (Stearman et al., 1996 ).
In a screen of genes which suppress the iron-limited lack of growth of fet3 fet4 mutants, Spizzo et al. (1997 ) isolated another multicopper oxidase from S. cerevisiae (Fet5p). Fet5p shows an identity of 47% to Fet3p and the same structural features. It has the same function as Fet3p in iron transport. However, the fet5 mutant is able to grow in iron-limited medium, in contrast to the fet3 mutant (Spizzo et al., 1997
). Two potential roles for Fet5p in iron metabolism have been proposed: FET5 may encode a Fet3p isoenzyme, or Fet5p may be involved in transport of iron across the membrane of an intracellular compartment such as the vacuole.
In this study we cloned a gene encoding a multicopper oxidase, Fet3p, in C. albicans and investigated whether Fet3p is essential for high-affinity iron uptake in vitro. We also examined its role in the pathogenicity of C. albicans.
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METHODS |
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Comparative studies of the growth of the homozygous fet3 mutant strain and the wild-type parental strain SC5314 were carried out as follows. Various concentrations of iron (FeCl3, 03 µM), equal numbers of cells from the fourth generation in LIM medium (containing approximately 0·2 µM iron as estimated by atom mass spectroscopy) (Eide & Guarente, 1992
) and 300 µM bathophenanthrolinedisulfonic acid (BPA; Sigma-Aldrich) were added to LIM medium supplemented with 300 µM BPA. In addition, similar growth experiments were performed in YNB medium supplemented with various iron concentrations (03 µM), 180 µM dipyridyl (Sigma-Aldrich) and Candida cells from a YNB culture [180 µM dipyridyl, 10 µM iron]. BPA only binds extracellular iron; dipyridyl chelates extra- and intracellular iron. S. cerevisiae DY1457 (MAT
ade6 can1 his3 leu2 trp1 ura3) and S. cerevisiae DEY1394 (MAT
ade6 can1 his3 leu2 trp1 ura3 fet3-2::HIS3) were used for the complementation experiments (Askwith et al., 1994
). These strains were grown in YNB supplemented with 40 or 80 µM BPA. For iron-deficiency growth experiments, only plastic bottles were used.
PCR.
The low-stringency PCR amplification reactions were carried out in a 100 µl reaction volume containing 10 mM Tris/HCl, pH 8·3, 1·5 mM MgCl2, 50 mM KCl, 0·2 mM dNTP and 0·2 µM of each primer. The reaction was started by addition of 1 unit Taq polymerase (Boehringer Mannheim) and the samples were overlaid with mineral oil. Thirty cycles were performed consisting of 30 s incubation at 96 °C, 1 min at 42 °C and 2 min at 72 °C using a Omn-E (Hybaid) thermal cycler. Degenerate oligonucleotide primers REBA (5'-TAKCAKATHTTYGAR-3') and REBE1 (5'-TCYTGNGARTCYTCNGT-3') were derived using the REB1 consensus sequences of S. cerevisiae REB1 (rDNA enhancer binding protein) and Kluyveromyces lactis REB1 (Morrow et al., 1993 ). The sequences of the primers were adapted to the codon usage in C. albicans (Lloyd & Sharp, 1992
) because the DNA of C. albicans is A/T-rich (the third position of triplets mostly contains A or T). The primers show insignificant homology to CaFET3. RT-PCR was performed by standard procedures (Ausubel et al., 1995
).
Cloning and sequence analysis of CaFET3.
The CaFET3 PCR probe was used to screen a fosmid library. A 7·0 kb PstI probe-reactive fragment was subcloned (pFOS1FET3). A 3·3 kb HindIII fragment of the insert DNA of pFOS1FET3 was cloned into pUC19, yielding plasmid pH3300, and sequenced (Fig. 1). The nucleotide sequences were determined by the dideoxy chain-termination method with synthetic oligonucleotide primers using the Sequenase version 2.0 kit (United States Biochemical). All other recombinant DNA procedures were carried out by standard protocols (Sambrook et al., 1989
). Alignments were performed using the GCG software package (Genetics Computer Group). Block searches were performed by the method of Henikoff & Henikoff (1994
) (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). Homology searches were performed using the blast computer programs (Altschul et al., 1990
).
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Transformation of C. albicans, isolation of heterozygous and homozygous multicopper oxidase mutants and selection of Ura- auxotrophs.
The methods were carried out by procedures described previously (Boeke et al., 1984 ; Swoboda et al., 1995
; Eck et al., 1997
).
Southern blot analysis and transformation of S. cerevisiae DEY1394 fet3.
Southern blot analysis was performed by standard procedures (Sambrook et al., 1989 ). Transformation was performed according to Klebe et al. (1983
).
Preparation of total RNA from C. albicans SC5314 and S. cerevisiae DY1457.
Because the S. cerevisiae FET3 gene was expressed in cells grown in low-iron LIM medium, CaFET3 mRNA was also isolated from cells grown in low-iron medium. Late-exponential-phase C. albicans SC5314 cells from the fourth passage (LIM medium, 300 µM BPA) were harvested by centrifugation and resuspended in two different media: 1, 300 ml LIM medium supplemented with 300 µM BPA and 0·1, 1, 10 or 100 µM iron; 2, 300 ml YNB medium supplemented with 180 µM bipyridyl and 0·1, 1, 10 or 100 µM iron. S. cerevisiae DY1457 cells from a late-exponential-phase YNB culture were harvested and resuspended in 300 ml LIM medium supplemented with 1 µM iron. After different periods of growth (C. albicans, 10 min to 24 h; S. cerevisiae, 3 h) the cells were harvested and resuspended in 10 ml 50 mM sodium acetate, 10 mM EDTA, pH 5·3, 10 ml phenol (Aqua-Roti-Phenol; Roth) 100 µl 10% (w/v) SDS and incubated twice for 5 min at 65 °C followed by 5 min in liquid nitrogen. After centrifugation, the aqueous phase was extracted with phenol and with phenol/chloroform (1:1, v/v). After ethanol precipitation the RNA was dissolved in 80% (v/v) ethanol and quantified on the basis of A260.
Northern blot analysis.
Northern analysis was performed using the internal 1·65 kb EcoRIMunI fragment of CaFET3 (+195 to +1836) and an internal PCR product of S. cerevisiae FET3 (+377 to +1600) as probes. RNA samples (20 µg) were separated in formaldehyde gels containing 1% (w/v) agarose. The RNA was blotted onto Hybond nylon membranes (Amersham). Membranes were hybridized and washed by standard techniques (Sambrook et al., 1989 ).
Characterization of the mutant strains.
The C. albicans SC5314 wild-type strain and the C. albicans fet3 mutant (fet3
::hisG/fet3
::hisG-URA3-hisG) were grown at 28 °C in 10 ml LIM medium with 300 µM BPA over five passages each of 5 d. The fifth passage did not grow. One hundred microlitres of cell suspension from the fourth passage was transferred into fresh LIM medium with different concentrations of iron (0, 0·01, 0·03, 0·05, 0·1, 0·3, 1 or 3 µM) and 300 µM BPA. The OD600 was measured after 48 h and 72 h. In further experiments both strains were incubated in YNB supplemented with 180 µM bipyridyl and 0, 0·1, 1 and 3 µM iron.
Pathogenicity assays.
We used the mouse candidiasis model described by Plempel (1984 ) to test C. albicans mutants for pathogenicity. Briefly, wild-type or mutant strains were grown for 24 h at 28 °C in YNB medium. Ten mice (eight week old, male, NMRI mice, Halan-Winkelmann, Paderborn, Germany) were infected by injection into the caudal vein of 5x106 or 5x104 cells in 0·2 ml PBS. Survival was monitored for 14 d.
Adherence assay.
Mouse L929 fibroblasts were kindly provided by H.-M. Dahse from our institute. The adherence fluorescence assay was carried out by the protocol of Borg-von Zepelin & Wagner (1995 ). Briefly, the fibroblasts were incubated in microtest plates; C. albicans cells (2x106 cells ml-1), preincubated and stained with calcofluor white, were then added, followed by incubation for 2 h at 37 °C. Calcofluor white binds predominantly to chitin and glucan in the fungal cell wall. Non-adherent Candida cells were removed. Finally, the amount of adherent fluorescent C. albicans cells was determined by an automatic fluorescence reader (FluoroScan; Labsystems) (absorption, 360 nm; emission, 460 nm).
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RESULTS |
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The two fosmids were digested with PstI. Southern analysis of one fosmid showed a 7·0 kb band that hybridized with the PCR probe. This fragment was subcloned into plasmid pUC18, yielding the plasmid pFOS1FET3. After digestion of pFOS1FET3 with HindIII and subsequent hybridization with the PCR probe, a 3·3 kb probe-reactive fragment was identified and cloned into pUC18, yielding plasmid pH3300. This fragment was subsequently found to contain the entire CaFET3 gene (Fig. 1).
Sequence analysis and characterization of CaFET3
The 3·3 kb insert of pH3300 contained one ORF of 1872 bp. This ORF encoded a hypothetical polypeptide of 624 amino acids with a predicted molecular mass of 70·5 kDa. The isoelectric point was predicted to be at a pH value of 4·63. Two TATA boxes at nucleotide positions -113 to -117 and -129 to -135 and a CAAT box at position -142 to -145 were located. In amino acid sequence comparisons, the product of the identified ORF from C. albicans showed the highest overall identity to the multicopper oxidase Fet3p from S. cerevisiae (55%). The ORF was therefore named CaFET3. Identities were also found between the putative CaFet3p and the multicopper oxidases Fet5p from S. cerevisiae (52%) and Fet3p from Schizosaccharomyces pombe (40%) (Fig. 2).
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The hydrophobicity plots (KyteDoolittle) of Fet3p and CaFet3p showed potential transmembrane domains. These were identified in the C-terminal regions (Fet3p, aa 560 to 589; CaFet3p, aa 556 to 585). Hydrophobic domains were identified in the N-terminal region of the proteins (Fet3p and CaFet3p: aa 1 to 21). These are potential secretory signal sequences (von Heijne, 1983 ). The putative transmembrane domains and the potential secretory signal sequences showed similarities of 70% and 45%, respectively (De Silva et al., 1995
).
Southern blot analysis was performed applying the same hybridization and washing conditions as used for the fosmid screening, and using the PCR product as a probe. The results implied that CaFET3 is present as a single copy per haploid genome in C. albicans SC5314 and that no other homologous genes occur in the C. albicans genome.
CaFET3-specific mRNA was not detected by Northern blot analysis of total RNA of C. albicans SC5314. Experiments with different time periods of growth (10 min to 24 h), concentrations of iron (0·1 to 100 µM) and media (LIM medium, YNB, YED) and with the iron chelators BPA and dipyridyl, did not lead to a detectable mRNA. According to expectations, the control Northern blot analysis of S. cerevisiae DY1457 RNA showed an approximately 2·1 kb hybridizing FET3 mRNA. To detect CaFET3 mRNA, RT-PCR was performed. We used primers 1 and 2 (see Methods, construction of pDV1) and total RNA from C. albicans SC5314 grown in LIM medium supplemented with 0·1, 1, 10 or 100 µM iron. We detected a 235 bp product in each RT-PCR experiment. This result indicated that the CaFET3 was expressed under both low- and high-iron conditions, although at low abundance.
Disruption of CaFET3
C. albicans CAI-4 was transformed with the hisG-URA3-hisG cassette flanked by 5' and 3' CaFET3 gene sequences obtained by digestion of pDV2 with SacI/HindIII (Fig. 1). DNA from a representative Ura+ colony was isolated and digested with PstI. Southern analysis with the 32P-labelled 3·3 kb HindIII insert of plasmid pH3300 (Fig. 1
) showed two hybridizing fragments (Fig. 3
, lane 2). One band is identical to the 7·0 kb hybridizing fragment of the parental strain CAI-4 shown in lane 1. The length of the second band of 10·1 kb is consistent with the replacement of 50·5% of the CaFET3 gene by the hisG-URA3-hisG cassette.
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The gene replacement was repeated to disrupt the second allele of CaFET3 gene in C. albicans. Five putative homozygous clones (fet3::hisG/fet3
::hisG-URA3-hisG) were obtained. Southern analysis of chromosomal DNA from a representative clone is shown in Fig. 3
, lane 4. One hybridizing fragment is identical to the 7·2 kb band of C. albicans CAI-4 FET3/fet3
::hisG shown in lane 3. The second hybridizing fragment is identical to the 10·1 kb band of C. albicans CAI-4 FET3/fet3
::hisG-URA3-hisG shown in lane 2. The 7·0 kb fragment of the parental strain CAI-4 was missing.
Characterization of the fet3 mutant
The wild-type strain C. albicans SC5314 and the mutant derivative C. albicans fet3 (fet3
::hisG/fet3
::hisG-URA3-hisG) showed different growth in iron-limited medium (0·013 µM iron) (Fig. 4
). The growth of C. albicans SC5314 and C. albicans fet3
was inhibited without addition of iron to the LIM medium supplemented with 300 µM BPA in the fifth passage. The addition of 0·010·3 µM iron facilitated the growth of C. albicans SC5314 but not that of C. albicans fet3
. The mutant strain grew after addition of at least 1·0 µM iron and an incubation period of 48 h. The chelator bipyridyl (180 µM in YNB medium), which bound extra- and intracellular iron, caused a dramatic decrease of growth in iron-limited medium. However, the results after addition of iron to YNB medium supplemented with bipyridyl were similar to those for BPA in LIM medium (data not shown). Together with the data from the comparison between Fet3p and Fet5p of S. cerevisiae and CaFet3p from C. albicans, it can be concluded that we have isolated a C. albicans multicopper oxidase gene involved in high-affinity iron uptake.
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Complementation of S. cerevisiae DEY1394 fet3 with CaFET3
Due to the role of Fet3p in high-affinity iron uptake, the knockout mutant strain S. cerevisiae DEY1394 fet3 is unable to grow on iron-limited media, either complex medium with 80 µM of the iron chelator BPA or LIM medium (Askwith et al., 1994
). This observation was the basis for our complementation experiments. We transformed S. cerevisiae DEY1394 fet3
with YCp50CaFET3 containing the 3·3 kb HindIII fragment of pH3300. Cells were plated on YNB plates without uridine so that only transformants bearing a plasmid were able to grow. Plasmid-rescue experiments were performed from three transformants. All three transformants contained YCp50CaFET3. One transformant (S. cerevisiae DEY1394 fet3
containing YCp50CaFET3), the mutant strain S. cerevisiae DEY1394 fet3
and the wild-type strain S. cerevisiae DY1457 were grown in YED medium with 40 µM and 80 µM BPA, respectively. If the medium contained 80 µM BPA only S. cerevisiae DY1457 grew. This result showed that at this concentration of BPA the CaFET3 gene was unable to restore the growth of S. cerevisiae DEY1394 fet3
in iron-limited medium. However, CaFET3 was able to complement the growth deficiency of S. cerevisiae DEY1394 fet3
in iron-limited medium with 40 µM BPA (Fig. 6
).
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DISCUSSION |
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The fact that wild-type C. albicans needed five passages before growth was inhibited in LIM medium (with approximately 0·2 µM iron) containing the extracellular chelator BPA indicated that an additional low-iron uptake mechanism exists in C. albicans. We assume that siderophores and the intracellular pool of iron contribute to the long survival of C. albicans in the iron-limited medium. Moreover, it is possible that our strain is unable to produce siderophores. Differences in siderophore production between C. albicans strains have been observed (Ismail et al., 1985 ; Sweet & Douglas, 1991
).
Unlike C. albicans fet3, S. cerevisiae fet3
showed growth inhibition in YPD medium containing 40 µM BPA, because it produces none of its own siderophores or/and it possesses no effective mechanism of iron storage.
We did not succeed in detecting CaFET3 mRNA following Northern analysis with a FET3-specific probe. However, RT-PCR experiments showed that CaFET3 mRNA was produced at low abundance. There are three possible reasons for these results: firstly, a fast turnover of CaFET3 mRNA; secondly, a low level transcription of CaFET3; and thirdly, unusual conditions for the initiation of transcription compared with S. cerevisiae FET3.
Comparative analysis of the pathogenicity of the wild-type strain C. albicans SC5314 and the mutant derivative C. albicans fet3 showed no differences in virulence, although the ability of pathogens to accumulate iron is considered as a virulence factor. Possible reasons for our results include the following: the storage of iron in the inoculated Candida cells may have been too high (first culture in LIM medium with 300 µM BPA); the mutant strain may have been able to produce enough siderophores; or the mutant may have used other means of iron uptake.
Fratti et al. (1998 ) found that chelation of endothelial cellular iron protected these cells from injury by C. albicans and that adherence was slightly enhanced. We also found a correlation between iron metabolism and adherence of C. albicans to mouse fibroblasts. Possibly the decreased adherence of C. albicans fet3
was mediated by modified production of reactive oxygen intermediates caused by an incomplete reduction of oxygen.
Two essential proteins of the high-affinity iron uptake system have now been identified in C. albicans. Morrissey et al. (1996 ) determined a cell-associated ferric reductase activity in C. albicans showing identical features to those of the homologue S. cerevisiae enzyme. Additionally, our results have shown that C. albicans FET3 encodes a homologous protein to the S. cerevisiae multicopper oxidase Fet3p which is responsible for the high-affinity transport of Fe(II). This study demonstrates for the first time the functional similarity between the high-affinity iron uptake systems of S. cerevisiae and C. albicans.Therefore, it seems that the basic elements of the high-affinity iron uptake system of the pathogenic yeast C. albicans and the non-pathogenic yeast S. cerevisiae are identical.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Askwith, C., Eide, D., Van Ho, A. V., Bernard, P. S., Li, L., Davis-Kaplan, S., Sipe, D. M. & Kaplan, J. (1994). The FET3 gene of Saccharomyces cerevisiae encodes a multicopper oxidase required for ferrous iron uptake. Cell 76, 403-410.[Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1995). Current Protocols in Molecular Biology, New York: Wiley.
Boeke, J. D., LaCroute, F. & Fink, G. R. (1984). A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197, 345-346.[Medline]
Borg-von Zepelin, M. & Wagner, T. (1995). Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates. Mycoses 38, 339-347.[Medline]
Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J. & Klausner, R. D. (1994). Molecular characterization of a copper transport protein in Saccharomyces cerevisiae: an unexpected role for copper in iron transport. Cell 76, 393-402.[Medline]
De Silva, D. M., Askwith, C. C., Eide, D. & Kaplan, J. (1995). The FET3 gene product required for high affinity iron transport in yeast is a cell surface ferroxidase. J Biol Chem 270, 1098-1101.
Dix, D., Bridgham, J., Broderius, M. & Eide, D. (1997). Characterization of the FET4 protein of yeast. J Biol Chem 272, 11770-11777.
Eck, R., Bergmann, C., Ziegelbauer, K., Schönfeld, W. & Künkel, W. (1997). A neutral trehalase gene from Candida albicans: molecular cloning, characterization and disruption. Microbiology 143, 3747-3756.[Abstract]
Eide, D. & Guarente, L. (1992). Increased dosage of a transcriptional activator gene enhances iron-limited growth of Saccharomyces cerevisiae. J Gen Microbiol 138, 347-354.[Medline]
Eide, D., Davis-Kaplan, S., Jordan, I., Sipe, D. & Kaplan, J. (1992). Regulation of iron uptake in Saccharomyces cerevisiae. J Biol Chem 267, 20774-20781.
Fonzi, W. A. & Irvine, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717-728.
Fratti, R. A., Belanger, P. H., Ghannoum, M. A., Edwards, J. E.Jr & Filler, S. G. (1998). Endothelial cell injury caused by Candida albicans is dependent on iron. Infect Immun 66, 191-196.
Georgatsou, E. & Alexandraki, D. (1994). Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in Saccharomyces cerevisiae. Mol Cell Biol 14, 3065-3073.[Abstract]
von Heijne, G. (1983). Pattern of amino acids near signal-sequence cleavage sites. Eur J Biochem 133, 17-21.[Abstract]
Henikoff, S. & Henikoff, J. G. (1994). Protein family classification based on searching a database of blocks. Genomics 19, 97-107.[Medline]
Holzberg, M. & Artis, W. M. (1983). Hydroxamate siderophore production by opportunistic and systemic fungal pathogens. Infect Immun 40, 1134-1139.[Medline]
Ismail, A., Bedell, G. W. & Lupan, D. M. (1985). Siderophore production by the pathogenic yeast, Candida albicans. Biochem Biophys Res Commun 130, 885-891.[Medline]
Klebe, I. K., Harris, I. V., Sharp, D. & Douglas, M. G. (1983). A general method for polyethylenglycol-induced genetic transformation of bacteria and yeast. Gene 25, 333-341.[Medline]
Lloyd, A. T. & Sharp, P. M. (1992). Evolution of codon usage patterns: the extent and nature of divergence between Candida albicans and Saccharomyces cerevisiae. Nucleic Acids Res 20, 5289-5295.[Abstract]
Morrissey, J. A., Williams, P. H. & Cashmore, A. M. (1996). Candida albicans has a cell-associated ferric-reductase activity which is regulated in response to levels of iron and copper. Microbiology 142, 485-492.[Abstract]
Morrow, B. E., Ju, Q. & Warner, J. R. . (1993). A bipartic DNA-binding domain in yeast Reb1p. Mol Cell Biol 13, 1173-1182.[Abstract]
Plempel, M. (1984). Antimycotic activity of Bay N 7133 in animal experiments. J Antimicrob Chemother 13, 447-463.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Spizzo, T., Byersdorfer, C., Duesterhoeft, S. & Eide, D. (1997). The yeast FET5 gene encodes a FET3-related multicopper oxidase implicated in iron transport. Mol Gen Genet 256, 547-556.[Medline]
Stearman, R., Yuan, D. S., Yamaguchi-Iwai, Y., Klausner, R. D. & Dancis, A. (1996). A permeaseoxidase complex involved in high-affinity iron uptake in yeast. Science 271, 1552-1557.[Abstract]
Sweet, S. P. & Douglas, L. J. (1991). Effect of iron concentration on siderophore synthesis and pigment production by Candida albicans. FEMS Microbiol Lett 80, 87-92.
Swoboda, R. K., Bertram, G., Budge, S., Gow, N. A. R., Gooday, W. & Brown, A. J. P. (1995). Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans. Infect Immun 63, 4506-4514.[Abstract]
Yamaguchi-Iwai, Y., Dancis, A. & Klausner, R. D. (1995). AFT1: a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae. EMBO J 14, 1231-1239.[Abstract]
Yuan, D. S., Stearman, R., Dancis, A., Dunn, T., Beeler, T. & Klausner, R. D. (1995). The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake. Proc Natl Acad Sci USA 92, 2632-2636.[Abstract]
Received 31 December 1999;
revised 6 May 1999;
accepted 14 May 1999.