The Aspergillus nidulans GATA Factor SREA Is Involved in Regulation of Siderophore Biosynthesis and Control of Iron Uptake*

Hubertus HaasDagger , Ivo Zadra, Georg Stöffler, and Klaus Angermayr

From the Department of Microbiologie Medical School, University of Innsbruck, Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria

    ABSTRACT
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Abstract
Introduction
References

A gene encoding a new GATA factor from Aspergillus nidulans, sreA, was isolated and characterized. SREA displays homology to two fungal regulators of siderophore biosynthesis: about 30% overall identity to SRE from Neurospora crassa and about 50% identity to URBS1 from Ustilago maydis over a stretch of 200 amino acid residues containing two GATA-type zinc finger motifs and a cysteine-rich region. This putative DNA binding domain, expressed as a fusion protein in Escherichia coli, specifically binds to GATA sequence motifs. Deletion of sreA results in derepression of L-ornithine-N5-oxygenase activity and consequently in derepression of the biosynthesis of the hydroxamate siderophore N,N',N''-triacetyl fusarinine under sufficient iron supply in A. nidulans. Transcription of sreA is confined to high iron conditions, underscoring the function of SREA as a repressor of siderophore biosynthesis under sufficient iron supply. Nevertheless, overexpression of sreA does not result in repression of siderophore synthesis under low iron conditions, suggesting additional mechanisms involved in this regulatory circuit. Consistent with increased sensitivity to the iron-activated antibiotics phleomycin and streptonigrin, the sreA deletion mutant displays increased accumulation of 59Fe. These results demonstrate that SREA plays a central role in iron uptake in addition to siderophore biosynthesis.

    INTRODUCTION
Top
Abstract
Introduction
References

All microorganisms, with the exception of certain lactobacilli that utilize manganese and cobalt as biocatalysts in place of iron, require iron for their growth (1). The electron transfer ability of the iron atom, modified by diverse ligand environments, makes it essential for redox reactions ranging from respiration to ribonucleotide synthesis. Although iron is the fourth most abundant element in the earth crust, the amount of naturally utilizable iron is very limited for organisms as most of this metal exists in extremely insoluble complexes of ferric hydroxide in nature. On the other hand, excess of free iron in the cell is detrimental, because iron can catalyze the production of cell-damaging hydroxyl radicals in the presence of oxygen. Therefore, the concentration of iron in biological fluids is tightly controlled. Because most species lack an excretory route for iron, control is primarily accomplished by regulating the rate of iron uptake. Thereby, diverse species, from Escherichia coli to human, have developed various and most often multiple iron transport mechanisms.

The genus Aspergillus is one of the most ubiquitous microorganisms worldwide and various Aspergillus species are facultative pathogens involved in human infections capable of causing severe diseases like allergic bronchopulmonary aspergillosis, aspergilloma, and invasive pulmonary aspergillosis. In particular, invasive pulmonary aspergillosis represents a life-threatening disease of increasing incidence in immunocompromised patients, which has been attributed to increasing iatrogenic immunosuppression and successful life-support measures for neutropenic patients. Because iron is tightly sequestered in mammalian hosts by high affinity iron-binding proteins, microbes require efficient iron-scavenging systems to survive and proliferate within the host. Under conditions of iron starvation, most fungi synthesize and excrete low molecular weight, iron-specific chelators called siderophores, which have therefore often been proposed as virulence factors. Siderophore biosynthesis and its impact on bacterial pathogenicity is well established (1). In contrast, the knowledge of the biochemical basis for siderophore biosynthesis and its regulation on the molecular level is rather fragmentary in eukaryotic organism. This may be in part because of the fact that the leading model organism for molecular genetic analysis, Saccharomyces cerevisiae, lacks the ability to synthesize siderophores, although it can utilize siderophores produced by other species (2). In the basidiomycete Ustilago maydis, a gene encoding a transcriptional repressor of siderophore biosynthesis belonging to the GATA protein family of transcription factors has been characterized (3). Subsequent genetic and molecular studies have shown that URBS1 acts as a transcriptional repressor by direct binding to GATA motifs in the promoter of at least sid1, encoding L-ornithine N5-oxygenase, the first committed step in the biosynthesis of the two Ustilago siderophores ferrichrome and ferrichrome A (4, 5).

In a general search for GATA factor-encoding genes, we have recently isolated a gene from the ascomycete Penicillium chrysogenum displaying significant homology to Ustilago urbs1, and the disruption of the corresponding gene from Neurospora crassa proved that this gene encodes a repressor of siderophore biosynthesis (6, 7). In this study, we report the cloning and characterization of sreA, a GATA factor-encoding gene responsible for repression of siderophore biosynthesis in Aspergillus nidulans and show its involvement in the control of iron uptake. This study represents a first important step in the investigation of the regulation of siderophore biosynthesis in Aspergilli and provides the basis for evaluation of the impact of iron metabolism on pathogenicity in these fungal species.

    EXPERIMENTAL PROCEDURES

Strains, Vectors, Growth Media, and General Molecular Techniques-- The vectors and plasmids were propagated in E. coli DH5alpha supplied from Life Technologies, Inc. Generally, A. nidulans strain A4 (Glasgow wild type) provided from the Fungal Genetic Stock Center, Kansas City, KS, was used. For fungal transformation, A. nidulans strain WG355 (biA1 bgaO argB2), kindly provided by Dr. A. Brakhage, was used (8). SRKO1 and SROE3, generated in this work (see below), represent A. nidulans strains with a deletion of the sreA gene and a strain overexpressing sreA, respectively. Generally, A. nidulans was grown in minimal medium according to Pontecorvo et al. (9) containing 1% glucose as the carbon source and 30 mM ammonium tartrate as the nitrogen source. High iron medium contained 10 µM FeSO4, and for preparation of low iron medium, addition of FeSO4 was omitted. If required, biotin (20 µg/liter) or L-arginine (200 mg/liter) was added to the media. YAG (2% glucose, 0.5% yeast extract, 2% agar, trace elements) was used as complete medium.

Standard molecular techniques were performed as described by Sambrook et al. (11). Fungal DNA was isolated according to Yelton et al. (12); for RNA isolation, RNAzolTM (Biotex Laboratories, Inc) was used.

Preparation of Maltose-binding Protein-SREA Fusion Proteins-- For expression of the N-terminal SREA zinc finger (NZF), a fragment encoding the SREA amino acids 80-175 was amplified from cDNA employing the primers 5'-TTTTGAATTCGAGACTCCAATGAACG (OSR1) and 5'-TTTTCTGCAGTTAACCTTCGCTTCCAGTA (OSR2), carrying add-on restriction enzyme cleavage sites for EcoRI and PstI. Subsequently, the amplified product was cleaved with EcoRI and PstI and ligated into the respective restriction sites of pMal-cRI (New England Biolabs). After verification of proper integration of the relevant fragment into the plasmid by sequencing, the resulting plasmid was transformed into E. coli DH5alpha . Expression and purification of the maltose-binding protein-NZF fusion protein were carried out according to the manufacturer's recommendations. For expression of the C-terminal SREA zinc finger (CZF, amino acid residues 229-315) and the peptide containing both zinc fingers (NCZF, amino acid residues 80-315), the same strategy was employed. For amplification of the CZF-encoding fragment primers, 5'-TTTTGAATTCCCGTCTCCAGAGGCTGA (OSR5) and 5'-TTTTCTGCAGTTAATGGGTAGCAGCAGTC (OSR6) were used, and the NCZF-encoding fragment was amplified employing the primers OSR1 and OSR6.

Northern Analysis-- Generally, 15 µg of total RNA was electrophoresed on 1.2% agarose-2.2 M formaldehyde gels and blotted onto Hybond N membranes (Amersham Pharmacia Biotech). Hybridization probes were generated by PCR1 using oligonucleotides 5'-CTCGCCCCCATACTAAA and 5'-CTTGCTATCATTCTTGC for the sreA probe A, 5'-TTTCGAGTCGCTAGGCT and 5'-TCCGTCCTCTCCCCTTT for the sreA probe B, 5'-TTCGCTCCGTACTCAAG and 5'-GAGTAGCGACAGCAATG for the argB probe C, 5'-ATCGCCAGAAGCATCGT and 5'-ACTGAAAAGGTTATCGCT for the sreA probe D (see Fig. 4), and 5'-CGGTGATGAGGCACAGT and 5'-CGGACGTCGACATCACA for actA (GenBankTM accession number U61733).

Electrophoretic Mobility Shift Assays of DNA-Protein Interactions-- The 130-bp fragment (F130) containing two GATA core elements described in Haas et al. (10) was labeled by end-filling of 5' overhangs with Klenow DNA polymerase (Boehringer Mannheim) and [alpha 32P]dATP (11). For assessing binding specificity, each oligonucleotide described in Fig. 3 was annealed with its complementary oligonucleotide by incubation in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl for 5 min at 90 °C and subsequent cooling to 4 °C. Radioactive end-labeling was performed by filling in with Klenow fragment of DNA polymerase I using [alpha -32P]dCTP (11). Electrophoretic mobility shift assays were carried out as described for NREB (10).

Gene Disruption and Overexpression of sreA-- For disruption of sreA, the 2.4-kbp NcoI-NsiI fragment of the sreA containing the 4.6-kbp EcoRI fragment was replaced by the 2.6-kbp PstI-BamHI argB-encoding fragment from pILJ16 (13) subsequent to blunt-ending of the NcoI and BamHI restriction sites by filling in using Klenow fragment of E. coli polymerase I. pILJ16 was provided by Dr. M. X. Caddick (University of Liverpool, UK). The resulting plasmid was digested with EcoRI, and the 4.8-kbp fragment was gel-purified before transformation of A. nidulans strain WG355.

For overexpression of sreA in A. nidulans, a translational fusion of the sreA-coding region with the gpdA promoter was constructed. Therefore, a 1.8-kbp sreA fragment was amplified from genomic DNA employing primers 5'-TTCTTTGGATCCACTTCTCTTGTCATTGA and 5'-TTCTTTCCATGGTAGCGTCCATCCCGCAC carrying add-on restriction enzyme cleavage sites for NcoI and BamHI, as primers. Subsequently, the amplified product was cleaved with NcoI and BamHI and ligated into the respective restriction sites of pAN52-1 (Ref. 14; GenBankTM accession number Z32697). pAN52-1 was provided by Dr. P. Punt (TNO, Nutrition and Food Research Institute, The Netherlands). The junctions as well as the coding regions of the fused product were verified by sequencing. The resulting plasmid was co-transformed into A. nidulans WG355, with the argB carrying vector pILJ16.

Transformation of A. nidulans was carried out according to Tilburn et al. (15). Screening of positive clones was performed by PCR from fungal colonies. For screening of sreA disruption strains, the primers Osre, 5'-CGCTAATCCCGCCATCG, and Oarg, 5'-TTCCTCTGCTGCGTCCG, were employed (see Fig. 4). To obtain homokaryotic transformants, colonies from single homokaryotic spores were picked, and genomic integration was analyzed by Southern blot analysis.

Identification and Quantification of Siderophores-- The siderophore concentration was determined by means of the ferric perchlorate assay, the chrome azurol S liquid assay, and quantitative HPLC analysis (16, 17). For reversed phase HPLC analysis of siderophores according to Konetschny-Rapp et al. (17), siderophores were isolated according to the procedure described for isolation of ferrichrome from Ustilago sphaerogena (16). Isolated ferric siderophores were photometrically quantified at 435 nm. The siderophores neocoprogen I, neocoprogen II, coprogen, and triacetylfusarinin C used as standards for HPLC analysis were a gift of Dr. G. Winkelmann (Universität Tübingen, Germany).

L-Ornithine-N5-oxygenase Enzyme Assay-- L-ornithine-N5-oxygenase enzyme activity was determined as already described (7).

Quantification of Resistance/Sensitivity to Drugs-- Quantification of resistance/sensitivity of A. nidulans to phleomycin and streptonigrin was performed as described by de Souza et al. (18). 109 conidia of the respective Aspergillus strain were spread onto a YAG plate and incubated for 12 h at 37 °C to produce a nonsporulating mycelial mat. Mycelial "plugs" were cut out from the mats using the wide end of a sterile Pasteur pipette and transferred in three repetitions onto plates containing various drugs or chelators mentioned below and incubated for 72 h at 37 °C. The diameters of the resulting colonies were measured, and the radial growth was compared with the average diameter of colonies on zero-dose plates expressed in %. Subsequently, the growth rate of the respective strain was then correlated to that of the A. nidulans wild type. This technique avoids problems of delayed germination and ensures measurement of true exponential-phase growth rate. Drugs were used in YAG with the following concentration: phleomycin (Sigma), 50 µg/ml; streptonigrin (Sigma), 10 µg/ml; benomyl (Sigma), 1 µg/ml; EDTA and bathophenanthrolinedisulfonic acid, 0.1 mM each. In the case of low iron conditions, minimal medium lacking iron was used, and growth was compared with that on minimal medium containing 10 µM FeSO4.

Iron Uptake-- For studying iron uptake, plates containing 50 nCi of 59FeCl3 (Amersham Pharmacia Biotech)/ml of medium were overlaid with a dialysis membrane (Serva, 67 mm). Subsequently, mycelial plugs of the different Aspergillus strains, prepared as described in Quantification of Resistance/Sensitivity to Drugs (18), were transferred on top of these dialysis membranes. After incubation for 72 h at 37 °C, the dialysis membranes carrying the fungal colonies were removed from the plates, and the 59Fe content of the colonies was determined using a Phosphor Storage Imaging System, model Storm 840 (Molecular Dynamics).

    RESULTS AND DISCUSSION

Isolation and Characterization of sreA-- The various members of the GATA protein family are characterized by a high degree of similarity within their DNA binding domain. To isolate the sreP homologous gene from A. nidulans, a PCR-aided strategy similar to the one used for the cloning of the GATA factor-encoding genes sreP and nreB from P. chrysogenum was employed (6, 10). Using the degenerated primers 5'-ACNCCNYTNTGGMG and 5'-APNCCPCANGCPTTPCA (Y = T or C; M = A or C; N = any nucleotide; P = A or G) derived from the amino acid sequences Thr-Pro-Leu-Trp-Arg and Cys-Asn-Ala-Cys-Gly-Leu, conserved in most fungal GATA factors, a 56-bp fragment was amplified from genomic Aspergillus DNA. The obtained fragments were subcloned into plasmid vectors and sequenced whereby the deduced amino acid sequence of one fragment displayed only a single amino acid exchange compared with the corresponding region of SREP. The encoding fragment was radiolabeled and utilized to probe the Aspergillus cosmid library constructed in pWE15 provided by the Fungal Genetic Stock Center (19). Five hybridizing clones, W04B12, W09A09, W09E09, W10A09, and W10E09, were detected. According to the information made available by the Fungal Genetic Stock Center, the inserts of all five cosmids originate from chromosome VIII, localizing sreA to chromosome VIII as well. Subsequently, a 4.6-kbp EcoRI fragment carrying sreA was subcloned from cosmid W09E09 into pBluescriptKS and sequenced in its entirety. Additionally, 0.4 kbp of the 3'-downstream region of sreA have been sequenced directly from the cosmid W09E09. The transcription start points and the polyadenylation site of sreA were mapped by 5'- and 3'-rapid amplification of cDNA ends protocols according to Frohman et al. (20). Two major transcription start points were localized 1073 bp and 211 bp upstream of the putative translation start codon. The polyadenylation site was mapped 435 bp downstream of the translation stop codon. Two introns were mapped by amplification of the coding region from mRNA by reverse-transcribed PCR.

Comparison of the genomic and cDNA sequences revealed an open reading frame of 1647 bp interrupted by two introns, 55 and 60 bp in length. The 5' and 3' borders of the introns as well as the putative lariat formation sites perfectly match the consensus sequences for fungal introns (21). The deduced SREA protein displays a calculated mass of 58.8 kDa. Searches in several data bases using the BLAST alignment computer program confirmed that the cloned gene encodes a member of the GATA protein family (38). In contrast to all other identified fungal GATA factors, SREA as well as SREP from P. chrysogenum, SRE from N. crassa, URBS1 from U. maydis, and GAF2p from Schizosaccharomyces pombe contain two zinc fingers (3, 6, 7, 22). Additionally, the sequence homology in these five proteins is not limited to the two zinc finger regions but also extends into the intervening region, where a conserved 27-amino acid residue sequence with four cysteines is present (Fig. 1). Moreover, SREA, SREP, and SRE share a common highly conserved C terminus predicted to form a coiled coil structure by computer analysis using the ExPASy tools software package, indicative of a putative protein-protein interaction domain (23). SREA displays the highest overall identity to Penicillium SREP and Neurospora SRE with 61% and 35%, respectively.


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Fig. 1.   Alignment of A. nidulans SREA, P. chrysogenum SREP, N. crassa SRE, U. maydis URBS1, and S. pombe GAF2p. Amino acid residues identical in at least three of the compared proteins are boxed in gray. The GATA-type zinc fingers (GTZ), the cysteine-rich region (CRR), and the conserved C terminus (CT) are boxed in black.

The positions of the two introns are perfectly conserved in A. nidulans sreA, P. chrysogenum sreP, and N. crassa sre, dating the intron origin before the evolutionary split of the ancestors of these three ascomycetes (6, 7). In contrast, urbs1 from U. maydis and GAF2p from S. pombe do not contain any introns (3, 22).

Expression of sreA-- Northern blot analysis indicates that expression of sreA is sensitive to the iron content of the medium (Fig. 2). The steady state sreA mRNA levels are significantly elevated in mycelia grown in high iron medium or transferred from low iron medium into high iron medium. In contrast, in mycelia grown in low iron medium or transferred from high iron medium into low iron medium, no sreA transcripts can be detected. In this respect, transcription of sreA differs significantly from that of Ustilago urbs1 and Neurospora sre, where it was found to be constitutive (3, 7). Analogous to the situation in P. chrysogenum, sreA is expressed via two transcripts, 3.2 kb and 2.4 kb in length. Northern blot analysis employing two different hybridization probes, probe A placed in the region between the two major transcription start points and probe B originating from the coding region, proved that the two transcripts are indeed because of different initiations of transcription found by the 5'-rapid amplification of cDNA ends procedure (Fig. 2).


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Fig. 2.   Northern analysis of sreA expression in A. nidulans wild type, SRKO1, and SROE3. A. nidulans strains were grown for 36 h in high iron minimal medium (Fe+) or low iron minimal medium (Fe-). Subsequently, mycelial pads were washed and transferred from Fe+ into Fe- media (Fe-S) or from Fe- into Fe+ media (Fe+S), respectively, and grown for another hour. Total RNA was isolated from mycelia of the different growth conditions, separated on an agarose/formaldehyde gel, blotted onto membrane filter, and hybridized with the radiolabeled sreA probe A or B. Probe A corresponds to the region between the two transcription start sites, and probe B originates from the coding region (see Fig. 5). As the control for loading and quality of RNA, blots were hybridized with the gamma -actin-encoding gene of A. nidulans (39).

sreA Encodes a GATA-binding Protein-- GATA factors are defined as GATA-binding proteins that recognize the core motif GATA. To investigate whether sreA in fact encodes a DNA-binding protein, three SREA peptides containing the NZF, the CZF, and both zinc fingers (NCZF), respectively, were expressed as fusion products with the maltose-binding protein in E. coli and purified by affinity chromatography. In A. nidulans, an SREA-regulated gene has not been isolated so far; therefore, no putative target binding sequence is available. Recently it was shown that NIT-2, WC-1, WC-2, and NGF1, four GATA-factors of N. crassa that are involved in diverse regulatory circuits, bind to the same nucleotide sequence containing GATA motifs, thus displaying little preference for flanking regions (24). Therefore, a fragment containing two GATA motifs, which was already shown to bind the Penicillium GATA factor NREB, was employed to test the DNA binding potential of the SREA peptides (10). As shown in Fig. 3, both zinc fingers are required for specific DNA binding, at least to the sequence provided, because only NCZF was able to shift the 130-bp DNA fragment (F130). In contrast to the SREA homologous proteins, all other fungal GATA factors identified so far contain only a single zinc finger, which proved to be sufficient for specific DNA binding. On the other hand, the requirement of two GATA-type zinc fingers for recognition of certain DNA sequences has been demonstrated for vertebrate GATA factors: in murine and chicken GATA-1, the C-terminal finger is sufficient for specific DNA binding and certain functions in vivo, but the N-terminal finger can help to achieve binding specificity and stability, whereby binding to palindromic GATA sites is dependent on the presence of both finger domains (25-28). Interestingly, a mutation of the conserved amino acid residue Arg-350 in the N-terminal URBS1 zinc finger does not affect regulation of siderophore biosynthesis in U. maydis, whereby the corresponding mutation in the C-terminal zinc finger abolishes the function of URBS1 (4). It cannot be ruled out that the C-terminal finger of SREA might recognize specific DNA sequences on its own, but for recognition of the provided sequence, obviously both fingers are necessary.


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Fig. 3.   Electrophoretic gel mobility shift analysis of SREA and target DNA. A, sequences of the 48-bp oligonucleotides used in gel mobility shift analysis. O1 contains both wild-type GATA motifs present in F130, O2 contains only the 5'-GATA motif, O3 contains only the 3'-site, and O4 lacks any GATA sequence. B, gel shift analysis employing 32P-labeled F130, the 130-bp fragment of the nreB 5'-upstream region, or radiolabeled O1-O4. NCZF represents a maltose-binding fusion protein containing both the N- and C-terminal zinc fingers of SREA. NZF and CZF contain only the N-terminal or the C-terminal zinc finger, respectively. The amount of fusion protein used was 0.2 µg.

To investigate the involvement of the two GATA motifs present in F130, 48 bp of double-stranded oligonucleotides containing both GATA motifs as well as mutations of one or both sites were employed in mobility shift analysis. As shown in Fig. 3, the mutation of either single GATA site has no apparent effect on the in vitro binding of NCZF, whereas the mutation of both GATA sites eliminates binding. These results prove that sreA indeed encodes a GATA-binding protein. In comparison, Ustilago URBS1 also specifically binds to a single GATA motif in the sid1 promoter in vitro, but for in vivo function, two clustered sites are necessary (5).

sreA Encodes a Repressor of Siderophore Biosynthesis-- Disruption of sreA was achieved using the 4.6-kbp EcoRI sreA genomic fragment in which the promoter region and the first two exons of sreA were replaced by the argB-encoding gene from A. nidulans (Fig. 4). The resulting 4.8-kbp EcoRI fragment was used to transform an argB- strain of A. nidulans (WG355). Aspergillus argB+/sreA- strains were isolated and characterized by PCR (using primers Osre and Oarg, see Fig. 4). Southern blot analysis proved that the sreA disruption strain, called SRKO1, does not contain the first two sreA exons (probe B) and that these exons are replaced by the argB gene, resulting in a 4.8-kbp EcoRI fragment instead of the original 4.6-kbp fragment (probe C), also confirmed by hybridization with probe D, originating from the third sreA exon (Fig. 4). Moreover, in SRKO1, no sreA transcripts could be detected (Fig. 2).


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Fig. 4.   Deletion of the sreA gene in A. nidulans. A and B, restriction maps of the 4.6- and 4.8-kbp EcoRI fragments containing the wild-type sreA sequence (A) and the construct used for sreA deletion (B), respectively. Open boxes represent sreA exons, and the gray box marks the argB gene. Osre and Oarg symbolize primers used for PCR screening of sreA deletion mutants. Hatched boxes represent the hybridization probes used in Northern and Southern analysis. tsp and pas mark the transcription start points and the polyadenylation site, respectively. C, Southern analysis of the sreA deletion strain SRKO1 in comparison to the wild type.

To check if sreA indeed encodes a regulator of siderophore biosynthesis, Aspergillus wild-type and SRKO1 strain were grown in high iron and low iron medium, respectively, and siderophore production was analyzed and quantified by reversed phase HPLC (Fig. 5). The amount of siderophores produced was confirmed employing the perchlorate assay and the chrome azurol S liquid assay (data not shown). As described by Charlang et al. (29), A. nidulans wild type was found to excrete exclusively the siderophore type N,N',N''-triacetyl fusarinine C in significant amounts, which holds for most other Aspergilli but only under iron starvation conditions (30). In contrast, SRKO1 produced siderophores irrespective of the iron content of the growth medium, whereby again only N,N',N''-triacetyl fusarinine C was found in the culture broth. However, siderophore production was not completely derepressed in SRKO1 under high iron conditions; it reached only about 10-20% the amount produced under iron starvation. These data indicate that in addition of lifting of repression by SREA, other factors are presumably involved in the expression of siderophore biosynthetic genes that are not active under sufficient iron supply as proposed for N. crassa (7).


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Fig. 5.   Reversed phase HPLC analysis of siderophore production of A. nidulans wild type, SRKO1, and SROE3. The Aspergillus strains were grown for 48 h in high iron minimal medium (Fe+) or low iron minimal medium (Fe-). Subsequently, siderophores were isolated from 50 ml of growth medium as described under "Experimental Procedures" and analyzed. As siderophore standards, neocoprogen I (NI), neocoprogen II (NII), coprogen (C), and N,N',N''-triacetylfusarinin C (TFC) were used. The absorption at 380 nm is denoted by milliabsorption units (mAU).

L-ornithine-N5-oxygenase represents the first committed step in the biosynthesis of most hydroxamate type siderophores (31). In Aspergillus wild type, only 10% of this enzyme activity can be detected under growth with high iron supply relative to growth under iron limiting conditions. In contrast, SRKO1 contains only about 70% L-ornithine-N5-oxygenase activity compared with wild type but similar enzyme activity in high and low iron conditions (data not shown), indicating that the expression of the encoding gene is under control of SREA, comparable with the situation in U. maydis (31).

Because transcription of sreA is confined to high iron conditions, it was tempting to speculate that constitutive expression of sreA might block siderophore biosynthesis under low iron conditions. Therefore, the sreA-coding region was placed under control of the promoter of the glycerine aldehyde 3-phosphate dehydrogenase (gpdA)-encoding gene using the A. nidulans expression vector pAN52-1 and co-transformed with a plasmid carrying the argB gene into WG355. Aspergillus strains carrying the sreA expression cassette were screened by Southern blot analysis after selection for arginine prototrophy. In SROE3, an Aspergillus strain containing a single copy of the sreA expression cassette (data not shown), siderophore production was unaffected under iron starvation despite high level constitutive sreA transcription, as proved by Northern analysis (Fig. 2). Important to note, the sreA mRNA is significantly shorter in SROE3, because in the sreA expression vector, the long sreA 5'-untranslated region is replaced by the small one of the gpdA gene. These data indicate that posttranscriptional regulation is involved in the activation of repressor function of SREA as proposed for the respective corresponding gene products in N. crassa and U. maydis. In the latter two organisms, transcription of sre and urbs1, respectively, was found to be constitutive. As with the prokaryotic functional homologue of siderophore repressors (FUR) or the iron regulatory element-binding protein, a regulator of iron homeostasis in mammals, SREA repressor function might be directly activated by iron (31-33). Such a mechanism has already been proposed for Ustilago, because URBS1 contains at least three motifs possibly participating in direct binding of iron: a histidine track at the C terminus, the zinc fingers themselves, and a cysteine-rich region intervening between the two finger domains. SREA, SREP, and SRE lack a histidine track, but the cysteine-rich region is perfectly conserved, making it a good candidate for such a function (Fig. 1). An alternative explanation is the absence of active co-factors for exertion of SREA function in low iron conditions. The requirement for interaction of a GATA factor and a pathway-specific factor for activation of target gene expression has been shown recently for NIT2 and NIT4 in N. crassa (24). A similar mechanism is also conceivable for repression of gene expression. Alternatively or additionally, it is possible that posttranscriptional control mediated by the long sreA 5'-untranslated region, which is missing in the constitutively expressed sreA in SROE3, is involved in the repression of siderophore biosynthesis by sreA.

sreA Deletion Leads to Increased Iron Uptake-- It was conceivable that SREA acts not only as a repressor of siderophore biosynthesis but also plays a more central role in iron homeostasis. Therefore, the link between iron and SREA was further investigated by the use of the antibiotics phleomycin and streptonigrin to assess iron levels in the SREA mutant and wild-type strains. Phleomycin and streptonigrin interact with intracellular iron pools, causing the formation of reactive oxygen species, which results in DNA damage and eventual cell death (34-36). Thus, sensitivity to these drugs can be used to estimate intracellular iron levels. The sensitivity of the A. nidulans strains WG355, SRKO1, and SROE3 was tested relative to the wild type by measuring the hyphal extension of germinating mycelial plugs on YAG or low iron minimal medium containing phleomycin and streptonigrin, respectively. The sreA deletion strain SRKO1 showed an approximate 50 and 60% reduced hyphal growth on phleomycin and streptonigrin, respectively, compared with the wild type. In contrast, both WG355 and the sreA-overexpressing strain SROE3 displayed growth rates similar to the wild type (Fig. 6). SRKO1 was offered some protection against phleomycin through limitation of available iron by the addition of the chelators EDTA and bathophenanthrolinedisulfonic acid to the medium or by growth on low iron medium. At growth on the chelators EDTA and bathophenanthrolinedisulfonic acid or the antibiotic benomyl, the growth rates of the four strains tested were similar. These results suggest that the effects of phleomycin and streptonigrin are indeed related to the levels of iron and that SRKO1 contains elevated levels of free intracellular Fe2+.


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Fig. 6.   Resistance/sensitivity of A. nidulans wild-type, SRKO1, SROE3, and WG355 to phleomycin and streptonigrin. Bars represent the growth rate normalized to the growth rate of the wild type on the respective medium. The data shown are mean values of three independent experiments (S.D. did not exceed 12%). BPDA, bathophenanthrolinedisulfonic acid.

To directly measure if SRKO1 assimilates higher levels of iron, SRKO1, SROE3, WG355, and the Aspergillus wild-type strain were grown 72 h at 37 °C on agar plates containing 59Fe(III), whereby the fungal colonies were separated from the growth medium by a dialysis membrane. Subsequently the content of radioactivity of the colonies was quantified using a Phosphor Storage Imaging System. Fig. 7 shows that SRKO1 colonies accumulate about 2.5 times more 59Fe compared with all other strains when grown under sufficient iron supply. Under iron-limiting conditions, no significant differences in the iron uptake of the four strains were found.


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Fig. 7.   Iron uptake in A. nidulans wild-type, SRKO1, SROE3, and WG355. A, mycelial plugs of Aspergillus strains (see "Experimental Procedures") were grown for 72 h on low and high iron minimal medium plates containing 50 nCi of 59FeCl3, whereby the fungal colonies were separated from the growth medium by a dialysis membrane. Subsequently, the 59Fe content of the fungal colonies was quantified using a Phospho-Imager. B, quantification of 59Fe uptake normalized with respect to the wild type on the respective medium using the PhosphorImager. Bars represent the mean values of three independent experiments (S.D. did not exceed 17%).

In summary, the results clearly demonstrate that the sreA deletion strain exhibits significantly enhanced iron uptake and increased intracellular iron content under conditions of sufficient iron supply, indicating that SREA controls iron uptake in addition to siderophore biosynthesis in Aspergillus. The simplest explanation for increased iron uptake in SRKO1 might be that not only the siderophore biosynthetic genes but also the gene(s) encoding the siderophore receptor(s) is subject to repression by SREA and therefore is expressed and active in an iron-rich environment in addition to the low affinity iron uptake system, which is probably active in both the sreA wild type and the mutant strain. Alternatively, siderophore-independent, high affinity iron uptake systems might be controlled by SREA. A theoretical third possibility is of course that the siderophore receptor is constitutively active under both iron-sufficient and iron-deficient conditions. In contrast to wild type, SRKO1 could take advantage of the siderophore-dependent iron transport system in addition to the low affinity system because it constitutively produces siderophores. However, because of the toxicity of excess intracellular iron, constitutive activation of such a high affinity iron transport system seems unlikely. In this respect it is important to note that in E. coli and probably most bacteria, all genes involved in high affinity iron uptake are negatively regulated by iron and probably by FUR or its functional homologs orthologs (1). This regulatory circuit is probably also conserved in fungi as well, e.g. transport rates for 55Fe-labeled siderophores were found to be enhanced about 5-fold in low iron-grown cells in N. crassa (37). The clarification of the exact mechanism requires the disruption of a siderophore biosynthetic gene in SRKO1. Because such a gene has not been identified in Aspergillus, this is not possible at the moment. Noteworthy, it has been impossible to isolate such a mutant strain, defective in both sid1 and urbs1, in U. maydis, suggesting that deregulation of iron uptake without a means of intracellular iron chelation by siderophores may be lethal (31).

The study presented shows that the mode of regulation of siderophore biosynthesis is conserved in the basidiomycete U. maydis and the ascomycetes A. nidulans, N. crassa, and probably also in P. chrysogenum. The cloning of Aspergillus sreA represents the first step in the molecular analysis of iron regulation in Aspergillus and will facilitate the isolation and characterization of the siderophore biosynthetic genes. The investigation of the siderophore-dependent iron uptake is also of significant medical importance, because this system might resemble a pathogenicity factor necessary for infection by the opportunistic genus Aspergillus.

    ACKNOWLEDGEMENTS

We are grateful to Dr. K. Haselwandter and Ing. M. Dexler for the siderophore analysis and D. Alber and H. Oberegger for technical assistance. We also thank Dr. A. Brakhage, Dr. M. X. Caddick, Dr. P. Punt, and Dr. G. Winkelmann for their gift of Aspergillus strains, vectors, and siderophore standards.

    FOOTNOTES

* This work was supported in part by Austrian Science Foundation Grant FWF-P13202-MOB (to H. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF095898.

Dagger To whom correspondence should be addressed. Tel.: 43-512-507-3608; Fax: 43-512-507-2866; E-mail: hubertus.haas{at}uibk.ac.at.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s); HPLC, high performance liquid chromatography; NZF, N-terminal SREA zinc finger; CZF, C-terminal SREA zinc finger; NCZF, both zinc fingers.

    REFERENCES
Top
Abstract
Introduction
References
  1. Guerinot, M. L. (1994) Annu. Rev. Microbiol. 48, 743-772[CrossRef][Medline] [Order article via Infotrieve]
  2. Askwith, C. C., de Silva, D., and Kaplan, J. (1996) Mol. Microbiol. 20, 27-34[Medline] [Order article via Infotrieve]
  3. Voisard, C., Wang, J., McEvoy, J. L., Xu, P., and Leong, S. A. (1993) Mol. Cell. Biol. 13, 7091-7100[Abstract]
  4. An, Z., Zhao, Q., McEvoy, J., Yuan, W. M., Markley, J. L., and Leong, S. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5882-5887[Abstract/Free Full Text]
  5. An, Z., Mei, B., Yuan, W. M., and Leong, S. A. (1997) EMBO J. 16, 1742-1750[Abstract/Free Full Text]
  6. Haas, H., Angermayr, K., and Stöffler, G. (1997) Gene 184, 33-37[CrossRef][Medline] [Order article via Infotrieve]
  7. Zhou, L., Haas, H., Feng, B., and Marzluf, G. A. (1997) Mol. Gen. Genet. 259, 532-540[CrossRef]
  8. Van Gorcom, R. F., Punt, P. J., Pouwels, P. H., and van den Hondel, C. A. (1986) Gene 48, 211-217[CrossRef][Medline] [Order article via Infotrieve]
  9. Pontecorvo, G., Roper, J. A., Hemmons, L. M., MacDonald, K. D., and Bufton, A. W. J. (1953) Adv. Genet. 5, 141-238
  10. Haas, H., Angermayr, K., Zadra, I., and Stöffler, G. (1997) J. Biol. Chem. 272, 22576-22582[Abstract/Free Full Text]
  11. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, NY
  12. Yelton, M. M., Hamer, J. E., and Timberlake, W. E. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1470-1474[Abstract]
  13. Upshall, A., Gilbert, T., Saari, G., O'Hara, P. J., Weglenski, P., Berse, B., Miller, K., and Timberlake, W. E. (1986) Mol. Gen. Genet. 204, 349-354[Medline] [Order article via Infotrieve]
  14. Punt, P. J., Zegers, N. D., Busscher, M., Pouwels, P. H., and van den Hondel, C. A. (1991) J. Biotechnol. 17, 19-33[CrossRef][Medline] [Order article via Infotrieve]
  15. Tilburn, J., Sarkar, S., Widdick, D. A., Espeso, E. A., Orejas, M., Mungroo, J., Penalva, M. A., and Arst, H. N., Jr. (1995) EMBO J. 14, 779-790[Abstract]
  16. Payne, S. M. (1994) Methods Enzymol. 235, 329-344[Medline] [Order article via Infotrieve]
  17. Konetschny-Rapp, S., Huschka, H. G., Winkelmann, G., and Jung, G. (1988) Biol. Met. 1, 9-17[Medline] [Order article via Infotrieve]
  18. de Souza, C. C., Pellizzon, C. H., Hiraishi, M., Goldman, M. H., and Goldman, G. H. (1998) Curr. Genet. 33, 60-69[CrossRef][Medline] [Order article via Infotrieve]
  19. Brody, H., Griffith, J., Cuticchia, A. J., Arnold, J., and Timberlake, W. E. (1991) Nucleic Acids Res. 19, 3105-3109[Abstract]
  20. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8998-9002[Abstract]
  21. Gurr, S. J., Uncles, S. E., and Kinghorn, J. R. (1987) in Gene Structure in Eucaryotic Microbes (Kinghorn, J. R., ed), pp. 93-193, IRL Press at Oxford University Press, Oxford
  22. Hoe, K. L., Won, M. S., Yoo, O. J., and Yoo, H. S. (1996) Biochem. Mol. Biol. Int. 39, 127-135[Medline] [Order article via Infotrieve]
  23. Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164[Medline] [Order article via Infotrieve]
  24. Feng, B., and Marzluf, G. A. (1998) Mol. Cell. Biol. 18, 3983-3990[Abstract/Free Full Text]
  25. Visvader, J. E., Crossley, M., Hill, J., Orkin, S. H, and Adams, J. M. (1995) Mol. Cell. Biol. 15, 634-641[Abstract]
  26. Martin, D. I., and Orkin, S. H. (1990) Genes Dev. 4, 1886-1898[Abstract]
  27. Trainor, C. D., Omichinski, J. G., Vandergon, T. L., Gronenborn, A. M., Clore, G. M., and Felsenfeld, G. (1996) Mol. Cell. Biol. 16, 2238-2247[Abstract]
  28. Yang, H. Y., and Evans, T. (1992) Mol. Cell. Biol. 12, 4562-4570[Abstract]
  29. Charlang, G., Horowitz, R. M., Lowy, P. H., Ng, B., Poling, S. M., and Horowitz, N. H. (1982) J. Bacteriol. 150, 785-787[Medline] [Order article via Infotrieve]
  30. Nilius, A. M. (1997) in Fungal Disease (Jacobs, P. H., and Nall, L., eds), pp. 401-411, Marcel Dekker, Inc., New York
  31. Leong, S. A., and Winkelmann, G. (1998) Met. Ions Biol. Syst. 35, 147-186[Medline] [Order article via Infotrieve]
  32. Bagg, A., and Neilands, J. B. (1987) Microbiol. Rev. 51, 509-518
  33. Rouault, T., and Klausner, R. (1997) Curr. Top. Cell. Regul. 35, 1-19[Medline] [Order article via Infotrieve]
  34. Moore, C. W. (1989) Cancer Res. 49, 6935-6940[Abstract]
  35. Moore, C. W. (1994) Antimicrob. Agents Chemother. 38, 1615-1619[Abstract]
  36. White, J. R., and Yeowell, H. N. (1982) Biochem. Biophys. Res. Commun. 106, 407-411[Medline] [Order article via Infotrieve]
  37. Huschka, H. G., and Winkelmann, G. (1989) Biol. Met. 2, 108-113[Medline] [Order article via Infotrieve]
  38. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
  39. Fidel, S., Doonan, J. H., and Morris, N. R. (1988) Gene 70, 283-293[CrossRef][Medline] [Order article via Infotrieve]


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