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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
Strains, Vectors, Growth Media, and General Molecular
Techniques--
The vectors and plasmids were propagated in E. coli DH5
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 DH5
.
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 [
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
[
-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).
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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.
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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 -actin-encoding gene of A. nidulans
(39).
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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.
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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.
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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).
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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.
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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%).
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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.