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The Intracellular Siderophore Ferricrocin Is Involved in Iron Storage, Oxidative-Stress Resistance, Germination, and Sexual Development in Aspergillus nidulans

ABSTRACT Iron is required by most organisms, but an excess of this metal is potentially toxic. Consequently, uptake and intracellular storage of iron are tightly controlled. The filamentous fungus A. nidulans lacks the iron storage compound ferritin but possesses an intracellular siderophore, which is accumulated in a highly regulated manner as iron-free desferri-ferricrocin or iron-containing ferricrocin via transcriptional regulation of the nonribosomal peptide synthetase SidC. Biosynthesis of desferri-ferricrocin was low during iron-replete conditions but up-regulated by both iron starvation and intracellular iron excess, the latter caused by either a shift from iron-depleted to high-iron conditions or deregulation of iron uptake. Consequently, ferricrocin constituted only about 5% of the total iron content under iron-replete conditions but up to 64% during conditions of intracellular excess. In contrast, during iron starvation, desferri-ferricrocin was accumulated, which appears to represent a proactive strategy to prevent iron toxicity. Accumulation of the intracellular siderophore was also up-regulated by oxidative stress, which underscores the intertwining of iron metabolism and oxidative stress. Lack of the intracellular siderophore causes pleiotropic effects, as SidC deficiency results in (i) less-efficient utilization of iron, indicated by reduced growth under iron-depleted conditions and a higher iron demand under iron-replete conditions, (ii) delayed germination under iron-depleted conditions, (iii) increased sensitivity of conidia to oxidative stress, and (iv) elimination of cleistothecia formation in homothallic conditions.

Iron is essential for all eukaryotes and most prokaryotes, as it serves as a cofactor in many enzymes and as a catalyst in electron transport systems. Excess or incorrect storage of iron, however, is toxic, as this metal has the capacity to reinforce the production of reactive oxygen species (14). Therefore, iron homeostasis is designed to maintain sufficient iron supply while, at the same time, preventing excess iron accumulation and minimizing cell-damaging effects.
In contrast to bacteria, plants, and animals (2,3), most fungi lack ferritin-mediated iron storage and detoxification and, therefore, have developed alternative strategies. Most fungi excrete siderophores, which are low-molecular-mass, ferriciron-specific chelators, to mobilize extracellular iron but also produce intracellular siderophores (11,36). The function of the latter has not been studied in great detail so far due to the fact that Saccharomyces cerevisiae, which serves as a paradigm for fungal metabolism, can utilize iron bound to siderophores produced by other organisms but lacks the ability to synthesize siderophores by itself (11,30,35). Therefore, the vast knowledge of iron homeostasis in this yeast is of limited value for our understanding of iron uptake and storage in siderophore-producing fungi.
The ascomycetes Aspergillus nidulans and Aspergillus fumigatus are typical siderophore-producing fungi and are attrac-tive for various aspects. A. fumigatus is the most common airborne fungal pathogen of humans, causing life-threatening invasive disease in immunocompromised patients (20); A. nidulans is a much rarer cause of disease but represents a model filamentous fungus because it possesses a sexual cycle and is therefore amenable to classical genetic analysis (6). Both Aspergillus species produce two major hydroxamate-type siderophores: desferri-triacetylfusarinine C (TAFC ϪFe ) to mobilize extracellular iron and desferri-ferricrocin (FC ϪFe ), which is involved in intracellular iron storage (26,32). TAFC ϪFe is a cyclic tripeptide consisting of three N 2 -acetyl-N 5 -cis-anhydromevalonyl-N 5 -hydroxyorhithine residues linked by ester bonds, and FC ϪFe is a cyclic hexapeptide with the structure Gly-Ser-Gly-(N 5 -acetyl-N 5 -hydroxyornithine) 3 (11). After uptake of iron-loaded TAFC ϪFe , termed ferri-triacetylfusarinine C (TAFC ϩFe ), by specific transporters (12), the iron is transferred to metabolic pathways or stored by conversion of FC ϪFe to ferricrocin (FC ϩFe ). TAFC ϪFe is hydrolyzed by an esterase, and the hydrolysis products are excreted (26).
The first committed step of biosynthesis of both siderophores is the hydroxylation of ornithine catalyzed by N 5ornithine-monooxygenase, which is encoded by the sidA gene (9,32). Deficiency in sidA causes the complete absence of siderophores in both fungal species. A. nidulans ⌬sidA mutants are unable to grow unless supplemented with siderophores or high amounts of ferrous iron. In contrast, sidA-deficient A. fumigatus strains are viable due to the presence of a reductive iron assimilatory system, which is missing in A. nidulans. However, siderophore biosynthesis, but not reductive iron assimi-lation, has been found to be essential for A. fumigatus virulence in a murine model of invasive aspergillosis (32). In A. nidulans, two other components of the siderophore metabolism have been characterized: the nonribosomal FC ϪFe synthetase SidC and the iron-regulatory GATA-type transcription factor SreA. SidC-deficient mutants still produce TAFC ϪFe but lack synthesis of FC ϪFe (9). SreA deficiency causes partial derepression of siderophore biosynthesis and iron uptake under ironreplete conditions (13,26,28).
Very little is known regarding the intracellular handling of iron in eukaryotes and, in particular, in ferritin-lacking fungi. We demonstrate that the accumulation of the intracellular siderophore is regulated by intra-and extracellular iron availability as well as oxidative stress in A. nidulans. The study revealed that FC ϩFe is a major iron storage compound during intracellular iron excess but also suggested the presence of an alternative iron storage mechanism. Moreover, the absence of the intracellular siderophore was found to impair both sexual and asexual reproduction, to cause increased sensitivity of conidia to oxidative stress, and to result in delayed germination of conidia under iron-depleted conditions.
Generally, fungal strains were grown at 37°C in minimal medium, according to the description of Pontecorvo et al. (31), containing 1% glucose as the carbon source, 20 mM glutamine as the nitrogen source, and 10 M FeSO 4 as the iron source supplemented with 20 g liter Ϫ1 biotin but lacking the addition of iron for iron-depleted conditions. For iron-replete and high-iron conditions, TAFC ϩFe was added to final concentrations of 10 M and 100 M, respectively. Shake flask cultures were obtained with 200 ml of medium in 1.0-liter Erlenmeyer flasks inoculated with 10 8 conidia. Conidia of wt, ⌬sreA, and ⌬sidC were derived from solid minimal medium containing either 1.5 mM FeSO 4 or 10 M FC ϩFe after growth for 96 h. Importantly, ⌬sidC conidia lack FC ϩFe when derived from FeSO 4 plates but have wt-like FC ϩFe content when derived from FC ϩFe plates.
Analysis of siderophores and iron. TAFC ϩFe and FC ϩFe were isolated from A. nidulans as described previously (26). Quantification of FC ϩFe and FC ϪFe was performed by reversed-phase high-performance liquid chromatography according to the method of Konetschny-Rapp et al. (18) as described previously (26). FC ϩFe and FC ϪFe were discriminated by analysis of the samples with and without the addition of ferric chloride to a final concentration of 1 mM, as photometric detection at 435 nm records only ferri-siderophores (Fig. 1). TAFC ϩFe and TAFC ϪFe can be discriminated just as well. TAFC ϩFe and FC ϩFe are distinguished by different retention times, as shown previously (26).
For determination of the total cellular iron content, 50 mg of freeze-dried mycelia was decomposed in closed polytetrafluorethylene vessels containing 2 ml of HNO 3 and 0.5 ml of hydrogen peroxide using a high-performance microwave digestion unit (mls-1200 mega). Appropriate dilutions were made with distilled water, and the total iron content was determined by graphite furnace atomic absorption spectrometry (Hitachi Polarized Zeeman AAS Z8200) according to standard methods.
Analysis of fungal damage by exogenous H 2 O 2 . The hydrogen peroxide diffusion assay was performed as described by Maerker et al. (23). Briefly, 10 8 conidia were plated on petri dishes containing iron-replete minimal medium. Holes of 1 cm in diameter were punched, and 100 l of a 3% hydrogen peroxide solution was applied either immediately or after preincubation for 7 h at 37°C. Inhibition zones were measured after further incubation for 24 h at 37°C.
The sensitivity of conidia to killing by hydrogen peroxide was assayed essentially as described by Han et al. and Kawasaki et al. (15,17). Briefly, 1 ml of conidial suspension containing approximately 10 5 spores was incubated for 30 min at 20°C with the indicated concentrations of hydrogen peroxide. To determine the number of surviving conidia, the spore suspensions were diluted 50-fold with sterile water and 0.1 ml was plated on iron-replete minimal medium. After incubation for 24 h at 37°C, colonies were counted and normalized to that without hydrogen peroxide treatment.
The sensitivity of hyphae to hydrogen peroxide was estimated by using a modification of the protocol of Kawasaki et al. (17). About 150 conidia were plated on iron-replete minimal medium and grown at 37°C for 19 h-at this time point, small colonies were countable. Subsequently, the plates were overlaid with 4.5 ml of the same medium as top agar but containing the indicated concentration of hydrogen peroxide. After further incubation for 24 h at 37°C, colonies able to resume growth were counted as survivors and normalized to the number before hydrogen peroxide treatment.
Analysis of sexual development. To analyze homothalic sexual development, complete medium-agar plates were point inoculated with 10 6 conidia and incubated for 48 h at 37°C. Subsequently, small mycelial sections were transferred to minimal medium containing the iron source indicated (see Fig. 6) and further incubated for 24 h at 37°C. After sealing with Parafilm, incubation of the plates was continued for another 10 days under dark conditions. Sexual development was analyzed using a Leica MZ16 stereomicroscope. Huelle cells and ascospores were photographed using a Zeiss Axioplan light microscope.
Analysis of germination. Approximately 10 4 conidia were incubated at 37°C in petri dishes containing 15 ml liquid minimal medium supplemented with different iron sources. Germination was scored microscopically at 0.5 h intervals. The time point when 45 out of 50 conidia developed a germ tube was defined as the time required for germination.

RESULTS
FC ؉Fe is a major iron compound during intracellular iron excess. As a first step toward the understanding of the function of the intracellular siderophore in iron metabolism of A. nidulans, we analyzed the respective amounts of FC ϪFe , FC ϩFe , and total cellular iron content during conditions of different iron availability. To this end, we compared A. nidulans wt and ⌬sreA strains that were grown for 24 h under iron-depleted, iron-replete (10 M TAFC ϩFe ), and high-iron (100 M TAFC ϩFe ) conditions (Table 1). A. nidulans ⌬sreA was analyzed because this mutant shows excess accumulation of iron under iron-replete conditions (13). TAFC ϩFe was used as an iron source because it is soluble even in high concentrations and is the natural iron source of A. nidulans (9). FC ϪFe binds iron stoichiometrically 1:1 (11), and therefore, the FC ϩFe content directly corresponds to the amount of iron bound to intracellular siderophores. Under iron-depleted conditions, A. nidulans wt contained about 0.23 mol iron and 1.78 mol FC ϪFe per g (dry weight) but did not contain detectable amounts of FC ϩFe . In comparison, iron-replete and high-iron conditions increased the iron content 4.3-fold and 6.8-fold, respectively. Under both conditions, FC ϩFe constituted about 5% of the total cellular iron content and FC ϪFe was absent.
A. nidulans ⌬sreA did not show any significant difference from the wt under iron-depleted conditions with respect to FC ϪFe and total iron content. However, under iron-replete conditions, the total iron content of ⌬sreA, compared to the wt, was increased 2.7-fold with 64% bound to FC ϩFe , which corresponds to a 34-fold increase in FC ϩFe content. In other words, the surplus in FC ϩFe made up for the entire surplus in iron excess in ⌬sreA compared to the wt under iron-replete conditions. During high-iron conditions, the total iron content of ⌬sreA was increased 11.6-fold with 31% consisting of FC ϩFe -a 72-fold increase in FC ϩFe compared to the wt under this condition. Remarkably, despite a 3.4-fold increase in FC ϩFe content from iron-replete to high-iron conditions in ⌬sreA, the ratio of FC ϩFe to total iron decreased from 64% to 31%. The latter indicates that, under high-iron conditions, the iron is stored to an increasing degree independently of FC ϩFe in ⌬sreA, which might be explained by a limited capacity of FC ϩFe -mediated iron storage. During high-iron conditions, the total iron content of ⌬sreA was increased 79-fold compared to iron-depleted conditions, which indicates a wide range in the content of this potentially toxic metal. Taken together, these data revealed that SreA deficiency causes an increase of total iron content accompanied by an increase of FC ϩFe content directly depending on ambient iron availability.
The high FC ϩFe accumulation in ⌬sreA might be a direct result of derepression of FC ϪFe biosynthesis due to deficiency in SreA or caused indirectly by the resulting iron excess. To address this question, we analyzed the role of FC ϩFe during iron excess in A. nidulans wt. The intracellular iron content cannot be raised significantly in A. nidulans wt by steady-state growth during high-iron conditions due to functional iron regulation. Therefore, the wt was grown for 22 h under irondepleted conditions to fully activate iron uptake mechanisms and subsequently shifted into high-iron conditions by addition of TAFC ϩFe to a final concentration of 100 M to induce excessive uptake of iron. FC ϪFe , FC ϩFe , and total iron contents were measured after 0.5, 2.0, 5.0, and 8.0 h ( Table 2). Within 0.5 h after the shift, the cellular iron content increased 25-fold and the complete FC ϪFe pool was converted to FC ϩFe , which constituted 44% of the total iron content. At 2 h after the shift, both the total iron and FC ϩFe contents showed a maximum. Remarkably, the total iron content increased 86fold compared to iron-depleted conditions and the FC ϩFe content increased 2.9-fold compared to the initial FC ϪFe pool. At this time point, the ratio of FC ϩFe to total iron decreased to about 30%, indicating that the iron was stored to an increasing extent independently of FC ϩFe . Within a further 3.0 h of incubation, both the total cellular iron and FC ϩFe contents decreased about 0.6-fold. This appears to be due to distribution by cell division because the total biomass increased 4.6-fold during the time course experiment, and in contrast to the cellular level, the content of both FC ϩFe and total iron of the entire culture increased. From the 5.0 h time point to the 8.0 h time point, the FC ϩFe pool was maintained, indicating slow metabolization.
The analyses of the FC ϩFe content of A. nidulans wt and ⌬sreA indicated that the intracellular siderophore is a major iron storage compound in hyphae during conditions of intracellular iron excess. Remarkably, the mycelial mat of A. nidulans wt and ⌬sreA displayed a brown coloration during conditions of high FC ϩFe accumulation (Fig. 2), which is likely to be a direct effect, as FC ϩFe has a brownish color.
Oxidative stress causes increased siderophore accumulation. As shown above, in the shift of A. nidulans wt from iron-depleted to high-iron conditions, the FC ϩFe content increased 2.9-fold within 2 h compared to the initial FC ϪFe pool ( Table 2). This might be caused directly by the intracellular   iron excess or indirectly by the oxidative stress provoked by the iron excess. To investigate a possible effect of oxidative stress on the accumulation of the intracellular siderophore, wt and ⌬sreA mycelia were exposed to hydrogen peroxide or the redox cycler paraquat (Fig. 3). Under iron-replete conditions, the wt strain responded to both modes of oxidative stress with a 2.9-fold increase in FC ϩFe content. In ⌬sreA, this treatment caused a 1.7-fold increase, and it has to be emphasized that this takes place at a 34-fold-higher level (compare FC ϩFe content of wt and ⌬sreA during growth in 10 M TAFC ϩFe in Table 1). Under iron-depleted conditions, the same hydrogen peroxide treatment caused killing of the wt and ⌬sreA mycelia, which indicates higher sensitivity of iron-starved mycelia toward hydrogen peroxide. However, wt and ⌬sreA responded to paraquat treatment by an increases in FC ϪFe content of 1.5-fold and 1.8-fold, respectively. Remarkably, treatment with neither paraquat nor hydrogen peroxide affected the total iron content of wt and ⌬sreA under iron-replete conditions. These data show that oxidative stress causes increased accumulation of the intracellular siderophore during both iron-replete and iron-depleted conditions independent of SreA and suggest that the increase in FC ϩFe under iron-replete conditions is due to redistribution of intracellular iron rather than iron uptake.
Both intracellular iron excess and oxidative stress up-regulate expression of sidC. To investigate at which level iron excess and oxidative stress regulate the synthesis of the intracellular siderophore, we performed expression analysis of sidC, which encodes the nonribosomal peptide synthetase involved in FC ϪFe biosynthesis.
Northern analysis demonstrated (Fig. 4A), as shown previously (28), that expression of sidC is repressed under ironreplete conditions and derepressed under iron-depleted conditions in the wt. The mirB gene, which encodes an SreAmediated iron-regulated transporter for uptake of TAFC ϩFe (12), exhibited an expression pattern similar to that of sidC. In ⌬sreA, however, the transcript level of sidC increased with elevating extracellular iron availability, which is directly related to the intracellular iron content in this mutant, whereas that of mirB decreased. Moreover, a shift from iron-depleted to highiron conditions further increased the expression of sidC but repressed expression of mirB in the wt (Fig. 4B). Taken together, the expression analysis of sidC and mirB suggests that the increase of FC ϩFe accumulation by intracellular iron excess in A. nidulans wt and ⌬sreA is mediated by transcriptional up-regulation of sidC expression. Moreover, intracellular iron excess caused inverse regulation of siderophore-mediated iron storage and siderophore-mediated iron uptake, two processes which are coregulated under iron-depleted conditions. Treatment with hydrogen peroxide and paraquat under ironreplete conditions up-regulated the transcript level of sidC, whereas no effect on the expression of mirB was found ( FC ؊Fe deficiency affects growth rate and iron content. We have previously found no significant difference in radial growth rate on solid medium between A. nidulans wt and ⌬sidC under iron-depleted and iron-replete conditions (9,32). In three parallel shake flask cultures, the wt produced dry weights of 0.51 Ϯ 0.08 g under iron-replete conditions and 0.47 Ϯ 0.07 g under iron-depleted conditions after 24 h at 37°C. The dry weight of ⌬sidC did not differ significantly under iron-replete conditions but was reduced by 27% under iron-depleted conditions. We have shown previously that TAFC ϪFe production is mildly increased during iron-replete growth (9), which indicates an increased demand for iron. Consistently, under iron-replete conditions, the iron content of ⌬sidC was found to be 1.88 Ϯ 0.12 mol g Ϫ1 (dry weight), which was 1.9-fold higher than that of the wt. Taken together, the decreased submerged growth rate under iron-depleted conditions and the increase of both TAFC ϪFe production and iron content during iron-replete growth of ⌬sidC suggest that the lack of the intracellular siderophore causes less-efficient utilization of intracellular iron.
The highest FC ϩFe content in A. nidulans wt was found during conditions of iron excess caused by the shift from irondepleted to high-iron conditions ( Table 2), suggesting that the intracellular siderophore plays a crucial role during this transition. However, the growth curve of ⌬sidC showed no significant difference to the wt in such an experiment (data not shown). At 5.0 h after the shift, the intracellular iron content of ⌬sidC was 1.5-fold higher than that of the wt. These results indicate that the lack of effect of FC ϩFe deficiency on growth rate is not due to reduced iron uptake and show that FC ϪFe is dispensable in hyphae even during intracellular iron excess. In cellular extracts of both the wt and ⌬sidC, TAFC ϩFe was detected only in trace amounts during the shift (data not shown), which demonstrates that ⌬sidC does not compensate FC ϩFe deficiency by increased retention of TAFC ϩFe .
Deficiency in SidC decreases the conidial iron content. A. nidulans wt conidia contain FC ϩFe , and FC ϪFe -deficient strains are able to take up FC ϩFe from the growth medium and store it in conidia (9). Therefore, conidia derived from ⌬sidC supplemented with FC ϩFe during sporulation have the same FC ϩFe content as wt conidia despite their inability to synthesize the intracellular siderophore. Determination of the iron content revealed that wt conidia contain about 2.6 times less iron when harvested from mycelia grown under iron-depleted conditions than that grown under high-iron conditions or in FC ϩFe -supplemented medium (Table 3). To our knowledge, these data indicate for the first time an influence of growth conditions on the characteristics of the formed conidia. The iron content of FC ϩFe -lacking ⌬sidC conidia was reduced by 34% compared to the wt, whereas FC ϩFe -containing ⌬sidC conidia showed a wt-like iron content.
FC ؉Fe -deficient conidia are sensitive to hydrogen peroxide. To analyze the role of FC ϩFe in oxidative-stress resistance, wt and ⌬sidC conidia derived from medium with or without supplementation with FC ϩFe were exposed to hydrogen peroxide in a plate diffusion assay as described in Materials and Methods. FC ϩFe -lacking ⌬sidC conidia displayed increased hydrogen peroxide sensitivity (inhibition zone of 25 mm compared to 20 mm of the wt), which suggests a role of FC ϩFe in oxidative-stress resistance. Preincubation for 7 h, to allow germination of the conidia before contact with hydrogen peroxide, decreased the sensitivity of both FC ϩFe -lacking ⌬sidC (inhibition zone of 19 mm) and the wt (inhibition zone of 17 mm). These data suggest that germination increases the hydrogen peroxide resistance and decreases the difference between FC ϩFe -lacking ⌬sidC and the wt. FC ϩFe -containing ⌬sidC conidia showed wt-like hydrogen peroxide resistance with and without preincubation, demonstrating that the hydrogen peroxide sensitivity of ⌬sidC is indeed due to the absence of FC ϩFe . To further analyze the impact of the intracellular siderophore on oxidative-stress resistance, the effect of hydrogen peroxide on conidial and hyphal survival was tested (Fig. 5). FC ϩFe -lacking ⌬sidC conidia were significantly more susceptible to killing by hydrogen peroxide, whereas FC ϩFe -containing ⌬sidC conidia were as resistant as wt conidia (Fig. 5A). In contrast, ⌬sidC hyphae displayed wt-like resistance to killing by hydrogen peroxide (Fig. 5B). These data suggest that FC ϩFe is more important for the oxidative-stress resistance of conidia than of hyphae.
FC ؉Fe deficiency causes delayed germination under irondepleted conditions. We analyzed the role of the conidial FC ϩFe pool by comparison of the germination of wt, FC ϩFecontaining ⌬sidC, and FC ϩFe -lacking ⌬sidC conidia under iron-replete conditions as well as iron-depleted conditions with and without the iron-specific extracellular chelator bathophenanthroline disulfonate in a final concentration of 200 M. Under all conditions, wt conidia formed germination tubes after 5.5 h of incubation at 37°C. However, the germination of FC ϩFe -lacking ⌬sidC conidia was delayed 1.5 h under irondepleted conditions and 2.5 h in the presence of bathophenanthroline disulfonate. The time required for germination of FC ϩFe -containing ⌬sidC conidia was wt-like, which underlines that the delay in germination of FC ϩFe -lacking ⌬sidC conidia is a direct effect of deficiency in conidial FC ϩFe . Under ironreplete conditions, FC ϩFe -lacking ⌬sidC conidia displayed no difference from the wt in the time required to germinate, indicating that the conidial FC ϩFe pool serves as an iron source during germination but that its absence can be compensated for by uptake of extracellular iron.
FC ؉Fe is required for homothallic sexual development. A. nidulans can form sexual fruiting bodies, termed cleistothecia, in both homothallic and heterothallic conditions (7). Mature black cleistothecia usually contain up to 10,000 asci in which eight red-pigmented ascospores (sexual spores) are present. Cleistothecia are surrounded by thick-walled globular cells, a Conidia were derived from iron-depleted solid minimal medium without the addition of iron (ϪFe) or containing 100 M FeSO 4 (hFe) or 20 M FC ϩFe (FC). The data represent the means Ϯ standard deviations of results from three independent measurements. b As shown previously (9), conidial production of ⌬sidC is drastically reduced under iron-depleted conditions, impeding iron content determination (-).
called Huelle cells, which are proposed to function in supporting fruiting body formation by providing necessary sources for energy and nutrients. During crossing experiments we found that ⌬sidC could be successfully crossed with other common laboratory strains (data not shown) but was unable to develop homothallic cleistothecia in iron-replete conditions. However, it produced normal Huelle cells (Fig. 6). Cleistothecia formation and production of ascospores was restored by supplementation with FC ϩFe but not high concentrations of iron, which demonstrates that self-sterility of ⌬sidC is indeed due to lack of FC ϩFe .

DISCUSSION
For a steady supply of cells with iron, not only uptake but also storage of this metal is of critical importance. Furthermore, the means of nontoxic iron storage is important in protection against iron-mediated oxidative stress. In organisms as diverse as bacteria, plants, and vertebrates, nontoxic storage of iron is conducted by ferritin and ferritin-like molecules (2,3). With the exception of zygomycetes, fungi lack ferritin-like molecules. However, many fungal species, like A. nidulans, contain an intracellular siderophore-the focus of this study. In response to extracellular and intracellular iron concentrations, A. nidulans accumulates its intracellular siderophore as FC ϪFe or FC ϩFe . Under iron-depleted conditions, A. nidulans wt accumulated FC ϪFe and did not contain FC ϩFe . Under iron-replete conditions and high-iron conditions, the total cellular iron content increased 4.3-fold and 6.8-fold, respectively, and FC ϩFe constituted about 5% of the total cellular iron content; FC ϪFe was absent. Compared to iron-depleted conditions, the cellular iron content increased about 80-fold during a shift from irondepleted to high-iron conditions or by derepression of iron uptake due to deficiency in the iron regulator SreA. This demonstrates the capacity of A. nidulans in coping with high amounts of this potentially toxic metal. Under these conditions of intracellular iron excess, FC ϩFe became a major iron compound and represented up to 64% of the cellular iron content. In agreement, FC ϩFe was also found to be the major iron compound under similar conditions in Neurospora crassa using Mössbauer spectroscopy (24). FC ϪFe accumulation during iron depletion at first sight appears inconsistent with a role in iron storage but might represent a proactive strategy to cope with a potentially high incoming iron load due to the activation of iron transport systems. In this respect, the up-regulation of FC ϪFe -dependent iron storage capacity under iron-depleted conditions might be comparable to induction of ZRC1 expression, which is essential for zinc tolerance by mediating vacuolar zinc storage, during zinc depletion in S. cerevisiae (22). Accumulation of FC ϪFe as well as FC ϩFe requires synthesis of FC ϪFe , which is subject to a remarkable regulation: FC ϪFe synthesis is low under iron-replete conditions and up-regulated by both iron starvation and intracellular iron excess. Accumulation of the intracellular siderophore correlated with the transcript level of sidC, which encodes the nonribosomal FC ϪFe synthetase, suggesting that accumulation of FC ϪFe and FC ϩFe is regulated at the level of expression of SidC. Comparison of expression of sidC and the TAFC ϩFe transporter-encoding mirB indicated that siderophore-mediated iron uptake and siderophore-mediated iron storage are simultaneously up-regulated under iron-depleted conditions, which is at least partly a consequence of coregulation by the iron regulator SreA (26,28). In contrast, intracellular iron excess caused up-regulation of siderophore-mediated iron storage but down-regulation of uptake. This inverse regulation seems appropriate to avoid iron-mediated oxidative stress and indicates an SreA-independent regulatory mechanism for up-regulation of FC ϪFe synthesis during iron excess. This regulation might be part of the oxidative-stress response, as hydrogen peroxide or the redox cycler paraquat up-regulated expression of sidC and production of FC ϪFe independent of SreA. Under iron-replete conditions, oxidative stress increased the FC ϩFe pool, which appears to represent a redistribution of intracellular iron because the total cellular iron content was unaffected.
A role of the intracellular siderophore in oxidative stress was suggested previously, as sidC deficiency results in an increased labile iron pool and slightly increased sensitivity to paraquat (9). In the present study, we found that FC ϩFe deficiency causes significantly increased sensitivity of conidia but not of hyphae to hydrogen peroxide. As iron can potentiate oxidative stress, FC ϩFe might be more important for the oxidative-stress resistance of conidia because FC ϩFe is the major iron compound of conidia (25) but represents only about 5% of the total cellular iron content of hyphae under iron-replete conditions. On the other hand, oxidative-stress resistance depends on iron because catalases require heme as a cofactor. Consequently, the reduced conidial iron content of ⌬sidC conidia might be responsible for the decreased hydrogen peroxide resistance. Compared to conidia, hyphal cells might be physiologically more versatile in the combat of oxidative stress by up-regulation of antioxidative enzymes. In support of the latter, deficiency in sidC results in up-regulation of the genes encoding hyphal catalase B and the Cu/Zn-superoxide dismutase (9). The lack of the siderophore system abrogates virulence of A. fumigatus (32), which might be due to the lack of production of TAFC ϪFe and/or FC ϪFe . The oxidative-stress sensitivity of ⌬sidC conidia indicates a possible role of FC ϩFe in virulence, as the killing of Aspergillus conidia by alveolar macrophages is mediated by reactive oxidant intermediates (29).
There are several indications that A. nidulans employs also FC ϪFe -independent mechanisms for iron storage and detoxification: (i) deficiency in synthesis of FC ϪFe is not lethal; (ii) during conditions of iron excess, a remarkably large FC ϩFeindependent iron pool was detected; (iii) a shift from iron-depleted to high-iron conditions resulted in even higher iron accumulation in ⌬sidC than in the wt but had no effect on the growth rate of ⌬sidC. Iron might be stored in proteins requiring iron-containing cofactors, as indicated by up-regulation of the respective genes in ⌬sreA during iron-replete growth (27) but which seems unlikely to account for buffering of vast overaccumulation of iron. A further possibility is vacuolar iron storage, as shown for S. cerevisiae (21). In S. cerevisiae, transport of iron into the vacuole is mediated by CCC1 and, in support of vacuolar iron storage in A. nidulans, a search of the A. nidulans genome (http://www.ncbi.nlm.nih.gov/BLAST/) revealed the presence of two putative CCC1 homologs, AN3681.2 and AN4990.2 (plastp E-values of 4e Ϫ32 and 3e Ϫ29 ). Nevertheless, the absence of the intracellular siderophore seems to cause a less-efficient utilization of iron, indicated by decreased growth in shake flask culturing under iron-depleted conditions as well as increased TAFC ϪFe production and cellular iron content under iron-replete conditions of ⌬sidC. The increased demand for iron might also be the reason for the reduced conidiation rate of ⌬sidC found previously (9).
In A. nidulans, N. crassa, and Penicillium chrysogenum, the conidial siderophore storage was found to be an important germination factor, as germination of conidia, which have lost cellular siderophores by treatment with high salt concentrations, fails or is greatly delayed unless a suitable siderophore is supplied (8,16). In agreement, germination of ⌬sidC conidia was delayed under iron-depleted conditions-most likely due to the reduced iron store. Under iron-replete conditions, no difference from the wt was found, indicating that the lack of FC ϩFe in ⌬sidC can be compensated for by uptake of iron.
A. nidulans possesses both asexual and sexual reproduction cycles. We have previously shown that deficiency in the intracellular siderophore causes a reduction of asexual conidiospore production, which can be partly cured by supplementation with high concentrations of iron (9). Here we show that the lack of FC ϪFe biosynthesis causes a defect in homothallic sexual development, which cannot be compensated by high iron supplementation. Consistent with the selection arena hypothesis, Bruggemann et al. (5) found that many auxotrophic mutants are self-sterile and produce only tiny cleistothecia lacking ascospores in homothallic conditions. SidC deficiency does not cause an auxotrophy and completely lacks cleistothecia formation, which might indicate a more specific role of FC ϩFe in sexual development. The intracellular siderophore might be essential for sexual development due to its role in iron metabolism or oxidative-stress resistance. In this respect, it is important that reactive oxygen species are not only harmful molecules but also function as signaling molecules required, among others, for sexual development in A. nidulans (1). And here again, iron metabolism and regulatory production of reactive oxygen species are interconnected, as the A. nidulans NADPH-oxidase NoxA, which is proposed to induce sexual development by production of reactive oxygen species, requires heme iron as a cofactor (19). Remarkably, another heme-containing oxidoreductase, TmpA, is involved in regulation of asexual development in A. nidulans (33).
Taken together, our studies revealed that the intracellular siderophore is a central component of the fungal physiology, as it is involved in iron storage, oxidative-stress resistance, asexual and sexual reproduction, and germination. The regulation of 1602 EISENDLE ET AL. EUKARYOT. CELL FC ϪFe biosynthesis by iron and oxidative stress resembles that of ferritin production in other organisms (34), emphasizing the similarity of these analogous mechanisms for storage and detoxification of iron.