Regulation of freA, acoA, lysF, and cycA Expression by Iron Availability in Aspergillus nidulans

In the filamentous fungus Aspergillus nidulans, iron homeostasisis regulated at the transcriptional level by the negative-actingGATA factor SREA. In this study the expression of a putativeheme-containing metalloreductase-encoding gene, freA, was foundto be upregulated by iron limitation independently of SREA,demonstrating the existence of an iron-regulatory mechanismwhich does not involve SREA. In contrast to freA, various othergenes encoding proteins in need of iron-containing cofactors—acoA,lysF, and cycA—were downregulated in response to irondepletion. Remarkably, SREA deficiency led to increased expressionof acoA, lysF, and cycA under iron-replete growth conditions.

INTRODUCTION

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Introduction

Virtually all organisms require iron for their growth. The electrontransfer ability of the iron atom makes it essential for redoxreactions ranging from respiration to ribonucleotide synthesis.Despite the fact that iron is the fourth most abundant elementin the earth's crust, the amount of bioavailable iron is verylimited since this metal is most commonly found as insolubleFe(III)-hydroxide. Thus, microorganisms need specialized ironmobilization systems (14). On the other hand, an excess of ironin the cell can be detrimental, because iron can catalyze theproduction of cell-damaging hydroxyl radicals in the presenceof oxygen. Therefore, the concentration of iron in biologicalfluids is tightly regulated, and control is accomplished primarilyby the rate of uptake.

Under iron starvation, most fungi synthesize and excrete low-molecular-weight,Fe(III)-specific chelators, termed siderophores, in order tosolubilize environmental iron. Subsequently, cells recover theiron from the ferrisiderophore complexes via specific uptakemechanisms (17). Furthermore, most fungi possess intracellularsiderophores as an iron storage compound. In this respect Saccharomycescerevisiae is an exception since it lacks the ability to synthesizesiderophores, although it can utilize siderophores producedby other species. This yeast employs two distinct high-affinityiron uptake systems which are both regulated by the paralogoustranscriptional activators Aft1p and Aft2p (2, 32). The firstmechanism—termed reductive iron assimilation—requiresthe action of surface metalloreductases with different substratespecificities (Fre1p to Fre4p) to reduce Fe(III) to Fe(II),which is subsequently transported into the cell by the permease-oxidasecomplex Ftr1p/Fet3p (1, 5, 27, 34). This system allows the uptakeof both siderophore-bound and unbound iron (33). The secondiron uptake system—called nonreductive iron assimilation—isspecialized for the uptake of siderophore-bound iron and dependson members of the major facilitator superfamily (16, 18, 33).

In ascomycetes and basidiomycetes, siderophore biosynthesisand siderophore-mediated iron uptake are controlled by orthologous,negative-acting GATA transcription factors, e.g., Aspergillusnidulans SREA, Neurospora crassa SRE, and Ustilago maydis URBS1(15, 29, 35). In A. nidulans, deletion of sreA results in derepressedintracellular and extracellular siderophore biosynthesis aswell as increased accumulation of iron under sufficient ironsupply due to derepressed siderophore uptake (21). Recentlyvarious members of the SREA regulon which are presumably involvedin biosynthesis, transport, and utilization of siderophoreshave been identified, e.g., mirA, which encodes an orthologueof the S. cerevisiae siderophore permeases (21, 22). Notably,neither the available A. nidulans cDNA and genomic sequencesnor the publicly accessible complete genomes of the close relativesAspergillus fumigatus (http://www.sanger.ac.uk/Projects/A_fumigatus/)and N. crassa (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/)seem to contain orthologues of S. cerevisiae AFT1 or AFT2. Furthermore,S. cerevisiae does not possess an orthologue of A. nidulansSREA. Thus, the question remains if SREA represents the majoriron regulator or if it is specific for control of siderophoremetabolism.

Up to now, it was not known if A. nidulans has the ability forreductive iron uptake. Searches for putative components of thissystem in various A. nidulans sequence databases led to theidentification of expressed sequence tag clone o5f06a1, whosetranslation product displayed significant similarity to metalloreductases.The sequence information was used to isolate corresponding genomicclones from a cosmid library provided by the Fungal GeneticStock Center (4). The five hybridizing clones, L4F02, L28H11,L25F03, L23A09, and L32A010, localized freA to chromosome IV,and the entire sequence of freA was sequenced directly fromcosmid L23A09. Comparison of the genomic and cDNA sequences,obtained by 5' and 3' rapid amplification of cDNA ends accordingto the protocols of Frohman et al. (8), revealed an open readingframe of 1,797 bp interrupted by five introns, 53, 50, 43, 47,and 45 nucleotides (nt) in length (Fig. 1A). Additionally, twointrons, 66 and 58 nt in length, are present in the 827-bp 5'untranslated region. The 3' untranslated region was found tobe 84 nt in length. The deduced FREA protein has a calculatedmolecular mass of 67.2 kDa and shows significant similarityto various metalloreductases, e.g., 24% identity (blastp E-valueof 8e^-32) to S. cerevisiae Fre2p. An alignment of A. nidulansFREA, S. cerevisiae Fre2p (10), Arabidopsis thaliana FRO2 (24),and the gp91^phox subunit of the NADPH oxidase (25), which iscritical for production of microbicidal oxidants in human neutrophils,is shown in Fig. 1B. FREA possesses all typical features ofmetalloreductases (7, 12): a flavin adenine dinucleotide cofactorbinding site, an NADPH binding motif, and four typically spacedhistidine residues predicted to coordinate a bis-heme structurebetween transmembrane domains of the protein (Fig. 1B). S. cerevisiaepossesses nine paralogous, metalloreductase-encoding genes whichdisplay different expression profiles: FRE1 is upregulated byiron and copper depletion, FRE2 to FRE6 are upregulated by ironstarvation only, FRE7 is specifically upregulated by copperlimitation, and YGL160w and YLR047c are regulated by neithercopper nor iron availability (12, 19). Iron regulation of thesegenes is mediated by Aft1p, and copper regulation is mediatedby Mac1p. Fre1p to Fre4p are involved in reduction of siderophore-boundand unbound iron (5, 34), Fre1p and Fre2p additionally functionin copper uptake (11), and the function of Fre5p to Fre7p isunknown. To study the expression pattern of freA, A. nidulanswild-type and sreA deletion strains were grown for 24 h at 37°Cunder standard conditions, iron limitation, and copper starvationas described previously (20). Northern blot analysis revealedthat the A. nidulans freA expression pattern resembles thatof Saccharomyces FRE2 to FRE4 by being iron but not copper regulated(Fig. 2). These data indicate that Aspergillus FREA is involvedin securing iron homeostasis. It might be a component of a possiblereductive iron assimilation system or function as an intracellularmetalloreductase. In contrast to typical members of the SREAregulon, e.g., mirA, SREA deficiency did not lead to derepressedfreA expression under iron-replete conditions. These data showthat in A. nidulans an iron-regulatory mechanism exists whichdoes not involve SREA.

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FIG. 1. A. nidulans freA. (A) Intron-exon structure of freA. (B) Alignment of A. nidulans FREA, S. cerevisiae Fre2p (P36033), A. thaliana FRO2 (CAA70770), and human gp91^phox (NP_000388). Amino acid residues identical in three of the four proteins are in boldface, and amino acid residues potentially involved in the bis-heme binding (H), NADPH binding (HPFT), and flavin adenine dinucleotide binding (GPYG) are shaded in gray.

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FIG. 2. Expression of freA, mirA, cycA, acoA, and lysF under standard (+Fe), iron depletion (-Fe), and copper depletion (-Cu) conditions in A. nidulans wild-type (wt) and SREA-deficient ( ${Delta}$ sreA) strains. Fungal strains were grown for 24 h in minimal medium containing 10 µm FeSO₄ and 10 µm CuSO₄ as described previously (20); for iron- and copper-depleted growth the addition of the respective metal was omitted. As a control for loading and RNA quality, blots were hybridized with the ${gamma}$ -actin-encoding acnA gene (6). (A and B) Northern blot analysis was performed with 1 µg of mRNA (A) or 10 µg of total RNA (B), respectively. (C) Quantification of mRNA levels normalized to acnA levels with a PhosphorImager. Bars represent mean values of two independent experiments; standard deviations did not exceed 20%.

Furthermore, SREA-independent expression of freA confirms thatSREA indeed acts as a direct repressor of extracellular siderophorebiosynthesis and uptake. SREA deficiency results in 20-fold-increasedaccumulation of the intracellular siderophore ferricrocin duringiron-replete growth (21). Therefore, it could have been alternativelyhypothesized that SREA acts only as a repressor of ferricrocinbiosynthesis and that SREA deficiency causes iron deprivationvia sequestration of intracellular iron. But in this case, theexpression of all iron starvation-induced genes, including freA,would be expected to be upregulated under iron-replete conditionsin an sreA deletion strain.

Iron depletion can lead to upregulation of expression, as inthe case of genes involved in high-affinity iron uptake. Butthe opposite regulatory pattern can also be found: expressionof catB, encoding a heme-containing catalase, is downregulatedat the transcript level under iron starvation (21). To investigateif this regulatory pattern is specific for catB or holds forother proteins in need of iron-containing cofactors, the expressionof the genes encoding the iron-sulfur cluster containing aconitase(acoA) and homoaconitase (lysF), as well as the heme-containingcytochrome c (cycA), was studied (23, 30). For partial analysisof the putative A. nidulans aconitase gene acoC, the expressedsequence tag clone c8d09 was sequenced. It contains the C-terminal398 amino acids of ACOC displaying 88 and 73% identity to theaconitases of Aspergillus terreus and S. cerevisiae, respectively.Northern blot analysis proved that expression of genes involvedin pathways as distinct as the citric acid cycle (acoA) andrespiration (cycA), as well as lysine and penicillin biosynthesis(lysF), is downregulated between two- and eightfold under ironlimitation in the wild-type and the SREA-deficient strains (Fig.2). With an eightfold-decreased transcript level, cycA was thegene most dramatically affected by iron depletion. Notably,CYCA-deficient A. nidulans mutants are viable, and it was suggestedpreviously that this is due to the ability of Aspergillus toferment and to use alternative respiratory pathways (3). Takentogether, these data suggest that, during iron depletion, decreasedexpression of cycA saves energy and iron for other processesessential for survival under iron limitation. Assuming thatFREA is involved in iron homeostasis, as has been shown previouslyfor four of the six iron-regulated S. cerevisiae paralogues(5, 10, 34), the opposite regulation of freA versus acoA, lysF,and cycA by iron availability suggests that under iron depletionthe flow of this limiting metal might be directed from variousmetabolic pathways to systems needed to secure iron homeostasis.

Interestingly, the transcript levels of acoA, lysF, and cycAwere elevated between two- (acoA) and ninefold (cycA) underiron-replete conditions in the sreA deletion strain (Fig. 2).Therefore, expression of these genes might be subject to SREAregulation. Alternatively, upregulation of these genes mightbe caused indirectly since SREA deficiency leads to increasediron accumulation and increased oxidative stress (21): (i) itmay reflect the increased bioavailability of iron within SREA-deficientcells, or (ii) it may represent an oxidative stress response.In the latter case, the increased expression of these genescould represent a compensatory response invoked to maintaincellular enzyme activities because, e.g., iron-sulfur cluster-containingenzymes are particularly sensitive to inactivation by oxidativeattack (9). In this respect it is noteworthy that, in Escherichiacoli, expression of aconitase-encoding acnA is specificallyinduced by iron and oxidative stress (13), and it was suggestedpreviously that the aconitase proteins serve as a protectivebuffer against oxidative stress by acting as a sink for reactiveoxygen species (28). The upregulation of cycA expression mightalso be a response to oxidative stress because cytochrome cplays an important role in the antioxidant system of mitochondria(26). Remarkably, the promoter region of lysF contains severalGATA motifs which potentially represent SREA binding sites.But since mutational analysis showed that at least two of theseGATA sites mediate a positive effect on lysF expression, a directinvolvement of the repressor SREA seems to be doubtful (31).

Hybridization probes.

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Hybridization probes.

The hybridization probes used in this study were generated byPCR with oligonucleotides 5'-AGCCCGGTGTGAAAAGAG and 5'-AACAGGAGGAGGATTGCGCCfor mirA, 5'-AGATCATGGGAGTTGACCTG and 5'-AGACGGATTGTATGGCGATGAGfor freA, 5'-ACCCTTTCTCTCTACCTC and 5'-CGCGATTAGACGAGATAA forcycA, 5'-TATCCATGTAGTCCGCCC and 5'-GGTCCCACTGTCCAATGC for acoA,5'-GCTGACGAACGAAGAAG and 5'-GCGTTCTTAACCCATTTC for lysF, and5'-CGGTGATGAGGCACAGT and 5'-CGGACGTCGACATCACA for ${gamma}$ -actin-encodingacnA.

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The freA and acnA sequences were assigned GenBank accessionno. AF515629 and AF515630, respectively.

ACKNOWLEDGMENTS

We are grateful to Bruce A. Roe et al. for the information suppliedby the A. nidulans cDNA sequencing project and to the WhiteheadInstitute/MIT Center for Genome Research for access to the N.crassa genome sequence, as well as to the Sanger Institute andits collaborators David Denning and Andrew Brass at the Universityof Manchester for access to the A. fumigatus genome sequence.We thank Axel Brakhage for a plasmid containing a lysF fragment.

This project was supported by Austrian Science Foundation grantFWF-P13202-MOB (to H.H.).

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology, Fritz-Pregl-Str. 3/II, A-6020 Innsbruck, Austria. Phone: 43-512-507-3605. Fax: 43-512-507-2866. E-mail: hubertus.haas{at}uibk.ac.at.

Present address: Institute of Medical Chemistry and Biochemistry,University of Innsbruck, Innsbruck, Austria.

REFERENCES

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