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Eukaryotic Cell, April 2004, p. 561-563, Vol. 3, No. 2
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.2.561-563.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biosynthesis and Uptake of Siderophores Is Controlled by the PacC-Mediated Ambient-pH Regulatory System in Aspergillus nidulans

Martin Eisendle,1,{dagger} Harald Oberegger,1,{dagger},{ddagger} Rudolf Buttinger,2 Paul Illmer,2 and Hubertus Haas1*

Department of Molecular Biology, Medical University of Innsbruck,1 Department of Microbiology, University of Innsbruck, A-6020 Innsbruck, Austria2

Received 20 November 2003/ Accepted 25 January 2004


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ABSTRACT
 
Biosynthesis and uptake of siderophores in Aspergillus nidulans are regulated not only by iron availability but also by ambient pH: expression of this high-affinity iron uptake system is elevated by an increase in the ambient pH. Mediation of this regulation by the transcriptional regulator PacC has been confirmed via acidity- and alkalinity-mimicking mutants.


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TEXT
 
To cope with variations in pH in their environment, fungi require an adapting regulatory system to ensure optimal function of numerous cellular activities. Such a system was originally identified and most extensively studied in the filamentous ascomycete Aspergillus nidulans, and the presence of a similar mechanism has subsequently also been described for other fungal species, e.g., Saccharomyces cerevisiae, Penicillium chrysogenum, Yarrowia lipolytica, Candida albicans, Acremonium chrysogenum, and Fusarium oxysporum (2, 4, 28). The pH regulatory system ensures that secreted enzymes (e.g., alkaline and acid phosphatases and xylanases) and metabolites (e.g., penicillin or aflatoxin), as well as membrane proteins (e.g., gamma-amino-n-butyrate [GABA] permease), are produced under conditions of pH where they can exert their full physiological function. In S. cerevisiae, it has been shown that extracellular pH also governs ion tolerance and differentiation programs, i.e., haploid invasive growth and sporulation (22). The wide-ranging physiological impact of pH changes is reflected by the diverse groups of pH-responsive genes identified in S. cerevisiae (18). In A. nidulans, pH regulation of gene expression is mediated by the wide-domain zinc finger transcription factor PacC. Under alkaline growth conditions, the pal ambient-pH signaling pathway, which consists of the gene products of palA, -B, -C, -F, -H, and -I, is responsible for the first of two proteolytic cleavages required for activation of the PacC protein. Activated PacC acts as both an activator of alkaline-expressed genes and a repressor of acid-expressed genes. Loss-of-function mutations in any of the six pal genes as well as in pacC cause an acidity-mimicking phenotype and result in increased expression of "acid" genes and reduced expression of "alkaline" genes. Gain-of-function, alkaline-mimicking mutations in pacC result in a phenotype opposite to that of acidity-mimicking mutations. The DNA consensus sequence recognized by PacC is 5'-GCCARG.

Under iron starvation conditions, most fungi excrete low-molecular-weight, ferric iron-specific chelators, termed siderophores, in order to mobilize environmental iron (11, 19). Subsequently, the iron from the ferrisiderophore complexes is recovered via specific uptake mechanisms. In A. nidulans, siderophore biosynthesis has been shown to be essential for viability (8, 25). Repression of synthesis and uptake of siderophores by iron is mediated by the GATA transcription factor SreA (13, 26, 27). Recently, we have functionally characterized three structural genes, sidA, mirA, and mirB, involved in siderophore metabolism of A. nidulans (8, 12). sidA encodes L-ornithine-N5-monooxygenase, which catalyzes the first committed step for biosynthesis of the hydroxamate-type siderophore triacetylfusarinine C. mirB and mirA encode transporters specific for the uptake of the native siderophore triacetylfusarinine C and the bacterial catecholate-type siderophore enterobactin, respectively. mirC, a paralogue of mirA and mirB, also shows iron- and SreA-dependent expression and potentially encodes another siderophore permease, but its function is not known so far (8, 12). The presence of consensus PacC binding sites in the promoter regions of sidA, mirA, and mirB, but not mirC, suggested control by pH regulation (data not shown), and in this study, we demonstrate pH regulation of siderophore biosynthesis and uptake.

For analysis of siderophore production and transcription of genes involved in siderophore production, A. nidulans strains were grown in iron-depleted minimal medium (29) containing the respective supplements and 50 mM potassium phosphate buffer adjusted to pH 4.7 and pH 7.0, respectively. Subsequent to orbital shaking for 24 h at 37°C, total RNA was prepared for Northern analysis, and the siderophore content of the culture supernatant was analyzed by reversed-phase high-pressure liquid chromatography as described previously (26). The A. nidulans strains used were a wild-type pacC strain, the alkalinity-mimicking pacCc200 strain, and the acidity-mimicking pacC+/–20205, pacC–6309, and palF15 strains; the relevant genotypes of these strains are given in Table 1. Siderophore production of the wild type increased 35.7-fold as the culture pH was raised from 4.2 to 7.0 (Table 2). The alkalinity-mimicking mutation pacCc200 caused upregulation of triacetylfusarinine C production at pH 4.2 and pH 7. In contrast, the acidity-mimicking pacC+/–20205, pacC–6309, and palF15 mutations downregulated siderophore production at pH 7.0. Taken together, the acidity- and alkalinity-mimicking mutations decreased the influence of pH on siderophore production 7.4- to 18-fold, which demonstrates a strong response of siderophore production to ambient pH mainly mediated by PacC. Nevertheless, all four pH regulatory mutants still showed weak pH regulation of siderophore biosynthesis, indicating PacC-independent effects.


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  		TABLE 1. Fungal strains used in this study


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  		TABLE 2. Siderophore production of A. nidulans wild type and pH regulatory mutants

pH regulation of genes involved in siderophore metabolism at the transcriptional level was studied by Northern analysis (Fig. 1). Consistent with PacC-mediated pH regulation of siderophore production, the transcript level of the siderophore biosynthetic gene sidA was higher in the wild type at pH 7 than at pH 4.2. The sidA transcript level was higher in the alkalinity-mimicking pacCc200 mutant at pH 4.2 and pH 7.0 than in the wild type and lower in the acidity-mimicking pacC+/–20205 mutant at pH 7.0 than in the wild type. Enterobactin transporter-encoding mirA and triacetylfusarinine C transporter-encoding mirB displayed the same expression pattern as sidA, revealing that in addition to siderophore biosynthesis, uptake of native and heterologous siderophores is also subject to PacC-mediated pH regulation (Fig. 1). In contrast, expression of mirC showed neither a pH response in the wild type nor deregulation in the pH regulatory mutants, which indicates that not all iron-regulated genes are subject to pH control.



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  		FIG. 1. Expression of mirA, mirB, mirC, and sidA in A. nidulans wild type and pH regulatory mutants during iron-replete (+Fe) (10 µM FeSO4) and depleted (– Fe) conditions at pH 7.0 and pH 4.2. Actin gene (acnA) expression is shown as a loading control.

The eukaryotic model organism S. cerevisiae lacks the ability to synthesize siderophores (24), although it can utilize siderophores produced by other species via four siderophore transporters, which are homologous to the Aspergillus siderophore transporters (14-16, 20, 21, 32, 33). The expression of two of the S. cerevisiae transporter-encoding genes, ARN3/SIT1 and ARN4/ENB1, has been shown to be induced by alkaline pH (18) and suggested to be mediated by the S. cerevisiae PacC orthologue Rim101p indirectly by functioning as a repressor of another repressor (17). In contrast to the situation in A. nidulans, the S. cerevisiae alkalinity-mimicking mutant was not able to upregulate expression of the siderophore transporters at acidic pH. These data point either to differences in the pH regulatory mode in these two fungal species or have to be ascribed to the different experimental setups: transcription of the siderophore transporter-encoding genes in S. cerevisiae was not monitored during iron-depleted conditions, and consequently, iron repression might have been stronger in acidic medium than in alkaline medium.

Consistent with direct control by PacC acting as a transcriptional activator, the promoters of sidA, mirA, and mirB contain at least one PacC consensus binding motif (data not shown). Alternatively, PacC may act indirectly, as suggested for Rim101p-mediated regulation of siderophore transporters in S. cerevisiae. During iron-replete conditions, neither the wild type nor the different pH regulatory mutants produced detectable levels of siderophores or showed expression of the analyzed genes (Fig. 1). Therefore, expression of the genes involved in siderophore biosynthesis and uptake is governed by both iron and pH control. It has been shown that PacC can regulate gene expression by competing with transcription factors recognizing overlapping binding sites (9). The PacC consensus sites in the promoter regions of sidA, mirA, and mirB do not overlap with sites for the iron regulator SreA, which suggests that PacC and SreA do not compete for binding to these promoters.

What might be the rationale for the siderophore system being subject to pH regulation in addition to iron control? There are at least four conceivable reasons. (i) Iron solubility is significantly elevated during acidic conditions because of stabilization of ferrous iron, which prevents autooxidation and formation of insoluble ferric oxyhydroxids. (ii) Due to competition from protons for the ferric iron binding sites, the affinity constants of siderophores for iron decrease in acidic conditions, which makes the siderophore system a less-efficient iron uptake tool at acidic pH (6). (iii) It has been suggested that uptake of siderophores requires cotransport with protons (31), providing the basis for cross talk between siderophore metabolism and pH sensing. (iv) Bacterial competition with fungi, in this case for the essential nutrient iron, is more intense during alkaline conditions than acidic conditions (5). In this respect, it is interesting that the iron-free form of the A. nidulans siderophore triacetylfusarinine C has also been purified from Aspergillus deflectus and shown to exhibit antibiotic activity against a range of bacterial species (1).


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ACKNOWLEDGMENTS
 
This work was supported in part by the Austrian Science Foundation (FWF P-15959-B11) and the Austrian National Bank (OENB-8750).

We are grateful to Miguel A. Penalva and Herbert N. Arst for kindly providing us with the A. nidulans mutants used in this work and for helpful suggestions and discussions. We acknowledge the excellent technical assistance of Gerlinde Häninger.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biology, Medical University of Innsbruck, Peter-Mayr-Str. 4b/III, A-6020 Innsbruck, Austria. Phone: 43 512 507 3605. Fax: 43 512 507 2866. E-mail: hubertus.haas{at}uibk.ac.at. Back

{dagger} M.E. and H.O. contributed equally to this work. Back

{ddagger} Present address: Sandoz GmbH, A-6250 Kundl, Austria. Back


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Eukaryotic Cell, April 2004, p. 561-563, Vol. 3, No. 2
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.2.561-563.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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