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Eukaryot Cell. Apr 2004; 3(2): 561–563.
PMCID: PMC387658

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

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.

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, pacC6309, and palF15 strains; the relevant genotypes of these strains are given in Table Table1.1. Siderophore production of the wild type increased 35.7-fold as the culture pH was raised from 4.2 to 7.0 (Table (Table2).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, pacC6309, 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.

TABLE 1.
Fungal strains used in this study
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. (Fig.1).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. (Fig.1).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.

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 ...

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. (Fig.1).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).

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.

REFERENCES

1. Anke, H. 1977. Desferritriacetylfusigen, an antibiotic from Aspergillus deflectus. J. Antibiot. 30:125-128. [PubMed]
2. Arst, H. N., and M. A. Penalva. 2003. pH regulation in Aspergillus and parallels with higher eukaryotic regulatory systems. Trends Genet. 19:224-231. [PubMed]
3. Brakhage, A. A., and G. Turner. 1992. l-Lysine repression of penicillin biosynthesis and the expression of penicillin biosynthesis genes acvA and ipnA in Aspergillus nidulans. FEMS Microbiol. Lett. 77:123-127. [PubMed]
4. Caddick, M. X., A. G. Brownlee, and H. N. Arst, Jr. 1986. Regulation of gene expression by pH of the growth medium in Aspergillus nidulans. Mol. Gen. Genet. 203:346-353. [PubMed]
5. Calvo, A. M., R. A. Wilson, J. W. Bok, and N. P. Keller. 2002. Relationship between secondary metabolism and fungal development. Microbiol. Mol. Biol. Rev. 66:447-459. [PMC free article] [PubMed]
6. Dhungana, S., M. J. Miller, L. Dong, C. Ratledge, and A. L. Crumbliss. 2003. Iron chelation properties of an extracellular siderophore exochelin MN. J. Am. Chem. Soc. 125:7654-7663. [PubMed]
7. Dorn, G. 1965. Genetic analysis of the phosphatases in Aspergillus nidulans. Genet. Res. 10:13-26. [PubMed]
8. Eisendle, M., H. Oberegger, I. Zadra, and H. Haas. 2003. The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC). Mol. Microbiol. 49:359-375. [PubMed]
9. Espeso, E. A., and H. N. Arst. 2000. On the mechanism by which alkaline pH prevents expression of an acid-expressed gene. Mol. Cell. Biol. 20:3355-3363. [PMC free article] [PubMed]
10. Fernandez-Martinez, J., C. V. Brown, E. Diez, J. Tilburn, H. N. Arst, Jr., M. A. Penalva, and E. A. Espeso. 2003. Overlap of nuclear localisation signal and specific DNA-binding residues within the zinc finger domain of PacC. J. Mol. Biol. 334:667-684. [PubMed]
11. Haas, H. 2003. Molecular genetics of fungal siderophore biosynthesis and uptake: the role of siderophores in iron uptake and storage. Appl. Microbiol. Biotechnol. 62:316-330. [PubMed]
12. Haas, H., M. Schoeser, E. Lesuisse, J. F. Ernst, W. Parson, B. Abt, G. Winkelmann, and H. Oberegger. 2003. Characterization of the Aspergillus nidulans transporters for the siderophores enterobactin and triacetylfusarinine C. Biochem. J. 371:505-513. [PMC free article] [PubMed]
13. Haas, H., I. Zadra, G. Stoffler, and K. Angermayr. 1999. The Aspergillus nidulans GATA factor SREA is involved in regulation of siderophore biosynthesis and control of iron uptake. J. Biol. Chem. 274:4613-4619. [PubMed]
14. Heymann, P., J. F. Ernst, and G. Winkelmann. 2000. A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae. Biometals 13:65-72. [PubMed]
15. Heymann, P., J. F. Ernst, and G. Winkelmann. 2000. Identification and substrate specificity of a ferrichrome-type siderophore transporter (Arn1p) in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 186:221-227. [PubMed]
16. Heymann, P., J. F. Ernst, and G. Winkelmann. 1999. Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily. Biometals 12:301-306. [PubMed]
17. Lamb, T. M., and A. P. Mitchell. 2003. The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae. Mol. Cell. Biol. 23:677-686. [PMC free article] [PubMed]
18. Lamb, T. M., W. Xu, A. Diamond, and A. P. Mitchell. 2001. Alkaline response genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway. J. Biol. Chem. 276:1850-1856. [PubMed]
19. Leong, S. A., and G. Winkelmann. 1998. Molecular biology of iron transport in fungi. Met. Ions Biol. Syst. 35:147-186. [PubMed]
20. Lesuisse, E., and P. Labbe. 1989. Reductive and non-reductive mechanisms of iron assimilation by the yeast Saccharomyces cerevisiae. J. Gen. Microbiol. 135:257-263. [PubMed]
21. Lesuisse, E., M. Simon-Casteras, and P. Labbe. 1998. Siderophore-mediated iron uptake in Saccharomyces cerevisiae: the SIT1 gene encodes a ferrioxamine B permease that belongs to the major facilitator superfamily. Microbiology 144:3455-3462. [PubMed]
22. Li, W., and A. P. Mitchell. 1997. Proteolytic activation of Rim1p, a positive regulator of yeast sporulation and invasive growth. Genetics 145:63-73. [PMC free article] [PubMed]
23. Mingot, J. M., J. Tilburn, E. Diez, E. Bignell, M. Orejas, D. A. Widdick, S. Sarkar, C. V. Brown, M. X. Caddick, E. A. Espeso, H. N. Arst, Jr., and M. A. Penalva. 1999. Specificity determinants of proteolytic processing of Aspergillus PacC transcription factor are remote from the processing site, and processing occurs in yeast if pH signalling is bypassed. Mol. Cell. Biol. 19:1390-1400. [PMC free article] [PubMed]
24. Neilands, J. B., K. Konopka, B. Schwyn, M. Coy, R. T. Francis, B. H. Paw, and A. Bagg. 1987. Comparative biochemistry of microbial iron assimilation, p. 3-34. In G. Winkelmann and D. R. Winge (ed.), Iron transport in microbes, plants and animals. VCH Verlagsgesellschaft, Weinheim, Germany.
25. Oberegger, H., M. Eisendle, M. Schrettl, S. Graessle, and H. Haas. 2003. 4′-Phosphopantetheinyl transferase-encoding npgA is essential for siderophore biosynthesis in Aspergillus nidulans. Curr. Genet. 44:211-215. [PubMed]
26. Oberegger, H., M. Schoeser, I. Zadra, B. Abt, and H. Haas. 2001. SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulans. Mol. Microbiol. 41:1077-1089. [PubMed]
27. Oberegger, H., I. Zadra, M. Schoeser, B. Abt, W. Parson, and H. Haas. 2002. Identification of members of the Aspergillus nidulans SREA regulon: genes involved in siderophore biosynthesis and utilization. Biochem. Soc. Trans. 30:781-783. [PubMed]
28. Penalva, M. A., and H. N. Arst, Jr. 2002. Regulation of gene expression by ambient pH in filamentous fungi and yeasts. Microbiol. Mol. Biol. Rev. 66:426-446. [PMC free article] [PubMed]
29. Pontecorvo, G., J. A. Roper, L. M. Hemmons, K. D. MacDonald, and A. W. J. Bufton. 1953. The genetics of Aspergillus nidulans. Adv. Genet. 5:141-238. [PubMed]
30. Tilburn, J., S. Sarkar, D. A. Widdick, E. A. Espeso, M. Orejas, J. Mungroo, M. A. Penalva, and H. N. Arst, Jr. 1995. The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH. EMBO J. 14:779-790. [PMC free article] [PubMed]
31. Winkelmann, G. 2001. Siderophore transport in fungi, p. 463-479. In G. Winkelmann (ed.), Microbial transport systems. Wiley-VCH, Weinheim, Germany.
32. Yun, C. W., T. Ferea, J. Rashford, O. Ardon, P. O. Brown, D. Botstein, J. Kaplan, and C. C. Philpott. 2000. Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake. J. Biol. Chem. 275:10709-10715. [PubMed]
33. Yun, C. W., J. S. Tiedeman, R. E. Moore, and C. C. Philpott. 2000. Siderophore-iron uptake in Saccharomyces cerevisiae. Identification of ferrichrome and fusarinine transporters. J. Biol. Chem. 275:16354-16359. [PubMed]

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