Siderophore synthesis in Magnaporthe grisea is essential for vegetative growth,conidiation and resistance to oxidative stress

The plant pathogenic fungus Magnaporthe grisea excretes siderophores of the coprogen-type for iron acquisition and uses ferricrocin for intracellular iron storage. In the present report we characterize mutants with defects in extracellular siderophore biosynthesis. Deletion of the M. grisea SSM2 gene, which encodes a non-ribosomal peptide synthetase, resulted in a loss of the production of all coprogens. The mutant strains had a reduced growth rate, produced fewer conidia and were more sensitive to oxidative stress. Ferricrocin production was not affected. Upon deletion of M. grisea OMO1, a gene predicted to encode an l-ornithine-N⁵-monooxygenase, no siderophores of any type were detected, the strain was aconidial, growth rate was reduced and sensitivity to oxidative stress was increased. Abundance of several proteins was affected in the mutants. The Δssm2 and Δomo1 mutant phenotypes were complemented by supplementation of the medium with siderophores or reintroduction of the respective genes.     

The siderophore ferricrocin produced by specific foliar endophytic fungi in vitro

Production of extracellular siderophores is typical for many plant-associated microbes, both mutualistic and antagonistic. Various strains of mycorrhizal fungi produce siderophores, and siderophore production by pathogenic fungi is typically associated with virulence. We analyzed extracellular siderophore production along with production of antibacterial and antioxidant compounds in foliar endophytic fungi of Scots pine (Pinus sylvestris L.) and Labrador tea (Rhododendron tomentosum Harmaja). The siderophore produced in vitro was ferricrocin, quantities ranging between 7.9 and 17.6 μg/l. Only the fungi with antibacterial activity produced ferricrocin and any well-known siderophores were not detected in the broths of antioxidant-producing fungi. Therefore, production of ferricrocin is typical for some, but not all foliar endophytic fungi. Ferricrocin was detected in the leaves of Labrador tea, which suggests that ferricrocin may play a role in vivo in the interaction between the endophyte and plant host.     

Siderophores produced by Magnaporthe grisea in the presence and absence of iron

An analysis of siderophores produced by Magnaporthe grisea revealed the presence of one intracellular storage siderophore, ferricrocin, and four coprogen derivatives secreted into the medium under iron depletion. Structural analysis showed that the compounds are coprogen, coprogen B, 2-N-methylcoprogen and 2-N-methylcoprogen B. Siderophore production under low and high iron conditions was quantified.     

The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC)

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l-ornithine N(5)-monooxygenase and sidC encoding a non-ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore-deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron-replete conditions. Partial restoration of the growth of siderophore-deficient mutants by high concentrations of Fe(2+) (but not Fe(3+)) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC-bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore-deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin-deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.     

Role of siderophores in iron storage in spores of Neurospora crassa and Aspergillus ochraceus.

Spores of Neurospora crassa 74A are lacking in ferritinlike iron pools, as demonstrated by M?ssbauer spectroscopic analysis. The cyclic hexapeptide siderophore ferricrocin constituted 47% of the total iron content in spores. After germination and growth, the ferricrocin iron pool disappeared, indicating that the metal was utilized. In spores of Aspergillus ochraceus, 74% of the total iron content was bound by ferrichrome-type siderophores. Siderophores may function as iron storage forms in fungal systems.     

Mycorrhizal Fungi: Siderophore Production

Abstract

Mycorrhizal fungi, which commonly occur in natural as well as agricultural soils, are known to enhance plant uptake of nutrients, including metal ions present as trace concentrations. As mycorrhizal infection is a widespread feature of plant communities, it seems appropriate to review the data on mycorrhizal fungi and their potential to produce siderophores.

Based on a bioassay with Aureobacteriumflavescens JG-9 it was shown that a number of ectomycorrhizal fungi (EM) produce hydroxamate siderophores. Also an arbuscular mycorrhizal (AM) grass species, which showed greater iron uptake than nonmycorrhizal controls, tested positively when bioassayed for hydroxamate siderophores. Encoid mycorrhizal fungi, too, have been demonstrated to be capable of producing hydroxamate-type siderophores. However, only in the case of the eridoid mycorrhizal fungi the main siderophores have been isolated and subsequently identified as ferricrocin and fusigen, respectively. The biotechnological and ecological significance of studies of the siderophore biosynthesis by mycorrhizal fungi is discussed.     

The membrane potential is the driving force for siderophore iron transport in fungi

Abstract Respiratory inhibitors and uncouplers severely impair [⁵⁵Fe]ferricrocin uptake by Neurospora crassa . parallel measurements of ATP decay and ferricrocin uptake, however, disprove the idea that direct input of metabolic energy in the form of ATP is required for transmembrane movement of siderophores. The role of the membrane potential for siderophore uptake was demonstrated using iron-deficient cells, which were derepressed in the glucose-II uptake system. Addition of high amounts of glucose (1 mM) to glu-II-derepressed cells leads to a membrane depolarization of about 120 mV, followed by a significant inhibition of ferricrocin uptake, which recovered after some minutes. Full transport inhibition occurred after membrane depolarization in the presence of plasma membrane ATP-ase inhibitors (DCCD or DES), indicating that the membrane potential is essential for siderophore transport in fungi.     

Ferricrocin - an ectomycorrhizal siderophore of Cenococcum geophilum

The ectomycorrhizal fungus Cenococcum geophilum was grown in low-iron medium and the excreted siderophores were extracted, purified and analyzed by HPLC. The principal hydroxamate siderophore produced, was identified as ferricrocin as confirmed by analytical HPLC, FAB-mass spectrometry and ¹H- and ¹³C-NMR spectra. Although the occurrence of ferricrocin has been shown earlier to occur in the ericoid mycorrhizal fungi, this is the first report of ferricrocin in a true ectomycorrhizal fungus which is taxonomically related to the ascomycetes.     

Ferricrocin functions as the main intracellular iron-storage compound in mycelia ofNeurospora crassa

Summary Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferrichrome C are predominantly found intracellularly. Mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-typeN.crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia ofN. crassa. After uptake of ferricrocin in both the wild-typeN. crassa 74A and the siderophore-free mutantN. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores ofN. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores.     

SreA-mediated iron regulation in Aspergillus fumigatus

Aspergillus fumigatus, the most common airborne fungal pathogen of humans, employs two high-affinity iron uptake systems: iron uptake mediated by the extracellular siderophore triacetylfusarinine C and reductive iron assimilation. Furthermore, A. fumigatus utilizes two intracellular siderophores, ferricrocin and hydroxyferricrocin, to store iron. Siderophore biosynthesis, which is essential for virulence, is repressed by iron. Here we show that this control is mediated by the GATA factor SreA. During iron-replete conditions, SreA deficiency partially derepressed synthesis of triacetylfusarinine C and uptake of iron resulting in increased cellular accumulation of both iron and ferricrocin. Genome-wide DNA microarray analysis identified 49 genes that are repressed by iron in an SreA-dependent manner. This gene set, termed SreA regulon, includes all known genes involved in iron acquisition, putative novel siderophore biosynthetic genes, and also genes not directly linked to iron metabolism. SreA deficiency also caused upregulation of iron-dependent and antioxidative pathways, probably due to the increased iron content and iron-mediated oxidative stress. Consistently, the sreA disruption mutant displayed increased sensitivity to iron, menadion and phleomycin but retained wild-type virulence in a mouse model. As all detrimental effects of sreA disruption are restricted to iron-replete conditions these data underscore that A. fumigatus faces iron-depleted conditions during infection.     

Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean (Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.     

Azotobacter vinelandii gene clusters for two types of peptidic and catechol siderophores produced in response to molybdenum

Aim: To characterize the complementary production of two types of siderophores in Azotobacter vinelandii. Methods and Results: In an iron‐insufficient environment, nitrogen‐fixing A. vinelandii produces peptidic (azotobactin) and catechol siderophores for iron uptake to be used as a nitrogenase cofactor. Molybdenum, another nitrogenase cofactor, was also found to affect the production level of siderophores. Wild‐type cells excreted azotobactin into molybdenum‐supplemented and iron‐insufficient medium, although catechol siderophores predominate in molybdenum‐free environments. Two gene clusters were identified to be involved in the production of azotobactin and catechol siderophores through gene annotation and disruption. Azotobactin‐deficient mutant cells produced catechol siderophores under the molybdenum‐supplemented and iron‐insufficient conditions, whereas catechol siderophore–deficient mutant cells extracellularly secreted excess azotobactin under iron‐deficient condition independent of the concentration of molybdenum. This evidence suggests that a complementary siderophore production system exists in A. vinelandii. Conclusions: Molybdenum was found to regulate the production level of two types of siderophores. Azotobacter vinelandii cells are equipped with a complementary production system for nitrogen fixation in response to a limited quantity of metals. Significance and Impact of the Study: This is the first study identifying A. vinelandii gene clusters for the biosynthesis of two types of siderophores and clarifying the relationship between them.     

SidL, an Aspergillus fumigatus transacetylase involved in biosynthesis of the siderophores ferricrocin and hydroxyferricrocin

The opportunistic fungal pathogen Aspergillus fumigatus produces four types of siderophores, low-molecular-mass iron chelators: it excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) for iron uptake and accumulates ferricrocin (FC) for hyphal and hydroxyferricrocin (HFC) for conidial iron distribution and storage. Siderophore biosynthesis has recently been shown to be crucial for fungal virulence. Here we identified a new component of the fungal siderophore biosynthetic machinery: AFUA_1G04450, termed SidL. SidL is conserved only in siderophore-producing ascomycetes and shows similarity to transacylases involved in bacterial siderophore biosynthesis and the N(5)-hydroxyornithine:anhydromevalonyl coenzyme A-N(5)-transacylase SidF, which is essential for TAFC biosynthesis. Inactivation of SidL in A. fumigatus decreased FC biosynthesis during iron starvation and completely blocked FC biosynthesis during iron-replete growth. In agreement with these findings, SidL deficiency blocked conidial accumulation of FC-derived HFC under iron-replete conditions, which delayed germination and decreased the size of conidia and their resistance to oxidative stress. Remarkably, the sidL gene is not clustered with other siderophore-biosynthetic genes, and its expression is not affected by iron availability. Tagging of SidL with enhanced green fluorescent protein suggested a cytosolic localization of the FC-biosynthetic machinery. Taken together, these data suggest that SidL is a constitutively active N(5)-hydroxyornithine-acetylase required for FC biosynthesis, in particular under iron-replete conditions. Moreover, this study revealed the unexpected complexity of siderophore biosynthesis, indicating the existence of an additional, iron-repressed N(5)-hydroxyornithine-acetylase.     

Intracellular siderophores are essential for ascomycete sexual development in heterothallic Cochliobolus heterostrophus and homothallic Gibberella zeae

Connections between fungal development and secondary metabolism have been reported previously, but as yet, no comprehensive analysis of a family of secondary metabolites and their possible role in fungal development has been reported. In the present study, mutant strains of the heterothallic ascomycete Cochliobolus heterostrophus, each lacking one of 12 genes (NPS1 to NPS12) encoding a nonribosomal peptide synthetase (NRPS), were examined for a role in sexual development. One type of strain (Delta nps2) was defective in ascus/ascospore development in homozygous Delta nps2 crosses. Homozygous crosses of the remaining 11 Delta nps strains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by NPS2 is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of C. heterostrophus. Functional conservation of NPS2 in both heterothallic C. heterostrophus and the unrelated homothallic ascomycete Gibberella zeae was demonstrated. G. zeae Delta nps2 strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to C. heterostrophus and G. zeae Delta nps2 strains, demonstrating that abnormal sexual development of Delta nps2 strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to C. heterostrophus and G. zeae Delta nps2 strains restored WT fertility. NPS1, a G. zeae NPS gene that groups phylogenetically with NPS2, does not play a role in sexual development. Overall, these data demonstrate that iron and intracellular siderophores are essential for successful sexual development of the heterothallic ascomycete C. heterostrophus and the homothallic ascomycete G. zeae.     

Evidence for a common siderophore transport system but different siderophore receptors in Neurospora crassa.

Uptake and competition experiments were performed with Neurospora crassa and Penicillium parvum by using 14C-labeled coprogen and 55Fe-labeled ferrichrome-type siderophores. Several siderophores of the ferrichrome family, such as ferrichrome, ferricrocin, ferrichrysin, and tetraglycyl-ferrichrome as well as the semisynthetic ferricrocin derivatives O-(phenyl-carbamoyl)-ferricrocin and O-(sulfanilyl-carbamoyl)-ferricrocin were taken up by N. crassa. The ferrichrome-type siderophores used vary in the structure of the peptide backbone but possess a common lambda-cis configuration about the iron center and three identical ornithyl-delta-N-acetyl groups as surrounding residues. This suggests that these ferrichrome-type siderophores are recognized by a common ferrichrome receptor. We also concluded that the ferrichrome receptor is lambda-cis specific from the inability to take up the synthetic enantiomers, enantio-ferrichrome and enantio-ferricrocin, possessing a delta-cis configuration about the iron center. On the other hand, we found that coprogen, possessing a delta-absolute configuration and two trans-anhydromevalonic acid residues around the metal center, was also taken up by N. crassa and was competitively inhibited by the ferrichrome-type siderophores. We therefore propose the existence of a common siderophore transport system but the presence of different siderophore receptors in N. crassa. In addition, ferrirubin, which is very slowly transported by N. crassa, inhibited both coprogen and ferrichrome-type siderophore transport. Contrary to the findings with N. crassa, transport experiments with P. parvum revealed the presence of a ferrichrome receptor but the absence of a coprogen receptor; coprogen was neither transported nor did it inhibit the ferrichrome transport.     

Isolation and identification of hydroxamate siderophores of ericoid mycorrhizal fungi

Three ericoid mycorrhizal fungi were grown in pure culture under iron deprivation: (i) the ascomyceteHymenoscyphus ericae, a characteristic endophyte of ericaceous plants on acid soils; (ii) the hyphomyceteOidiodendron griseum, an ericoid mycorrhizal fungus which is also a soil-borne fungus able to colonize wood; and (iii) an endophyte of the calciculous ericaceous plantRhodothamnus chamaecistus. All three fungi produced several hydroxamate siderophores which were isolated in the ferric form by adsorption to Amberlite XAD-2, gel chromatography on Sephadex LH20 and by HPLC on a C₁₈ reversed-phase column. Siderophores were identified by (i) co-chromatography with known fungal siderophores, (ii) ion spray mass spectrometry after semi-preparative HPLC and (iii) analyzing their electrophoretic behavior. WhileH. ericae andO. griseum were similar in producing ferricrocin as their principal siderophore, the endophyte ofR. chamaecistus produced mainly fusigen.     

Chemical properties and NMR spectroscopic identification of certain fungal siderophores

Siderophores of six fungi viz. Aspergillus sp. ABp4, Aureobacidium pullulans, Penicillium oxalicum, P. chrysosporium, Mycotypha africana and Syncephalastrum racemosum were examined for their (1) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di-, or trihydroxamate; (3) amino acid composition; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicum, P. chrysosporium, and M, africana) exhibited net basic charge, siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racemosum) were acidic and 1 fungus (A. pullullans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporium was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, histidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporium, and valine in M. africana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. pullulans) as asperchrome F1, and AB-513 (M. africana) as rhizoferrin. The peak obtained for siderophore AB-5 (Aspergillus sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception.     

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H]⁺). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C₃₃H₅₂N₆O₁₃Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.