Characterization of siderophores from Ustilago maydis

Two siderophores, ferrichrome and ferrichrome A, were found in cultures of Ustilago maydis (DC) Corda. Both siderophores were found intracellularly and extracellularly. Their authenticity was confirmed by thin layer chromatography, HPLC, UV-visible spectrometry, paper electrophoresis, amino acid analysis, NMR and fast atom bombardment mass spectroscopy. Regulation of siderophore production by iron was examined. Repression of biosynthesis of extracellular siderophores occurred at 10^–5 M iron. Ferrichrome was found intracellularly at all iron concentrations employed; in general, ferrichrome A was not found to be cell-associated.     

Ferrichrome in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> – an iron transport and iron storage compound

Schizosaccharomyces pombe has been assumed not to produce siderophores. Nevertheless, the genomic sequence of this fission yeast revealed the presence of siderophore biosynthetic genes for hydroxamates. Applying a bioassay based on an Aspergillus nidulans strain deficient in siderophore biosynthesis, and using reversed-phase HPLC and mass spectrometry analysis, we demonstrate that S. pombe excretes and accumulates intracellularly the hydroxamate-type siderophore ferrichrome. Under iron-limiting conditions, the cellular ferrichrome pool was present in the desferri-form, while under iron-rich conditions, in the ferri-form. In contrast to S. pombe, hydroxamate-type siderophores could not be detected in two other yeast species, Saccharomyces cerevisiae and Candida albicans.     

Iron Uptake in Ustilago maydis: Tracking the Iron Path

In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.     

Role of two siderophores in Ustilago sphaerogena: Regulation of biosynthesis and uptake mechanisms

Under iron-deficient conditions the smut fungus Ustilago sphaerogena produces two kinds of siderophores, ferrichrome and ferrichrome A. Regulation of ligand biosyntheses and uptake mechanisms of the iron chelates were studied to determine the role of each chelate in U. sphaerogena. The biosynthesis of each ligand was differentially regulated. Ferrichrome A, the more effective chelate, was preferentially synthesized under more extreme conditions of iron stress, but completely repressed when the cell was supplied with sufficient iron. In contrast, biosynthesis of ferrichrome was strongly but not completely repressed by iron. The mechanism of repression was examined using a newly developed in vivo synthesis assay. Chromium and gallium-containing siderophore analogs had no effect on siderophore ligand biosynthesis. Iron, added as siderophores, resulted in increased oxygen uptake and amino acid transport, which was soon followed by decreased ligand biosynthesis, suggesting that regulation may be indirect and related to oxidative metabolism. Uptake experiments were used to rule out a ligand-exchange mechanism for ferrichrome A-iron transport. The data suggest that ferrichrome A-iron is taken up at a specific site that results in a rapid distribution of iron inside the cell.     

Role of two siderophores in Ustilago sphaerogena. Regulation of biosynthesis and uptake mechanisms

Under iron-deficient conditions the smut fungus Ustilago sphaerogena produces two kinds of siderophores, ferrichrome and ferrichrome A. Regulation of ligand biosyntheses and uptake mechanisms of the iron chelates were studied to determine the role of each chelate in U. sphaerogena. The biosynthesis of each ligand was differentially regulated. Ferrichrome A, the more effective chelate, was preferentially synthesized under more extreme conditions of iron stress, but completely repressed when the cell was supplied with sufficient iron. In contrast, biosynthesis of ferrichrome was strongly but not completely repressed by iron. The mechanism of repression was examined using a newly developed in vivo synthesis assay. Chromium and gallium-containing siderophore analogs had no effect on siderophore ligand biosynthesis. Iron, added as siderophores, resulted in increased oxygen uptake and amino acid transport, which was soon followed by decreased ligand biosynthesis, suggesting that regulation may be indirect and related to oxidative metabolism. Uptake experiments were used to rule out a ligand-exchange mechanism for ferrichrome A-iron transport. The data suggest that ferrichrome A-iron is taken up at a specific site that results in a rapid distribution of iron inside the cell.     

Analysis of ferrichrome biosynthesis in the phytopathogenic fungus Ustilago maydis: cloning of an ornithine-N5-oxygenase gene

By using a non-enterobactin-producing enb-7 mutant of Salmonella typhimurium LT2 as a biological indicator, a novel screening method was developed for identifying mutants of Ustilago maydis defective in the biosynthesis of the siderophores ferrichrome and ferrichrome A. Two classes of siderophore mutations, both recessive, were isolated after mutagenesis of haploid cells of the corn smut fungus. Class I mutants no longer produced ferrichrome while retaining the ability to produce ferrichrome A; class II mutants were defective in the production of both ferrichrome and ferrichrome A. Genetic and biochemical data suggest that class II mutants are defective in the ability to hydroxylate L-ornithine to delta-N-hydroxyornithine, the first step in the biosynthesis of these siderophores. A genomic library of wild-type U. maydis DNA was constructed in the cosmid transformation vector pCU3, which contains a dominant selectable marker for hygromycin B resistance. Two cosmids, pSid1 and pSid2, were identified in this library by their ability to complement class II siderophore auxotrophs. The production of both siderophores was concomitantly restored in the majority of the resultant transformants. Transforming DNA could be recovered from the fungal, cosmid-containing transformants by in vitro packaging with lambda bacteriophage extracts. Alternatively, the clones could be identified by a sib selection procedure. Cotransformation was found to occur at a high frequency in the fungus and was used to determine that a 2.5-kilobase HindIII-NruI fragment in pSid1 was responsible for complementing the class II siderophore biosynthetic mutation.     

Module evolution and substrate specificity of fungal nonribosomal peptide synthetases involved in siderophore biosynthesis

Background  

Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches.     

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O‐glycosidically linked to 3,15,16‐trihydroxypalmitic acid. The sugar moiety is acylated with 2‐hydroxy‐octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl‐transferase and is lacking a gene homologous to the α‐hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl‐transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl‐transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.     

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.     

Identification of extracellular siderophores and a related peptide from the endophytic fungus Epichloë festucae in culture and endophyte-infected Lolium perenne

A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three N^δ-trans-anhydromevalonyl–N^δ-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.     

Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes

Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe³⁺-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe³⁺-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe³⁺ bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe³⁺ or Fe²⁺ forms or Fe³⁺ associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.     

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.     

Siderophore production in Azotobacter vinelandii in response to Fe‐, Mo‐ and V‐limitation

Azotobacter vinelandii is a terrestrial diazotroph well studied for its siderophore production capacity and its role as a model nitrogen fixer. In addition to Fe, A. vinelandii siderophores are used for the acquisition of the nitrogenase co‐factors Mo and V. However, regulation of siderophore production by Mo‐ and V‐limitation has been difficult to confirm and knowledge of the full suite of siderophores synthesized by this organism has only recently become available. Using this new information, we conducted an extensive study of siderophore production in N₂‐fixing A. vinelandii under a variety of trace metal conditions. Our results show that under Fe‐limitation the production of all siderophores increases, while under Mo‐limitation only catechol siderophore production is increased, with the strongest response seen in protochelin. We also find that the newly discovered A. vinelandii siderophore vibrioferrin is almost completely repressed under Mo‐ and V‐limitation. An examination of the potential nitrogen ‘cost’ of siderophore production reveals that investments in siderophore N can represent as much as 35% of fixed N, with substantial differences between cultures using the Mo‐ as opposed to the less efficient V‐nitrogenase.     

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

Nonribosomal peptide synthase is responsible for the biosynthesis of siderophore in Vibrio vulnificus MO6-24/O

Vibrio vulnificus produces siderophores, lowmolecular- weight iron-chelating compounds, to obtain iron under conditions of iron deprivation. To identify genes associated with the biosynthesis of siderophore in V. vulnificus MO6-24/ O, we screened clones with mini-Tn5 random insertions for those showing decreased production of siderophore. Among 6,000 clones screened, nine such clones were selected. These clones contain the transposon inserted in VV2_0830 (GenBank accession number) that is a homolog of a nonribosomal peptide synthetase (NRPS). There is an another NRPS module, VV2_0831, 49-bp upstream to VV2_0830. We named these two genes vvs (Vibrio vulnificus siderophore synthetase) A and B, respectively. Mutation of either vvsA or vvsB showed a decreased production of siderophore. The expression of an NRPS-lux fusion was negatively modulated by the presence of iron, and the regulation was dependent on Fur (ferric uptake regulator). However, the expression of the NRPS genes was still not fully derepressed in the iron-rich condition, even in fur-null mutant cells, suggesting that some other unknown factors are involved in the regulation of the genes. We also demonstrated that the NRPS genes are important for virulence of the pathogen in a mice model.     

The mineral weathering ability of Collimonas pratensis PMB3(1) involves a Malleobactin-mediated iron acquisition system

Mineral weathering by microorganisms is considered to occur through a succession of mechanisms based on acidification and chelation. While the role of acidification is established, the role of siderophores is difficult to disentangle from the effect of the acidification. We took advantage of the ability of strain Collimonas pratensis PMB3(1) to weather minerals but not to acidify depending on the carbon source to address the role of siderophores in mineral weathering. We identified a single non-ribosomal peptide synthetase (NRPS) responsible for siderophore biosynthesis in the PMB3(1) genome. By combining iron-chelating assays, targeted mutagenesis and chemical analyses (HPLC and LC-ESI-HRMS), we identified the siderophore produced as malleobactin X and how its production depends on the concentration of available iron. Comparison with the genome sequences of other collimonads evidenced that malleobactin production seems to be a relatively conserved functional trait, though some collimonads harboured other siderophore synthesis systems. We also revealed by comparing the wild-type strain and its mutant impaired in the production of malleobactin that the ability to produce this siderophore is essential to allow the dissolution of hematite under non-acidifying conditions. This study represents the first characterization of the siderophore produced by collimonads and its role in mineral weathering.     

De novo synthesis of ferrichrome by Fusarium oxysporum f. sp. cubense TR4 in response to iron starvation

Manipulation of iron bioavailability in the banana rhizosphere may suppress Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc). However, iron starvation induced by application of synthetic iron chelators does not effectively suppress Fusarium wilt. It is unclear whether Foc can subvert iron chelators and thereby evade iron starvation through the synthesis of iron-scavenging secondary metabolites, called siderophores. In vitro studies were conducted using iron-deficient growth medium and medium supplemented with a synthetic iron chelator, 2,2′-dipyridyl, to mimic iron starvation in Foc Tropical Race 4 (Foc TR4). Concentration of extracellular siderophores increased three-fold (p < 0.05) in the absence of iron. Liquid chromatography-mass spectrometry analysis detected the hydroxamate siderophore, ferrichrome, only in the mycelia of iron-starved cultures. Moreover, iron-starved cultures exhibited a reduction in total cellular protein concentration. In contrast, out of the 20 proteinogenic amino acids, only arginine increased (p < 0.05) under iron starvation. Our findings suggest that iron starvation does not cause a remodelling of amino acid metabolism in Foc TR4, except for arginine, which is required for biosynthesis of ornithine, the precursor for siderophore biosynthesis. Collectively, our findings suggest that biosynthesis of siderophores, particularly ferrichrome, could be a counteractive mechanism for Foc TR4 to evade iron starvation.     

Progress in pathogenesis research of Ustilago maydis,and the metabolites involved along with their biosynthesis

Ustilago maydis is a pathogenic fungus that causes corn smut. Because of its easy cultivation and genetic transformation, U. maydis has become an important model organism for plant-pathogenic basidiomycetes. U. maydis is able to infect maize by producing effectors and secreted proteins as well as surfactant-like metabolites. In addition, the production of melanin and iron carriers is also associated with its pathogenicity. Here, advances in our understanding of the pathogenicity of U. maydis, the metabolites involved in the pathogenic process, and the biosynthesis of these metabolites, are reviewed and discussed. This summary will provide new insights into the pathogenicity of U. maydis and the functions of associated metabolites, as well as new clues for deciphering the biosynthesis of metabolites.