Under iron deprivation Omphalotus olearius was found to produce the hydroxamate siderophore ferrichrome A. A gene cluster consisting of three genes: fso1, a nonribosomal peptide synthetase whose expression is enhanced in the absence of iron; omo1, a l-ornithine-N5-monooxygenase; and ato1, an acyltransferase probably involved in the transfer of the methylglutaconyl residue to N5-hydroxyorinithine was identified. The fso1 sequence is interrupted by 48 introns and its derived protein sequence has a similar structure to the homologous genes of Ustilago maydis and Aspergillus nidulans. This is the first report of a nonribosomal peptide synthetase gene and a biosynthetic gene cluster in homobasidiomycetes.
Iron is an essential element for the growth and proliferation of nearly all organisms. Under iron starvation, most microorganisms excrete siderophores, low-molecular-mass compounds with a very high affinity for iron. Their function is to mediate iron uptake by microbial cells. Most fungal siderophores are of the hydroxamate group , divided into three structural families: fusarinines, coprogens and ferrichromes . All share the basic structural unit N5-acyl-N5-hydroxyornithine. Ferrichrome A (Fig. 1) consists of a hexapeptide ring made up of one glycine, two serine, and three N5-hydroxyornithine amino acid residues, the latter acylated by trans-(α-methyl)-glutaconic acid residues . Ferrichrome A was first isolated from the smut fungus Ustilago sphaerogena.
Hydroxamate siderophore biosynthesis takes place in at least three steps. The first is the hydroxylation of l-ornithine catalysed by a l-ornithine-N5-monooxygenase. The second is the acylation of N5-Hydroxy-l-ornithine to form N5-acyl-N5-hydroxy-l-ornithine, catalysed by an acyl-CoA:N5-hydroxy-l-ornithine N-acyl transferase. The third step comprises the linking of the hydroxamic acids and, in the case of ferrichromes, the incorporation of the other amino acids by a modular nonribosomal peptide synthetase (NRPS) . Three domains are necessary for a basic NRPS module: an adenylation domain (A) that selects the amino acid and activates it as amino acyl adenylate, a peptidyl carrier protein domain (PCP or T domain) that binds the co-factor 4′-phosphopantetheine and to which the amino acid is attached, and a condensation domain (C) that catalyses the peptide bond formation [6,7].
Production of siderophore biosynthetic enzymes was shown to be repressed by iron in cell-free experiments for rhodotorulic acid , fusigen  and ferrichrome . In Ustilago maydis (heterobasidiomycete) and Aspergillus Emericella nidulans (ascomycete), the expression of siderophore biosynthetic genes is enhanced in iron depleted medium, and is under the control of the GATA family transcription factors Urbs1  and SREA , respectively.
We describe here the first NRPS gene as well as the first iron regulated siderophore biosynthetic gene cluster in a higher basidiomycete.
2Materials and methods
2.1Organisms, media, and cultivation conditions
Omphalotus olearius TA90170 was isolated from spore prints of fruiting bodies. The culture is deposited at the IBWF. The strain was cultivated and maintained in YMG medium (glucose 4 g/l, yeast extract 4 g/l, malt extract 10 g/l; pH 5.5). For cultures on solid media, 1.5% of agar were added. Fermentation up to 2 l were carried out in Erlenmeyer flasks with agitation (120 rpm) at 28°C. Larger fermentations were carried out in a Braun Biostat A-20 fermenter containing 20 l of YMG medium with aeration (3 l air/min) and agitation (120 rpm) at 28°C. For iron depletion studies mycelia of O. olearius were washed four times with Sundström minimal medium  (20 g/l glucose, 1.4 g/l l-asparagine, 0.35 g/l KH2PO4, 0.15 g/l K2 HPO4· 3H2O, 0.5 g/l Na2S04· 10H2O, 0.1 mg/l thiamine, 5 mg/l CaCl2, 20 mg/l MgCl2· 6H2O, 0.27 g/l sodium citrate, 0.26 g/l citric acid, 0.22 g/l MnSO4· 4H2O, 0.20 g/l ZnSO4· 7H2O), transferred to 0.25–2 l flasks with Sundström minimal medium and incubated with agitation (120 rpm) at 28°C.
The Escherichia coli strain DH5α (Gibco BRL, Rockville, MD, USA) was used for cloning and plasmid propagation. E. coli XL1 b [P2] was used as host for bacteriophage lambda FIX II (Stratagene) and was grown in NZY medium. DNA from lambda clones was purified by standard techniques and cloned into pUC18 .
2.2DNA and RNA isolation
For genomic DNA isolation, lyophilized mycelium of O. olearius was ground to a fine powder using a mortar. Extraction buffer (1 M Tris–HCl, pH 8.0, 0.1 mM EDTA, 1% SDS, 200 μg/ml proteinase K and 100 μg/ml DNase-free RNase) was added and the mixture incubated for 50 min at 56°C. The mixture was extracted once with phenol and three times with phenol/chloroform/isoamylalcohol (25:24:1 v/v/v) followed by a final extraction with chloroform/isoamylalcohol (24:1 v/v). To remove proteins, 0.1 vol of 5 M potassium acetate was added to the supernatant, the mixture incubated 1 h on ice and centrifuged. DNA was precipitated with 2 vol of ethanol and washed with 70% ethanol.
RNA was isolated by grinding lyophilized mycelium under liquid nitrogen. The fine powder was extracted with the Total RNA Isolation System (Promega) according to the manufacturer's instructions. DNase treatment was performed by dissolving the RNA pellets in 200 μl buffer (0.1 M sodium acetate, 5 mM MgSO4, pH 5.0) containing 2 units DNase and incubated for 20 min at room temperature, followed by a final phenol/chloroform extraction. RNA was precipitated with 2 vol ethanol and dissolved in 500 μl of RNase-free water. mRNA was isolated from total RNA using the “PolyATract mRNA Isolation System” (Promega).
2.3Isolation and characterization of ferrichrome A
Mycelia and culture broth were separated by filtration from 6 to 8 week old culture of O. olearius, grown in iron depleted medium. FeCl3 was added to the culture filtrate which was then loaded onto a XAD-16 column and washed with three column volumes of water. Ferrichrome A was eluted with methanol. After evaporation of the solvent, the extract was dissolved in methanol and subjected to gel filtration on Sephadex LH20 in methanol. The fractions containing ferrichrome A were pooled, concentrated and purified by preparative HPLC (RP-18 250 × 4 mm, elution with 20% acetonitrile, 80% of 0.1% H3PO4). For MS–MS analysis, samples and standards were dissolved in 50% methanol 0.1% formic acid to a final concentration of 0.1 μg/μl. Experiments were performed on a Q-TOF2 (Micromass) with direct application of the sample in a nano-ESI capillary, 1 kV cone voltage, 80°C source temperature and collision energies from 10 to 80 eV. Ferrichrome A standard was purchased from Biophore Research Products (Tübingen, Germany).
2.4Cloning and analysis of fso1, ato1 and omo1
A GST (genome sequence tags) project was started using genomic DNA from O. olearius digested with Bsp143 I to obtain fragments of 0.5–1.5 kb. DNA fragments were cloned into pBluescript KS(−) and a total of 1500 clones were sequenced. Sequence analysis was performed with the GCG Wisconsin Package 10.3, (Accelrys) using BlastX and FastX.
A genomic library of O. olearius was constructed using the “Lambda FIX II/Xho I Partial Fill-in Vector Kit” and the “Gigapack III XL extract” (Stratagene) according to the manufacturer's instructions. One GST clone showing NRPS homology was labeled with DIG-dUTP (Roche) using the HexaLabel DNA labeling Kit (Fermentas). Sreening of the genomic library was performed using standard protocols . Lambda phages with positive hybridization were amplified and DNA was extracted and digested with Sal I, Sac I and Bam HI. DNA fragments were subcloned in pUC19 and sequenced after generating a population of DNA sequencing templates with randomly interspersed primer-binding sites (GPS-1 Genome Priming System, NEB).
cDNA was generated using mRNA isolated from O. olearius using the Qiagen OneStep RT-PCR kit. Gene-specific primers which covered the whole genomic sequence of fso1, omo1 and ato1 were used for the amplification of cDNA fragments which were subsequently compared with the genomic sequence using Seqman from the DNASTAR package.
For the expression analysis of fso1, mycelia from O. olearius were cultivated in Sundström medium with and without supplementation of 10 μM FeSO4. After cultivation, the mycelium was harvested and washed with 0.6 M MgCl2 and sterile water. RNA was extracted from lyophilized mycelia using the “RNagents Total RNA Isolations-Kit” (Promega). For real-time PCR analysis cDNA was synthesized with 5 μg total RNA using the “Revert Aid H Minus First Strand cDNA Synthesis Kit” (Fermentas) following manufacturer's instructions. For the PCR reaction the “QuantiTect SYBR Green PCR Kit” (Qiagen) was used following manufacturer's instructions. Primers used were fso1 LC for 5′-GCTAGTATCTTGCTCGCCTACTC-3′, fso1 LC rev 5′-GCTGACATTGC GTACACGAAC-3′, gpd LC for 5′-CTAACAAAGACTGGCGTGGAGGAC-3′, gpd LC rev 5′-GAAGGAGAGACCAGTGAGCTTTCC-3′. Experiments were carried out in the LightCycler (Roche) as follows: initial activation step (95°C for 15 min), amplification and quantification program repeated 60 times (94°C for 15 s, 55°C for 25 s, 72°C for 20 s with a single fluorescence measurement), melting curve program (52–95°C with a heating rate of 0.1°C per second and a continuous fluorescence measurement).
3.1Ferrichrome A from O. olearius
Ferrichrome A was isolated from culture filtrates of O. olearius when grown on Sundström medium and identified by its MS–MS fragmentation pattern which was identical to that of authentic ferrichrome A. No other siderophore could be detected. Ferrichrome A could not be detected when the medium was supplemented with iron.
3.2Molecular characterization of fso1, omo1 and ato1
In order to identify nonribosomal peptide synthetase (NRPS) genes in the homobasidiomycete O. olearius a GST project (Genome Sequence Tag) was performed. A fragment with high homology to known NRPS genes was used to screen a genomic Lambda bank of O. olearius. The sequence of the entire NRPS gene isolated (fso1; Accession No. AY929618) was determined directly from overlapping Lambda clones and was found to have a length of 16,282 bp. Comparison with peptide synthetase genes and BlastX searches led to the conclusion that the fso1 ORF was likely to be interrupted by introns. Therefore, the entire cDNA sequence was analysed by reverse transcribed PCR using RNA isolated from O. olearius cultivated in YMG medium. The comparison of cDNA and genomic sequences confirmed the existence of 48 introns ranging in size from 47 to 72 bp which are distributed over the whole length of the gene (Fig. 2). The 5′ and 3′ boundaries in most cases match the consensus sequences for fungal introns .
The predicted protein encoded by the fso1 gene comprises 4548 amino acids and has a mass of approximately 500 kDa. Sequence analysis of the predicted product of fso1 led to the identification of multiple core motifs which are characteristic for NRPS and allowed the prediction of the modular structure of Fso1. The putative peptide synthetase contains three complete modules with an adenylation (A) domain, an peptidyl carrier protein (T) and a condensation (C) domain (Fig. 3). Downstream of the three complete modules, two additional incomplete modules consisting of a T and a C-domain were identified. The predicted domain structure of Fso1 is identical to that of SidC from A. nidulans, and similar to that of Sid2 from U. maydis (Fig. 3). Furthermore, Fso1 displays the highest similarity to fungal peptide synthetases and particulary to SidC (50% similarity) and Sid2 (46% similarity).
As genes responsible for the biosynthesis of fungal secondary metabolites are usually clustered [16,17], further DNA sequence analysis of flanking lambda clones was performed. This led to the identification of other genes close to the putative NRPS gene (Fig. 3(a)) including two other genes whose predicted function is consistent with a possible role in siderophore biosyntheses. These two genes, ato1 and omo1, are directly adjacent to fso1 with spacer regions of only 352 and 785 bp, respectively.
Ato1 (Accession No. AY929616), the gene proximal to fso1, has a genomic sequence length of 1362 bp and is interrupted by three introns. The predicted amino acid sequence of Ato1 shows 60% similarity to a hypothetical protein from U. maydis (UM01432.1) and 54% to a hypothetical protein from Giberella zeae (FG11027.1). The predicted product of ato1 also displays significant homology to various acetylases including an acetylase from Aspergillus oryzae (Ace1) with 49% similarity and putative siderophore biosynthesis proteins/acyltransferases from various bacteria with up to 54% similarity.
The second gene, omo1 (Accession No. AY929617), which is divergently transcribed from the 785 bp intergenic region shared with ato1 consists of 2067 bp and is interrupted by five putative introns. The derived amino acid sequence shows homology to l-ornithine-N5 monoogygenases of various species, including PvdA of Pseudomonas aeruginosa (51% similarity) and Sid1 of U. maydis (48% similarity). O. olearius Omo1 was aligned with diverse l-ornithine-N5-monoogygenases proving that signature sequences for ω-amino acid hydroxylases could be identified (data not shown). The FAD binding site (GLGFGP) has the typical fingerprint sequence of siderophore biosynthetic monooxygenases with the last glycine of the consensus sequence GXGXXG changed to proline .
3.3Regulation of fso1 by iron
The sequence analysis described above and the finding that O. olearius produces ferrichrome A under iron deprivation led to the assumption that fso1, omo1 and ato1 are responsible for the production of ferrichrome A. Siderophore biosynthetic genes are usually negatively regulated by iron. Therefore, real-time PCR analysis was performed using RNA isolated from O. olearius cultivated in Sundström medium with or without supplementation of FeSO4. The corresponding real-time PCR efficiency (E) of one cycle in the exponential phase was calculated according to the equation: E= 10[−1/slope].
For the calculation of the real-time PCR efficiencies cDNA input from the two different RNA populations in triplicate experiments with several points in the range of 0.10 to100 ng was investigated. The efficiencies were determined to be 1.98 for fso1 and 1.75 for gpd (glyceraldehyde-3-phosphate dehydrogenase from O. olearius, Accession No. AJ439986, used as internal normalisation control). The fso1 transcript expression in the abscence of iron was upregulated 62-fold, normalised with the expression of the gpd gene .
A large number of NRPS enzymes and NRPS coding genes have been described from bacteria and ascomycetes [12,13]. NRPS are large enzymes organized in modules, each responsible for the incorporation of one element into the product. Linking of the hydroxamate groups and, in the case of ferrichromes, additional incorporation of amino acids, is carried out by NRPSs. Deletion mutants of the NRPS coding gene sidC in A. nidulans lack ferricrocin , whereas defective sid2 mutants of U. maydis are not able to produce ferrichrome  but still can produce ferrichrome A .
fso1, the presumed ferrichrome A synthetase gene, encodes a single protein of 4548 amino acids with homology (up to 50%) to fungal and bacterial NRPS. Sequence alignments revealed similarities to the consensus sequences defined by Konz and Marahiel . The predicted modular organization of Fso1 is identical to that of the ferricrocin synthetase SidC of A. nidulans which consists of three complete modules (A–T–C) and two additional modules each lacking the adenylation domain (Fig. 3). Although in Fso1 the A domain of the third module is not very similar to the consensus, it is still highly homologous to the third A domains of Sid2 and SidC. The U. maydis ferrichrome synthetase (Sid2) shows a similar domain organisation, but with only one additional incomplete module consisting of a T-domain and a truncated C domain (Fig. 3). Most likely, these enzymes including Fso1 are responsible for the formation of the complete hexapeptides via repeated use of one or more modules, in which the additional T- and C-domains could be involved [1,20,21].
Interestingly, the 16,282 bp genomic sequence of fso1 was found to be interrupted by a total number of 48 introns (which is more than 2 kb). Most fungal peptide synthetase genes characterized so far do not contain any introns  and only few exeptions are described for NRPS genes interrupted by 1–7 introns [25–28]. This high number of introns is not usual for NRPS genes but it is a fact known for basidiomycetous genes in general [29,30]. To our knowledge fso1 is the first NRPS gene described in a higherbasidiomycete. The divergence of ustilaginomycetes (including U. maydis) from hymenomycetes, to which the homobasidiomycetes belong, occurred at an early stage in evolution at least 500 millions years ago .
Real-time PCR experiments showed a 62-fold overexpression of fso1 under iron deprivation compared to iron supplemented conditions. This correlates with the production of ferrichrome A, supporting evidence for an involvement of the fso1 gene product in ferrichrome A biosynthesis.
The first step in hydroxamate siderophore biosynthesis is the N5-hydroxylation of l-ornithine to N5-hydroxy-l-ornithine . This reaction is catalysed by a specific monooxygenase. Two genes have been assigned encoding l-ornithine N5-monooxygenases in fungi: sid1 in U. maydis whose disruption leads to a lack of ferrichrome and ferrichrome A production , and sidA in A. nidulans, which is essential for viability .
Two genes were found downstream of fso1: ato1 and omo1, separated by 352 and 785 bp, respectively (Fig. 3). omo1 encodes for a l-ornithine-N5-monooxygenase similar to sid1 and sidC. In U. maydis, the NRPS gene (sid2) and sid1 are clustered but separated by an intergenic region of 3.7 kb. As in O. olearius and U. maydis, in Schizosaccharomyces pombe and Aureobasidium pullulans the putative siderophore synthetase encoding genes are clustered with sid1 orthologs .
The second step, the acylation of N5-hydroxy-l-ornithine is catalysed by an acyl-CoA:N5-hydroxy-l-ornithine N-acyltransferase. Such activities were detected for the first time in U. sphaerogena and Fusarium cubense, but, to our knowledge, no corresponding genes have been reported.
ato1 encodes a putative acyltransferase with homology to an acyltransferase from A. oryzae (Accession No. BAC78653) and to N6-hydroxylysine acetyltransferases from bacteria producing the hydroxamate siderophore aerobactin . Moreover, ato1 is placed between fso1 and omo1, strongly suggesting a direct involvement in ferrichrome A synthesis.
To our knowledge this is the first report of a NRPS gene and biosynthetic gene cluster within the higher basidiomycetes. Recently we established a transformation system for O. olearius (manuscript in preparation) and the construction of deletion mutants of fso1, ato1 and omo1 is in progress.
We thank Michael Becker and Anja Meffert for excellent technical assistance and Andrew Foster for critical reading of the manuscript. This project was funded by the BMBF (Förderkennzeichen 0312833A). K. Welzel was supported by the Landesgraduiertenförderung des Landes Rheinland-Pfalz.