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.     

Hydroxamate siderophore synthesis by Phialocephala fortinii, a typical dark septate fungal root endophyte

The siderophore production of various isolates of Phialocephala fortinii was assessed quantitatively as well as qualitatively in batch assays under pure culture conditions at different pH values and iron(III) concentrations. We found a distinct effect of both of these parameters on siderophore synthesis and as well as on fungal growth. In comparative analyses of two of the isolates, maximum siderophore production was found at a pH in the range of pH 4.0 to 4.5 while, under the experimental conditions employed, the optimal concentration of ferric iron was determined to be between 20–40 g iron (III) l^–1 (0.36–0.72 M, respectively). HPLC analysis of the culture filtrate of most of the isolates of P. fortinii revealed the excretion of ferricrocin as main hydroxamate siderophore, followed by ferrirubin and ferrichrome C. The pattern of release of these three substances proved to be dependent on pH and iron(III) concentration of the culture medium, and to be specific for each isolate under investigation.     

Fusarinines and dimerum acid, mono- and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants

Cucumber, as a strategy I plant, and Maize as a strategy II plant, were cultivated in hydroponic culture in the presence of a ferrated siderophore mixture (1 M) from a culture of Penicillium chrysogenumisolated from soil. The siderophore mixture significantly improved the iron status of these plants as measured by chlorophyll concentration to the same degree as a 100-fold higher FeEDTA supply. Analysis of the siderophore mixture from P. chrysogenum by HPLC and electrospray mass spectrometry revealed that besides the trihydroxamates, coprogen and ferricrocin, large amounts of dimerum acid and fusarinines were present which represent precursor siderophores or breakdown products of coprogen. In order to prove the iron donor properties of dimerum acid and fusarinines for plants, purified coprogen was hydrolyzed with ammonia and the hydrolysis products consisting of dimerum acid and fusarinine were used for iron uptake by cucumber and maize. In short term experiments radioactive iron uptake and translocation rates were determined using ferrioxamine B, coprogen and hydrolysis products of coprogen. While the trihydroxamates revealed negligible or intermediate iron uptake rates by both plant species, the fungal siderophore mixture and the ammoniacal hydrolysis products of coprogen showed high iron uptake, suggesting that dimerum acid and fusarinines are very efficient iron sources for plants. Iron reduction assays using cucumber roots or ascorbic acid also showed that iron bound to hydrolysis products of coprogen was more easily reduced compared to iron bound to trihydroxamates. Ligand exchange studies with epi-hydroxymugineic acid and EDTA showed that iron was easily exchanged between coprogen hydrolysis products and phytosiderophores or EDTA. The results indicate that coprogen hydrolysis products are an excellent source for Fe nutrition of plants.     

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.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

Purification and characterization of rhodobactin: a mixed ligand siderophore from Rhodococcus rhodochrous strain OFS

The siderophore produced by Rhodococcus rhodochrous strain OFS, rhodobactin, was isolated from iron-deficient cultures and purified by a combination of XAD-7 absorptive/partition resin column and semi-preparative HPLC. The siderophore structure was characterized using 1D and 2D ¹H, ¹³C and ¹⁵N NMR techniques (DQFCOSY, TOCSY, NOESY, HSQC and LR-HSQC) and was confirmed using ESI-MS and MS/MS experiments. The structural characterization revealed that the siderophore, rhodobactin, is a mixed ligand hexadentate siderophore with two catecholate and one hydroxamate moieties for iron chelation. We further investigated the effects of Fe concentrations on siderophore production and found that Fe limiting conditions (Fe concentrations from 0.1 μM to 2.0 μM) facilitated siderophore excretion. Our interests lie in the role that siderophores may have in binding metals at mixed contamination sites (containing metals/radionuclides and organics). Given the broad metabolic capacity of this microbe and its Fe scavenging ability, R. rhodochrous OFS may have a competitive advantage over other organisms employed in bioremediation. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Siderophore transport through Escherichia coli outer membrane receptor FhuA with disulfide-tethered cork and barrel domains

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.     

A Catecholic Siderophore Produced by the Thermoresistant Bacillus licheniformis VK21 Strain

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for the producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe³⁺, in response to addition of manganese(II) salts. The growth of the producer on a minimal medium containing MnSO₄ under the conditions of iron deficiency is accompanied by the accumulation of a catecholic product, the content of which reaches maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl₃, the amount of the catecholic product in the medium considerably decreases. The siderophore, called S_VK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to the amino acid analysis and NMR spectrometry, the metabolite S_VK21 is 2,3-dihydroxybenzoyl-glycyl-threonine.     

Evaluation of bacteria isolated from rice rhizosphere for biological control of charcoal rot of sorghum caused by <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> (Tassi) Goid.

A total of 360 bacteria, isolated from the rhizospheres of a system of rice intensification (SRI) fields, were characterized for the production of siderophore, fluorescence, indole acetic acid (IAA), hydrocyanic acid (HCN) and solubilization of phosphorus. Of them, seven most promising isolates (SRI-156, -158, -178, -211, -229, -305 and -360) were screened for their antagonistic potential against Macrophomina phaseolina (causes charcoal rot in sorghum) by dual culture assay, blotter paper assay and in greenhouse. All the seven isolates inhibited M. phaseolina in dual culture assay, whereas six isolates solubilized phosphorous (except SRI-360), all seven produced siderophore, four produced fluorescence (except SRI-178, -229 and -305), six produced IAA (except SRI-305) and five produced HCN (except SRI-158 and -305). In the blotter paper assay, no charcoal rot infection was observed in SRI-156-treated sorghum roots, indicating complete inhibition of the pathogen, while the roots treated with the other isolates showed 49–76% lesser charcoal rot infection compared to the control. In the antifungal activity test (in green house on sorghum), all the isolates increased shoot dry mass by 15–23% and root dry mass by 15–20% (except SRI-158 and -360), over the control. In order to confirm the plant growth-promoting (PGP) traits of the isolates, the green house experiment was repeated but, in the absence of M. phaseolina. The results further confirmed the PGP traits of the isolates as evidenced by increases in shoot and root dry mass, 22–100% and 5–20%, respectively, over the control. The sequences of 16S rDNA gene of the isolates SRI-156, -158, -178, -211, -229, -305 and -360 were matched with Pseudomonas plecoglossicida, Brevibacterium antiquum, Bacillus altitudinis, Enterobacter ludwigii, E. ludwigii, Acinetobacter tandoii and P. monteilii, respectively in BLAST analysis. This study indicates that the selected bacterial isolates have the potential for PGP and control of charcoal rot disease in sorghum.     

Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica

Clinical isolates of Yersinia enterocolitca, which belong to mouse-lethal serotypes, produce the siderophore yersiniophore. Siderophore production was shown to be iron regulated and to reach maximum production in late log phase. Yersiniophore is a fluorescent siderophore with maximum excitation at 270 nm and a major emission peak at 428 nm. Absorption maxima were seen at 210 and 250 nm with a low broad peak from 280 to 320 nm. Purification of unchelated yersiniophore for structural analysis was made difficult by low yields (1–2 mg mg^-1), and susceptibility to acid hydrolysis, oxidation and possibly polymerization. Yersinophore was therefore purified as an Al³⁺ chelate, which was found to be stable in solution for several weeks. To purify Al³⁺-yersiniophore, unchelated yersiniophore was first extracted from culture supernatants with dichloromethane, concentrated by rotary evaporation and adsorbed to a DEAE-sephacel column. Al³⁺-yersiniophore was eluted with 0.01 m AlCl₃ and further purified by HPLC. The structure was established by a combination of elemental analysis, high resolution mass spectrometry and two-dimensional NMR experiments. Yersiniophore is a phenolate-thiazole siderophore with the formula C₂₁H₂₄N₃O₄S₃Al and a molecular weight of 505.07404 when chelated to Al³⁺. The structure of yersiniophore was determined to be closely related to the structures of pyochelin, produced by Pseudomonas aeruginosa, and anguibactin, produced by Vibrio anguillarum.     

Isolation and characterization of the siderophore N-deoxyschizokinen from Bacillus megaterium ATCC 19213

N-Deoxyschizokinen, a novel siderophore, was isolated from stationary phase cultures of Bacillus megaterium ATCC 19213 and identified as 4-[(3(acetylhydroxyamino)propyl)amino]-2-[2-[(3-(acetylamino)propyl)amino]-2-oxoethyl]-2-hydroxy-4-oxo-butanoic acid. The siderophore was purified by HPLC and its structure determined using ¹H and ¹³C NMR, ¹H-¹H COSY and electrospray mass spectroscopy. The monohydroxamate siderophore has the same carbon skeleton as schizokinen but the hydroxyl group on one hydroxamate is replaced by a hydrogen. A detailed ¹H NMR study of schizokinen, N-deoxyschizokinen and their imides, schizokinen A and N-deoxyschizokinen A is presented.     

Purification and chemical characterization of staphyloferrin B,a hydrophilic siderophore from staphylococci

This paper describes the chemical characterization of staphyloferrin B, a new complexone type siderophore isolated from low iron cultures of Staphylococcus hyicus DSM 20459. Purification of the very hydrophilic metabolite was achieved by anion exchange high performance liquid chromatography HPLC. Mass spectrometry showed a molecular mass of 448 amu. Hydrolysis with 8 mHCl revealed the presence of l-2,3-diaminopropionic acid, citrate, ethylenediamine and succinic semialdehyde. The connections between the four building blocks were determined by two-dimensional nuclear magnetic resonance measurements. UV/Vis and circular dichroism spectra are consistent with the proposed structure, which could also be confirmed by precursor feeding. The siderophore activity of staphyloferrin B was demonstrated by iron transport measurements.     

Ornibactin production and transport properties in strains of Burkholderia vietnamiensis and Burkholderia cepacia (formerly Pseudomonas cepacia)

Several strains of Burkholderia vietnamiensis, isolated from the rhizosphere of rice plants, and four strains formerly known as Pseudomonas cepacia including two collection strains and two clinical isolates were compared for siderophore production and iron uptake. The B. vietnamiensis (TVV strains) as well as the B. cepacia strains (ATCC 25416 and ATCC 17759) and the clinical isolates K132 and LMG 6999 were all found to produce ornibactins under iron starvation. The two ATCC strains of B. cepacia additionally produced the previously described siderophores, pyochelin and cepabactin. Analysis of the ratio of isolated ornibactins (C4, C6 and C8) by HPLC revealed nearly identical profiles. Supplementation of the production medium with ornithine (20 mm) resulted in a 2.5-fold increase in ornibactin synthesis. Ornibactin-mediated iron uptake was independent of the length of the acyl side chain and was observed with all strains of B. vietnamiensis and B. cepacia, but was absent with strains of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, known to produce pyoverdines or desferriferrioxamines as siderophores. These results suggest that ornibactin production is a common feature of all Burkholderia strains and that these strains develop an ornibactin-specific iron transport system which is distinct from the pyoverdine-specific transport in Pseudomonas strains.     

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.     

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.     

The detection of <Emphasis Type="Italic">hip</Emphasis>O gene by real-time PCR in thermophilic <Emphasis Type="Italic">Campylobacter</Emphasis> spp. with very weak and negative reaction of hippurate hydrolysis

A total of 190 Campylobacter spp. isolates, of which 34 gave the result of very weak activity, and 156 gave the negative activity in the test for hippurate hydrolysis were characterized. The genomic DNA was isolated from a fresh culture of each isolate and the real-time PCR, targeting the hipO gene, was used to confirm the species distribution of Campylobacter isolates. The hipO gene was detected in 17 isolates (11%) within the total of 156 negative isolates for hippurate hydrolysis. Out of 34 isolates with very weak activity, 19 isolates (56%) were also found to be positive for hipO gene and characterized as C. jejuni. The real-time PCR assay used in this study could be employed for more accurate diagnosis of Campylobacter infections at species level after the biochemical characterization based on hippuricase activity of the isolates. This could also provide important data for the epidemiology of infections associated with these zoonotic pathogens.     

Rhizoferrin — a novel siderophore from the fungusRhizopus microsporus var.rhizopodiformis

Summary From a strain ofRhizopus microsporus var.rhizopodiformis a novel siderophore, named rhizoferrin, was isolated by ion-exchange column chromatography, gel filtration and preparative HPLC. Hydrolysis with 6 M HCl and subsequent gas chromatography/mass spectrometry (GUMS) of the esterified/trifluoroacetylated derivatives indicated that citric acid and diaminobutane were the only constituents. From positive fastatom-bombardment (FAB) and ion-spray tandem mass spectrometry, a molecular mass of 436 Da and the assignment of several daughter ion fragments could be obtained, which indicated the presence of two citric acid residues and one diaminobutane residue. NMR studies finally confirmedN ¹,N ⁴-bis(1-oxo-3-hydroxy-3,4-dicarboxybutyl)-diaminobutane as the structure of rhizoferrin. The iron-binding property was demonstrated on chromeazurol S plates and its siderophore activity was confirmed by iron transport measurements in young mycelia ofR. microsporus. While rhizoferrin and also ferrioxamines B and E proved to be effective siderophores, coprogen was a poor siderophore in this fungus.     

Cellular and extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum.

Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active, unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied and complex.     

First reported isolation from New Zealand pasture ofPithomyces chartarum unable to produce sporidesmin

A total of 676 isolates ofPithomyces chartarum, recovered from pasture at a single site, were examined for their ability to produce sporidesmin. Two isolates did not produce sporidesmin in levels detectable by HPLC despite their ability to spore profusely. This is the first report of sporulatingP. chartarum isolated from New Zealand pasture which does not produce sporidesmin.Abbreviations RCA rabbit chow agar     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.