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Siderophore-mediated acquisition of iron has been shown to be indispensable for the virulence of several fungal pathogens, the siderophore transporter Sit1 was found to mediate uptake of the novel antifungal drug VL-2397, and siderophores were shown to be useful as biomarkers as well as for imaging of fungal infections. However, siderophore uptake in filamentous fungi is poorly characterized. The opportunistic human pathogen Aspergillus fumigatus possesses five putative siderophore transporters. Here, we demonstrate that the siderophore transporters Sit1 and Sit2 have overlapping, as well as unique, substrate specificities. With respect to ferrichrome-type siderophores, the utilization of ferrirhodin and ferrirubin depended exclusively on Sit2, use of ferrichrome A depended mainly on Sit1, and utilization of ferrichrome, ferricrocin, and ferrichrysin was mediated by both transporters. Moreover, both Sit1 and Sit2 mediated use of the coprogen-type siderophores coprogen and coprogen B, while only Sit1 transported the bacterial ferrioxamine-type xenosiderophores ferrioxamines B, G, and E. Neither Sit1 nor Sit2 were important for the utilization of the endogenous siderophores fusarinine C and triacetylfusarinine C. Furthermore, A. fumigatus was found to lack utilization of the xenosiderophores schizokinen, basidiochrome, rhizoferrin, ornibactin, rhodotorulic acid, and enterobactin. Taken together, this study characterized siderophore use by A. fumigatus and substrate characteristics of Sit1 and Sit2.

Aerobic anoxygenic phototrophs have been isolated from a rich variety of environments including marine ecosystems, freshwater and meromictic lakes, hypersaline springs, and biological soil crusts, all in the hopes of understanding their ecological niche. Over 100 isolates were chosen for this study, representing 44 species from 27 genera. Interactions with Fe3+ and other metal(loid) cations such as Mg2+, V3+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Se4+ and Te2+ were tested using a chromeazurol S assay to detect siderophore or metallophore production, respectively. Representatives from 20 species in 14 genera of α-Proteobacteria, or 30% of strains, produced highly diffusible siderophores that could bind one or more metal(loid)s, with activity strength as follows: Fe &gt, Zn &gt, V &gt, Te &gt, Cu &gt, Mn &gt, Mg &gt, Se &gt, Ni &gt, Co. In addition, γ-proteobacterial Chromocurvus halotolerans, strain EG19 excreted a brown compound into growth medium, which was purified and confirmed to act as a siderophore. It had an approximate size of ~341 Da and drew similarities to the siderophore rhodotorulic acid, a member of the hydroxamate group, previously found only among yeasts. This study is the first to discover siderophore production to be widespread among the aerobic anoxygenic phototrophs, which may be another key method of metal(loid) chelation and potential detoxification within their environments.

Fungi are producing many secondary compounds such as siderophores that play an important role in the agriculture field. Siderophores are an essential compound produced by some fungal communities such as plant growth-promoting fungi (PGPF), biocontrol agent, and phytopathogen during iron deficiency. Iron is an essential compound for the fungi physiology. Indeed, a few studies mention the role of fungal siderophores in agriculture. Many types of siderophores are detected such as fusarinines, rhodotorulic acid, coprogens, and ferrichrome that are secreted by fungi. The ability to produce siderophores is an interesting trait for some species of fungi which can be utilized in agriculture instead of synthetic chemicals. Many important fungi can produce siderophores including mycorrhiza, Trichoderma, Fusarium, Aspergillus, and Penicillium. However, the mechanism of iron chelating by producing siderophores, is possible applying in control of plant pathogens, weeds, and promoting plant growth. This mechanism showed high efficacy in competing and affecting the activity and growth of several plant pathogens that can cause inhibition for growth. This mechanism can provide iron easy to absorb for plants which are promoting plant growth. However, it must determine the ability of fungal strains to produce siderophores at an appropriate level. It is helpful in enhancing the traits of producing siderophores by these these fungi under the different environments that potential increase in their efficiency for using in agriculture.

International audience; Actinide-based mineral phases occurring in contaminated soils can be solubilized by organic chelators excreted by plants, such as citrate. Herein, the efficiency of citrate towards U and Pu extraction is compared to that of siderophores, whose primary function is the acquisition of iron(III) as an essential nutrient and growth factor for many soil microorganisms. To that end, we selected desferrioxamine B (DFB) as an emblematic bacterial trishydroxamic siderophore and a synthetic analog, abbreviated (LCy,Pr)H2, of the tetradentate rhodotorulic acid (RA) produced by yeasts. Firstly, the uranyl speciation with both ligands was assessed in the pH range 2–11 by potentiometry and visible absorption spectrophotometry. Equilibrium constants and absorption spectra for three [UO2(DFB)Hh](h–1)+ (h = 1–3) and five [UO2(LCy,Pr)lHh](2+h–2l)+ (–1  h  1 for l = 1 and h = 0–1 for l = 2) solution complexes were determined at 25.0 °C and I = 0.1 M KNO3. Similar studies for the Fe3+/(LCy,Pr)2– system revealed the formation of five species having [Fe(LCy,Pr)]+, [Fe(LCy,Pr)OH], [Fe(LCy,Pr)(OH)2]–, [Fe(LCy,Pr)2H], and [Fe2(LCy,Pr)3] compositions. Then, the ability of DFB, (LCy,Pr)H2, and citrate to solubilize either U or Pu from pitchblende-rich soils (soils 1 and 2) or freshly plutonium-contaminated soils (LBS and PG) was evaluated by performing batch extraction tests. U was extracted significantly only by citrate after a day. After one week, the amount of U complexed by citrate only slightly exceeded that measured for the siderochelates, following the order citrate > (LCy,Pr)H2  DFB  H2O, and were comparatively very low. Pu was also more efficiently extracted by citrate than by DFB after a day, but only by a factor of ~2–3 for the PG soil, while the Pu concentration in the supernatant after one week was approximately the same for both natural chelators. It remained nearly constant for DFB between the 1st and 7th day, but drastically decreased in the case of citrate, suggesting chemical decomposition in the latter case. For the Fe-rich soils 1 and 2, the efficiencies of the three chelators to solubilize Fe after a day were of the same order of magnitude, decreasing in the order DFB > citrate > (LCy,Pr)H2. However, after a week DFB had extracted ~1.5 times more Fe, whereas the amount extracted by the other chelators stayed constant. For the less Fe-rich LBS and PG soils contaminated by Pu, the amounts of extracted Fe were higher, especially after 7 days, and the DFB outperformed citrate by a factor of nearly 3. The higher capacity of the hexadentate DFB to extract Pu in the presence of Fe and its lower ability to mobilize U qualitatively agree with the respective complexation constant ratios, keeping in mind that both Pu-containing soils had a lower iron loading. Noticeably, (LCy,Pr)H2 has roughly the same capacity as DFB to solubilize U, but it mobilizes less Fe than the hexadentate siderophore. Similarly, citrate has the highest capacity to extract Pu, but the lowest to extract Fe. Therefore, compared to DFB, (LCy,Pr)H2 shows a better U/Fe extraction selectivity and citrate shows a better Pu/Fe selectivity.

The objective of this research is to elucidate the coordination chemistry and the structure of complexes formed with siderochelates organic ligands and the physico-chemical studies of iron(III) complexation. Three dihydroxamic acids synthesized ((LCyEt)2−, (LCyPr)2− and (LO)2−) mimicking a fungal siderophore, rhodotorulic acid. They were evaluated with iron(III) chelation by potentiometric and spectrophotometric titrations in 0.1 M KNO3. These measurements revealed the formation of dileptic and trileptic complexes in excess ligand conditions. The chemical model includes five species of [Fem(L)lHh](3m−2l+h)+ with general formula: [Fe(L)]+, [Fe(L)(OH)], [Fe(L)(OH)2]−, [Fe(L)2H] and [Fe2(L)3]. The numerical treatment of the spectrophotometric data collected in the visible range led us to propose the electronic absorption spectrum for each of the identified species. Moreover, electrospray ionization mass spectroscopy (ESI-MS) confirmed the formation of the monoleptic ([FeL]+) and dileptic ([FeL2H]) complexes.

Siderophores are a diverse group of low molecular weight biogenic metallophores with a particular affinity for Fe(III) but they also have potential to complex a number of other polyvalent metal cations, including Cr(III). Here we show that two hydroxamate siderophores, desferrioxamine B and rhodotorulic acid, at environmentally relevant concentrations, facilitate the dissolution of hydroxy-Cr(III) precipitates from a common layer silicate. Desferrioxamine B and rhodotorulic acid induced maximum initial Cr dissolution rates of 11.3 ± 1.7 × 10− 4 and 9.03 ± 0.68 × 10− 4 µmol m− 2 h− 1, respectively, yielding maximum solution Cr concentrations of 0.26 ± 0.01 and 0.20 ± 0.02 µmol m− 2, respectively. These data demonstrate that hydroxamate siderophores may play an important role increasing the dispersal of Cr in natural environments, thus facilitating greater bioavailability of this potential toxin.

This article focuses on our investigation of the molecular structure characteristics of diketopiperazines (DKPs), and application of these findings to the development of novel functional molecules. DKPs bearing a benzyl moiety are known to adopt a folded conformation, in which the benzyl moiety is folded over the DKP ring. In order to investigate the driving force behind the folded conformation, we synthesized DKPs bearing a benzyl moiety with different para-substituents, and demonstrated that the folded conformation likely arose from intramolecular CH/π interactions, based on the electronic effects of para-substituents on the benzyl group in 1H NMR spectroscopy. On the other hand, N4-methylation of DKPs bearing a benzyl moiety was found to change their folded conformation to an extended conformation, based on single crystal X-ray crystallography and 1H NMR spectroscopy analysis. Next, we attempted to synthesize both hydroxamate-type siderophores containing the DKP ring: rhodotorulic acid and erythrochelin. Facile synthesis of rhodotorulic acid and its N,N'-dimethylated derivative was achieved by microwave-assisted cyclization of the corresponding dipeptide precursors. Interestingly, N,N'-dimethylated rhodotorulic acid was found to be more soluble in various organic solvents than rhodotorulic acid. Moreover, erythrochelin was synthesized for the first time, and its metal-chelating ability with not only Fe(III) but also Mg(II) was confirmed based on electrospray ionization mass spectrometry (ESI-MS) analysis. Finally, we synthesized DKPs bearing a primary amino group, and found that they could catalyze the asymmetric aldol reaction between hydroxyacetone and p-nitrobenzaldehyde.

Synthetic iron chelates are the most efficient iron fertilizers. The stability in solution of the iron chelates in the presence of other metal cations that may compete with iron for the chelating agent in nutrient solutions is crucial for its effectiveness in supplying iron to plants. In this work, the chelating properties of N,N-dihydroxy-N,N′-diisopropylhexanediamide (DPH), a biological model of the natural siderophore rhodotorulic acid, were evaluated in order to assess its potentialities for being used, as an iron chelate, in hydroponic cultures. For this purpose, the complexation for the metals [Ca(II), Cu(II), Mg(II), Mn(II) or Zn(II)]–DPH–OH systems has been studied using pH potentiometry and the overall stability constants have been determined for the first time. For all M–DPH–OH systems, MHL and ML species were identified. For [Cu(II), Mn(II) or Zn(II)]–DPH–OH systems, the M2L3 species was detected, whereas formation of ML(OH) species was found in the [Ca(II) or Mg(II)]–DPH–OH systems. Finally, the chemical stability of iron chelated with DPH in hydroponic conditions was assessed by computer chemical simulations and compared with the one predicted when iron is chelated with ethylenediamine-N,N′-bis(o-hydroxyphenyl)acetic acid (o,o-EDDHA) in similar conditions.

Resistance to antibiotics is an emerging phenomenon and a major medical problem. The resistance of Gram-negative bacteria such as Pseudomonas aeruginosa and the Burkholderia group to conventional antibiotics leads to therapeutic failure and requires new antibiotic therapies. The use of iron transport systems is a promising strategy to overcome this phenomenon. These TonB-dependent receptors, essential for the survival of microorganisms, allow specific recognition of ferric siderophore complexes in order to transport iron within bacteria1. Bacteria, according to their kind, express different types of receptors that allow them to recognize their endogenous siderophores but also xenosiderophores. Pseudomonas aeruginosa and Burkholderia pseudomallei in particular possess FptA receptors allowing the recognition of pyochelin.2 These specific systems may allow the introduction of antibacterial agents by forming antibiotic-siderophore conjugates or toxic complexes such as gallium complexes, in the bacteria to kill it. Siderophores have three types of chelating function: catechols, hydroxamates and hydroxy-carboxylates. Previous work in the laboratory has shown that piperazine 1,4-dicatechol structures (MPPS0225) could be recognized by Pseudomonas aeruginosa strains. In order to further investigate this piperazine platform, we have synthesized iron chelators bearing 3-hydroxypyridin-4-ones and 1,3-dihydroxypyridin-4-one ligands. At the same time, we were interested in the synthesis of a more complex 2,5-dioxopiperazine platform, part of the rhodotorulic acid (RA), a natural siderophore produced by Rhodotorula pilimanae showing an interesting iron affinity (pFe = 21,8). Two RA synthesis strategies will be developed as well as the corresponding 3,6-disubstituted analogs. Through the synthesis of these chelators, we would like to study the influence, on the iron complexation, of: i) the nitrogen platform (piperazine or dioxopiperazine), ii) the presence of stereogenic centers (3,6-disubstituted dioxopiperazine vs 1,4 -disubstituted piperazines) and iii) the nature of the iron ligands (hydroxypyridinone vs catechol). An evaluation of the siderophore-like potential and a measurement of the complexing force will be carried out. We would like to thank the DGA and the Haut de France region for their financial support. References Miethke M.; Marahiel MA. Microbiology and Molecular Biology Reviews. 2007, 71, 413-451. Butt AT.; Thomas MS. Frontiers in Cellular and Infection Microbiology. 2017, 7.

Antibiotic resistance is an emerging disease and a real problem of health. Resistance of Gram negative bacteria such as Acinetobacter baumannii and Escherichia coli to conventional antibiotics lead to therapeutic failure and require new antibiotherapies. The use of the iron transport systems is one of the most promising strategies to overcome this resistance phenomenon. These specific routes of entry, essential for the survival of the microorganisms, allow ferric siderophore complexes to carry iron within the bacteria. These systems allow the introduction of antibacterial agents (conjugates antibiotic-siderophore) or toxic complexes (gallium complexes) into the bacteria to kill them. Rhodotorulic acid (RA) is a siderophore transported by TonBox dependant Fhu receptors. These kinds of receptors are expressed by Acinetobacter baumannii and Escherichia coli. RA is dioxopiperazine iron chelater with hydroxamate as iron ligands and two asymmetric centers (S,S configuration). This spatial orientation is essential for the Fhu receptors recognition. We have previously reported the asymmetric synthesis of 3-substituted 2-oxopiperazines. Herein, we present an original and a convergent strategy to synthesize RA and corresponding 3,6-disubstituted analogues. Siderophore-like test and measurement of the complexing strength of these compounds will be carried out.

In this study, we found that Acinetobacter baumannii utilized exogenously supplied desferricoprogen, rhodotorulic acid, and desferrioxamine B for growth under iron-limiting conditions. The ferric uptake regulator (Fur) titration assay method was then successfully applied to select iron-regulated genes in A. baumannii genomic libraries. Part of the nucleotide sequence homologous to Escherichia coli, fhuE, obtained from one of the positive clones allowed us to clone the entire gene, which was named fhuE. The fhuE gene had an amino acid sequence consistent with the N-terminal amino acid sequence of the 76-kDa iron-repressible outer membrane proteins in A. baumannii. Reverse transcription-polymerase chain reaction analysis demonstrated that fhuE mRNA is transcribed under iron-limiting conditions, consistent with the presence of a sequence homologous to the consensus Fur box in the promoter region. Disruption of fhuE resulted in the loss of expression of the 76-kDa protein. In addition, the double disruptant of fhuE and basD, which encodes one of the biosynthetic genes for the cognate siderophore acinetobactin, was unable to grow in the presence of desferricoprogen, rhodotorulic acid or desferrioxamine B. However, growth of the double disruptant was restored by complementation with fhuE, demonstrating that A. baumannii FhuE functions as the receptor common to coprogen, ferric rhodotorulic acid and ferrioxamine B.

EDTA, calcium chloride, and two siderophores (rhodotorulic acid produced by Rhodotorula glutinis BNM 0524, and enterochelin from the bacterium Rahnella aquatilis BNM 0523) were evaluated as possible inhibitors of polygalacturonase (PG) and laccase (LC) from Botrytis cinerea. The aim was to apply them to the control of this pathogen, taking into account the fact that these enzymes are related to the invasion and installation of the fungus in the host. Two B. cinerea Pers.:Fr strains (BNM 0527 and BNM 0528) were used. Enzyme activities were measured in the supernatant of 7-day-old cultures. EDTA, calcium chloride, rhodotorulic acid, or enterochelin were added in the reaction mixture. Laccase activity from two strains was more affected by enterochelin (70–80% inhibition) than by the other compounds, while polygalacturonase was more inhibited (45% inhibition) by calcium chloride. The inhibitors were added to the growth medium and after 7 days of culture, the activities of the enzymes were measured in the supernatants. The production of PG and LC in both strains was lower when enterochelin or calcium chloride was added. In the third step, when the inhibitors were tested on apple, all them provided both effects, preventive and curative, against infections caused by B. cinerea, with EDTA and rhodotorulic acid exhibiting more preventive effects while calcium chloride and enterochelin provided more control of pre-existing infections (curative effect), coinciding with their ability to inhibit the production of polygalacturonase and laccase.

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 DeltafhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.

The effect of different drugs which affect lysosomal function (chloroquine, methylamine) and endocytosis (colchicine) or of the iron chelators desferrioxamine B, rhodotorulic acid, bipyridyl, 2,3-dihydroxybenzoic acid, isoniazid pyridoxal hydrazone on the uptake of 59Fe-loaded 3H-labelled transferrin by cultured rat embryo fibroblasts has been studied. All these substances, except isoniazid pyridoxal hydrazone reduce the uptake of iron by fibroblasts, without any significant effect on the uptake of [3H]transferrin, nor of its specific binding to the cells. Cell fractionation experiments indicate that in the presence of methylamine and chloroquine, most of the cell associated 3H label is no longer transferred into lysosomes and remains associated with subcellular compartments which have a 5'-nucleotidase activity (a marker enzyme of plasma membrane and related structures such as endocytic vesicles). Short-term kinetic experiments with fibroblasts incubated for 1 min at 37 °C with doubly labelled transferrin show that on reincubation in fresh medium almost all of the 59Fe initially bound remains associated with the cells whereas the 3H-labelled transferrin is released with a t1/2 of about 1.5 min. In the presence of desferrioxamine B both 59Fe and transferrin, the former in part as ferrioxamine, are released from the cells with the same kinetics as is observed for transferrin in the control experiments. Similar studies with cells preincubated for 16 h with chloroquine or methylamine indicate that the release of transferrin from the cells is slowed down considerably. Chloroquine-treated cells also release large amounts of 59Fe. These results are consistent with the model for iron release from transferrin proposed previously [1] involving receptor mediated endocytosis of transferrin followed by release of iron during a rapid passage of the transferrinreceptor complex in lysosomes and recycling of the complex to the plasma membrane where the iron-depleted transferrin is released. The inhibition of iron uptake by iron chelators such as desferrioxamine is consistent with complexation of iron by the chelator subsequent to its release from transferrin inside the cells and transfer of the iron-chelate into the extracellular medium.

Siderophores are low-molecular weight organic molecules secreted by plants and micro-organisms in response to Fe stress. With stability constants commonly exceeding 10 30 , siderophores are considered to have higher affinities for Fe(III) than for any other major or trace element dissolved in soil solution. However, several siderophores have affinities for trace metals that approach those for Fe(III), and certain actinides form siderophore complexes of surprisingly high stability. The purpose of this study was to examine the role of hydroxamate siderophores in controlling Pb sorption to an Fe(III) oxide adsorbent. Goethite [α-FeOOH], prepared by standard methods and identified by X-ray diffraction, gave a specific surface of 36 m 2 g −1 as determined by N 2 multipoint BET analysis. Adsorption experiments were performed aseptically using a batch method with a goethite concentration of 1.0 g l −1 and an ionic strength of 0.01 M NaClO 4 . Soluble Pb and Fe were measured between pH 3 and 8 by first adding Pb (10 μM) and then siderophore (10, 20, or 40 μM) to the goethite suspension. Three hydroxamate siderophores were employed: desferrioxamine B (DFB), ferrichrome (FC), and rhodotorulic acid (RA). Following 20 h reaction, Pb and Fe in solution were measured by ICP–MS and ICP–AES, respectively. The efficacy of siderophore-mediated Pb desorption varied with siderophore type and generally increased with pH and siderophore/Pb molar ratio. Desferrioxamine B, at pH 6.5 and a DFB/Pb molar ratio of 4, solubilised nearly 25% of the total sorbed Pb. In the presence of 10 μM FC, Pb adsorption largely mimicked that for the siderophore-free system, whereas significant amounts of Pb were desorbed with 20 μM FC at pH >5.5. The dihydroxamate siderophore, RA, was the least effective Pb chelator, requiring 20 μM to desorb detectable amounts of Pb.

To better understand Fe cycling in marine and freshwater systems, we have developed a biomolecular tool to track the perceived bioavailability of Fe to heterotrophic bacteria. Bioluminescent reporters, constructed by fusing the fepA–fes promoter of Escherichia coli (an Enterobactin biosynthesis gene regulated by the ferric uptake regulatory [Fur] system) to a luxCDABE cassette, were integrated into the chromosome of a halotolerant Pseudomonas putida, which uses the Fur system to regulate high-affinity Fe uptake. The resultant P. putida bioreporter has been successfully tested both in lab and field studies. Laboratory cultures were maintained at a range of concentrations of total Fe (0–25 nM) or limited by the addition of concentrations of wellcharacterized siderophores (desferrioxamine B [DFB], ferrichrome, 2,2V-dipyridyl [DP] and Rhodotorulic acid [RA], 0–200 nM) and used to establish the dynamic range of this reporter system. Analysis of sample incubations after only 4 h suggest that both of the trihydroxamate-type siderophores DFB and ferrichrome efficiently reduced Fe availability, resulting respectively in a 1.77- and 1.88-fold increase in luminescence relative to Fe-replete conditions. In contrast, additions of the dihydroxamate-type siderophore RA and the synthetic chelator DP resulted in no response from the system, suggesting that cells could access Fe complexed to these compounds without activating high-affinity Fe transport systems. Field studies were performed in the central basin of Lake Erie, which has previously been shown to undergo sporadic Fe limitation during summer stratification. DFB concentrations were titrated across a range of 0–50 nM into unfiltered water to manipulate Fe availabilities. Bioreporters expressed Fe stress (ca. a 2-fold increase in luminescence) at concentrations of DFB equivalent to the total (dissolved+particulate) Fe in the system (c30 nM), indicative of the concentration of bioavailable Fe. In a similar experiment with 0.2-Am pre-filtered water (2.25–5.24 nM Fe), a 6-fold increase in luminescence (relative to controls) was observed at the lowest (15 nM) concentration of chelators. The results of this study demonstrate the validity of bioreporters as a complimentary tool to measurements of total Fe. Moreover, these results suggest that a significant source of

Carrier-facilitated bulk liquid membrane (BLM) transport from an aqueous source phase through a chloroform membrane phase to an aqueous receiving phase was studied for various hydrophilic synthetic and naturally occurring Fe(III)-siderophore complexes using first coordination sphere recognition. Iron transport systems were designed such that two cis coordination sites on a hydrophilic Fe(III) complex are occupied by labile aquo ligands, while the other four coordination sites are blocked by strong tetradentate ligands (siderophores). The labile aquo coordination sites can be "recognized" by a liquid membrane-bound hydrophobic bidentate ligand, which carries the hydrophilic Fe(III)-siderophore complex across the hydrophobic membrane to an aqueous receiving phase. The system is further designed for uphill transport of Fe(III) against a concentration gradient, driven by anti-port H(+) transport. Three tetradentate siderophore and siderophore mimic ligands were investigated: rhodotorulic acid (H(2)L(RA)), alcaligin (H(2)L(AG)), and N,N'-dihydroxy-N,N'-dimethyldecanediamide (H(2)L(8)). Flux values for the transport of Fe(L(x))(OH(2))(2)(+) (x = RA, AG, 8) facilitated by the hydrophobic lauroyl hydroxamic acid (HLHA) membrane carrier were the highest when x = 8, which is attributed to substrate lipophilicity. Ferrioxamine B (FeHDFB(+)) was also selectively transported through a BLM by HLHA. The process involves partial dechelation of ferrioxamine B to produce the tetradentate form of the complex (Fe(H(2)DFB)(OH(2))(2)(2+)), followed by ternary complex formation with HLHA (Fe(H(2)DFB)(LHA)(+)) and transport across the membrane into the receiving phase. Uphill transport of ferrioxamine B was confirmed by increased flux as [H(+)](source phase) < [H(+)](receiving phase). The membrane flux of ferrioxamine B occurs near neutral pH, as evidence that ternary complex formation and ligand exchange are viable processes at the membrane/receptor surface of microbial cells.

Rhodotorula mucilaginosa produces the siderophore rhodotorulic acid (RA) when grown in iron-limited conditions. R. mucilaginosa grew at rates between 0.10 and 0.19 h(-1) in iron-restricted conditions, depending on the carbon source, and at 0.23 h(-1) in iron-sufficient conditions. In bioreactors inoculated with iron-starved pre-cultures, initial specific growth rates in batch culture were dependent on the iron concentration. The critical dilution rate (Dcrit, at which steady state cultures cannot be sustained) in continuous cultures was also dependent on the iron concentration and was lower than mu(max) in batch culture. Sucrose was the best carbon source for RA production [287+/-11 micromol (g biomass)(-1)] and production could be further increased by supplementing the medium with the precursors acetate [460+/-13 micromol (g biomass)(-1)], ornithine [376+/-6 micromol (g biomass)(-1)], or both [539+/-15 micromol (g biomass)(-1)]. Citric acid was an effective suppresser of RA production. RA was produced in a growth rate dependent manner and was optimally produced at pH 6.5.

Nearly all of the dissolved iron in the ocean is bound in very strong organic complexes, but how heterotrophic bacteria obtain Fe from these uncharacterized natural ligands is not under- stood. We examined Fe uptake from model ligand/ 55 Fe complexes by cultures of gamma and alpha marine proteobacteria grown under Fe-replete and Fe-limited conditions, and by natural bacterio- plankton communities from the Sargasso Sea and California upwelling region. Model chelators included siderophores and porphyrins chosen to represent the possible structures of the unknown natural ligands. Complexed-Fe uptake was compared to uptake of Fe added in inorganic form. In all cases, the Fe-stressed cultures had higher uptake rates than the Fe-replete bacteria. The gamma proteobacterium Vibrio natriegens was best able to use siderophore-bound Fe, especially from the catecholate siderophore enterobactin and the dihydroxamate rhodotorulic acid, but had very little success at utilizing porphyrin-bound Fe. In contrast, the alpha proteobacterium IRI-16 could use very little of the Fe bound to any of the model ligands in comparison to iron added in inorganic form. We observed variable degrees of Fe availability relative to ligand structure in natural communities. In some cases, Fe uptake by the prokaryotic size-class was most efficient from siderophores; in other cases, porphyrin-bound iron was more available. To better understand organically-bound Fe utiliza- tion by natural bacterioplankton communities, further studies combining ligand bioavailability measurements with detailed analyses of bacterioplankton community composition are needed.

The ability of five different ligands to solubilize α-FeO(OH) (goethite), ThO2.2H2O and γ-UO3 was investigated at pH 3, 5 and 7: the hydroxamates acetohydroxamate (AHA), glutarodihydroxamate (GDA), rhodotorulate (RA), desferoxamine B (DFO) and the dicarboxylate iminodiacetate (IDA). Solubilization in the absence of ligand and solubilization by DFO (pH 5) as a function of time was also investigated. RA was the most effective at solubilizing α-FeO(OH) at all three pH values and ThO2.2H2O at pH 5 and 7. IDA solubilized most ThO2.2H2O at pH 3. GDA was the least effective ligand for α-FeO(OH) and ThO2.2H2O. Most γ-UO3 was solubilized by DFO (pH 3 and 5) and GDA (pH 7). AHA was the least effective. For dissolution over time, solubilization of α-FeO(OH) appears to be a diffusion-controlled process, whilst for ThO2.2H2O and γ-UO3 it is controlled by first-order chemical kinetics.

Soils contain siderophores produced by bacteria and fungi; however, the role of siderophores in Fe nutrition of plants is uncertain. The Strategy I plant cucumber (Cucumis sativus L.) was used in an investigation of ferric chelate reduction activity and uptake and transport of Fe from ferric hydroxyethylethylenetriacetic acid (FeHEDTA) and ferric N,N′–di–(2–hydroxybenzoyl)–ethylenediamine– N,N′-diacetic acid (FeHBED) and the hydroxamate siderophores, ferric rhodotorulic acid (FeRA) and ferric ferrioxime B (FeFOB). Cucumber seedlings were grown in a hydroponic medium without Fe or supplied with 10 μM FeHEDTA. Iron-deficient cucumber roots readily reduced FeHEDTA, while Fe-sufficient roots had low levels of ferric chelate reduction activity. The siderophore FeRA was reduced by Fe-deficient roots at 8% of the rate of FeHEDTA, while FeFOB was not reduced. The highly stable synthetic chelate FeHBED was reduced at 16% the rate of FeHEDTA. Fe transport to shoots by Fe-deficient seedlings from the slowly reducible complexes 59FeRA and 59FeHBED was, respectively, 74% and 73% of that transported from 59FeHEDTA. The ferrous complexing agent, bathophenanthrolinedisulfonic acid (BPDS), had a strong inhibitory effect on uptake and transport of Fe from 59FeHEDTA or 59FeRA into shoots. An average of 11% as much Fe was transported to shoots of Fe-deficient seedlings from 59FeFOB as from 59FeHEDTA. Neither the Fe nutritional status of the seedlings nor the presence of BPDS influenced the uptake and transport of Fe from 59FeFOB. It is concluded that cucumber roots may take up substantial amounts of Fe from FeRA and FeHBED following reduction, while small amounts of Fe may be taken up from FeFOB by a mechanism not involving reduction of the ferric siderophore at the root surface.

The mammalian host specifically limits iron duringHistoplasma capsulatuminfection, and fungal acquisition of iron is essential for productive infection.H. capsulatumexpresses several iron acquisition mechanisms under iron-limited conditions in vitro. These components include hydroxamate siderophores, extracellular glutathione-dependent ferric reductase enzyme, extracellular nonproteinaceous ferric reductant(s), and cell surface ferric reducing agent(s). We examined the relationship between these mechanisms and a potential role for the extracellular ferric reductase in utilization of environmental and host ferric compounds through the production of free, soluble Fe(II). Siderophores and ferric reducing agents were coproduced under conditions of iron limitation. TheH. capsulatumsiderophore dimerum acid and the structurally similar basidiomycete siderophore rhodotorulic acid acted as substrates for the ferric reductase, and rhodotorulic acid removed Fe(III) bound by transferrin. The mammalian Fe(III)-binding compounds hemin and transferrin served both as substrates for the ferric reductase and as iron sources for yeast-phase growth at neutral pH. In the case of transferrin, there was a correlation between the level of iron saturation and efficacy for both of these functions. Our data are not consistent with an entirely pH-dependent mechanism of iron acquisition from transferrin, as has been suggested to occur in the macrophage phagolysosome. The foreign siderophore ferrioxamine B also acted as a substrate for the ferric reductase, while the foreign siderophore ferrichrome did not. Both ferrioxamine and ferrichrome served as iron sources for yeast- and mold-phase growth, the latter presumably by some other acquisition mechanism(s).

The synthesis of four bipodal dihydroxamic acids containing an apical C atom and amide linkages is described, where Ia,b represent "normal" and "retro" hydroxamate isomers: (R)CH[C(=O)NH(CH(2))(2)NHC(=O)(CH(2))(n)()R'](2) (Ia, R = CH(3), R' = N(OH)(C=O)CH(3), n = 2; Ib, R = CH(3), R' = (C=O)N(OH)CH(3), n = 2; Ic, R = CH(3), R' = (C=O)N(OH)CH(3), n = 3; Id, R = C(4)H(9), R' = (C=O)N(OH)CH(3), n = 2.). The pK(a1) and pK(a2) values in aqueous solution are reported, and some degree of cooperativity is noted. Complexation equilibria with Fe(aq)(3+) are described, and values for stepwise and overall equilibrium constants are reported. log beta(230) values for Ia-d are 59.22, 59.45, 58.91, and 58.46, slightly lower than for rhodotorulic acid, although the pFe values for the synthetic siderophores are comparable to that for rhodotorulic acid.

Aqueous solutions of Fe3+ complexes of cyclic (alcaligin) and linear (rhodotorulic acid) dihydroxamate siderophores and synthetic linear eight-carbon-chain and two-carbon-chain dihydroxamic acids ([CH3N(OH)C=O)]2(CH2)n; H2Ln; n = 2 and 8) were investigated by electrospray ionization mass spectrometry (ESI-MS). Information was obtained relevant to the structure and the speciation of various Fe(III)-dihydroxamate complexes present in aqueous solution by (1) comparing different ionization techniques (ESI and FAB), (2) altering the experimental parameters (Fe3+/ligand ratio, pH, cone voltage), (3) using high-stability hexacoordinated Fe(III) siderophore complex mixtures (ferrioxamine B/ferrioxamine E) as a calibrant to quantify intrinsically neutral (H+ clustered or protonated) and intrinsically charged complexes, and (4) using mixed-metal complexes containing Fe3+, Ga3+, and Al3+. These results illustrate that for all dihydroxamic acid ligands investigated multiple tris- and bis-chelated mono- and di-Fe(III) species are present in relative concentrations that depend on the pH and Fe/L ratio.

A family of four putative transporters (Arn1p–4p) in Saccharomyces cerevisiae is expressed under conditions of iron deprivation and is regulated by Aft1p, the major iron-dependent transcription factor in yeast. One of these, Arn3p/Sit1p, facilitates the uptake of ferrioxamine B, a siderophore of the hydroxamate class. Here we report that ARN family members facilitated the uptake of iron from the trihydroxamate siderophores ferrichrome, ferrichrome A, and triacetylfusarinine C. Uptake of siderophore-bound iron was dependent on either the high-affinity ferrous iron transport system or the ARNfamily of transporters. The specificity of each siderophore for individual transporters was determined. Uptake of ferrichrome and ferrichrome A was facilitated by both Arn1p and Arn3p. Uptake of triacetylfusarinine C was facilitated by Arn2p, although small amounts of uptake also occurred through Arn1p and Arn3p. In contrast to the trihydroxamates, uptake of iron from the dihydroxamate rhodotorulic acid occurred only via the high-affinity ferrous iron system. Epitope-tagged Arn1p was expressed in intracellular vesicles in a pattern that was indistinguishable from that of Arn3p, whereas Ftr1p, a component of the high-affinity ferrous system, was expressed on the plasma membrane. These data indicate that S. cerevisiaemaintains two systems of siderophore uptake, only one of which is located on the plasma membrane.

Rhodotorulic acid produced by Rhodotorula glutinis strains improved the biological control of blue rot caused by Penicillium expansum in harvested apples. The production of the siderophore was closely associated with the iron concentration in the medium. Thus, very low additions of the metal reduced the siderophore production considerably. The antagonistic effect of R. glutinis and rhodotorulic acid was studied by using in vitro and in vivo assays. In the in vitro assays, rhodotorulic acid reduced the growth of P. expansum, whereas the chelate (rhodotorulic acid plus iron) did not. Siderophore antagonism was then related to competition for iron. In biocontrol assays on apple wounds, the blue mold was more effectively controlled by the antagonistic agent plus siderophore than by the antagonistic agent alone. The disease incidence (DI: percentage of treated wounds that developed rot) was 34% when apples were protected by R. glutinis alone, whereas it was 6% when the fruits were protected by R. glutinis plus rhodotorulic acid.

Siderophore production is an integral component of high-affinity iron uptake by many microorganisms. We examined the extracellular production of the siderophore rhodotorulic acid (RA) in 16 field-collected and seven herbarium strains of Microbotryum violaceum. These strains were collected from a variety of caryophyllaceous host plant species, from many geographical locations, over a period of years. All of the tested strains, when starved for iron, produced detectable levels of RA, which is consistent with a critical function of the high-affinity iron uptake system. We found normal patterns of siderophore secretion accompanying iron starvation as well as repression of siderophore secretion when cells were grown in culture containing adequate levels of iron. We detected a fivefold variation in the extracellular siderophore level (0.33 mM-1.71 mM) among the 23 strains. Levels of extracellular siderophore accumulation correlated with cell density; strains that grew to a greater density (cells/mL) in liquid c...

Naturally occurring biological molecules are of considerable interest as possible corrosion inhibitors because of increased attention on the development of environmentally compatible, nonpolluting corrosion inhibitors. A hydroxamate yeast siderophore (rhodotorulic acid), a catecholate bacterial siderophore (parabactin), an adhesive protein from the blue mussel Mytilus edulis, and two metal-binding compounds isolated from the tomato and sunflower roots, namely, chlorogenic and caffeic acid, respectively, were adsorbed from solution onto pure aluminum (99.9995%) and their effect on the critical pitting potential and polarization resistance in deaerated 0.1 M NaCl was measured. These measurements were made using anodic polarization and ac impedance spectroscopy. The catechol-containing siderophore has an inhibitive effect on the critical pitting potential of aluminum in 0.1 M NaCl and increases the polarization resistance of the metal over time. The adhesive protein from the blue mussel is also effective in inhibiting the pitting of aluminum.

The hydrolysis of hydroxamic acids to hydroxylamine is an important first step in the quantitative analysis of hydroxamate siderophores. The rate of this hydrolysis can be increased by the addition of ferric iron and the metals Mn(II), Cu(II), Ni(II), Cd(II), Cr(III), and Al(III). The maximum hydrolysis rate is obtained at iron to desferal ratios greater than 40:1. Using these conditions, >75% hydrolysis of the siderophores desferal, rhodotorulic acid, and schizokinen was obtained in 2 h at temperatures as low as 70 °C. These low temperatures make possible the direct analysis of hydroxamates in culture media containing high concentrations of carbohydrates. An example of this technique showing increased siderophore formation in low-iron cultures of Bacillus megaterium upon exposure to aluminum is presented.

Four siderophores isolated from bacteria have been studied as possible corrosion inhibitors for iron in deaerated 1N HCl. These compounds are aerobactin, enterobactin, parabactin, and rhodotorulic acid. Polarization measurements and ac impedance studies show that all four compounds are inhibitors, with the order of effectiveness being rhodotorulic acid < aerobactin < enterobactin < parabactin. X-ray photoelectron spectroscopy surface analysis with one of the siderophores, rhodotorulic acid, shows that it chemisorbs at the iron/HCl interface. Enterobactin and parabactin are the most effective corrosion inhibitors of the four because they have the optimum combination of relatively high values of pK{sub a1} and the largest molecular cross-sectional areas.

This study describes the yeast Rhodotorula mucilaginosa strain LM9 isolated from copper-bearing, organic-rich Kupferschiefer black shale and its role in copper biotransformation. Strain LM9 exhibited great ability to simultaneously mobilize and immobilize copper from this sedimentary rock. In addition, it showed considerable resistance to copper and high uptake of this metal. Moreover, malic and oxalic acid as well as siderophore (rhodotorulic acid) produced by this strain enhanced its resistance by promoting the mobilization and complexation of copper from black shale. These processes, characterized here under laboratory conditions, are assumed to play a role in copper cycling in black shale as well as in the adaptation of strain LM9 to the conditions prevailing in its natural mine habitat. The findings of this study indicate that yeast strain LM9 might be used for the recovery of copper particularly from alkaline or slightly neutral ores in a non-chemical environmentally-friendly procedure.

Numerous analogous dihydroxamates of rhodotorulic acid have been prepared, and their mode of complexation of iron(III) and the stability of these complexes studied as a function of pH, for comparison with ferrioxamine B and the complex of acetohydroxamic acid with iron(III). This study has shown that the stoichiometry of the complexes is 2:3 (at a pH of 7) and 1:1 (at a pH of 2), and that, in spite of a complete complexation, their electrochemical behaviour varies greatly with pH. The very strong complexation constant observed (log β110 = 21.3) at a pH of 1 with the ligand N,N′-bis(acetylhydroxamino-3′ propyl)-pentyloxy-4 pyridine-2,6-dicarboxamide confirms the strong complexation capacity of iron(III) by these complexes. The high relaxivity of the complex of iron(III) with the N,N′-bis(acetylhydroxyamino-3′ -propyl)-(amino-4″ -butyloxy)-4-pyridine-2,6-dicarboxamide suggests that this product could be a useful contrasting agent for nuclear magnetic imaging. [Journal translation]

Nine iron chelators were tested in five systems for their effects on radical-generation and conversion at chelator: iron molar ratios from 0.1 to 10. Stimulatory actions might distinguish toxic from safer chelators. Radical-generating reactions which represent different aspects of iron (ferrous and ferric) availability were studied: a) the reaction with hydrogen peroxide to hydroxylate benzoate; b) the oxidation of ascorbate; c) the reaction with hydrogen peroxide to fragment proteins; d) the reaction with hydrogen peroxide to permit amplified chemiluminescence; and e) the induction of peroxidation of mitochondrial membrane lipids. The compounds used were HBED, CP130, Desferal, EDTA, pyridine-hydrazone (CGP 43′902B), Ferrozine, CP 94 (CGP 46′700), LI (CGP 37 391) and rhodotorulic acid (CGP 45 274). Only the hexadentate compounds HBED, CP130 and Desferal were uniformly inhibitory (“protective”). The protective compounds were also apparently more stable during radical fluxes than the other chelators.

A new cyclic diaza-dihydroxamic acid, 1,4-diazacycloheptane- N, N ′-bis( N -methyl-acetohydroxamic acid) (DACHDMAHA) has been synthesized and studied. This paper is the second one devoted to finding synthetic ferric sequestering agents suitable for mimicking chemical and biological properties of the naturally occurring rhodotorulic acid. This ligand, having the hydroxamate arms bound to a seven-membered ring diamine, presents some biological activity, as was previously found for the corresponding six-membered compound (PIPDMAHA). Stability constants with iron(III) and iron(II) as well as with copper(II) and copper(I) have been determined by potentiometry, spectrophotometry and cyclic voltammetpy, this latter technique being also useful for the interpretation of the electrochemical reaction and the determination of the rate constant of dissociation of iron(II) complexes. Comparing the complexes obtained with DACHDMAHA and PIPDMAHA it has been noticed that the complexes with the first ligand are more stable and that the rate constant of dissociation of the iron(II) complex is higher, showing that this ligand has more of the characteristics of a siderophore.

The relative rates of ferric‐iron (Fe3+) reduction and uptake by two citrus rootstocks were measured for a series of synthetic Fe3+ chelates and microbial siderophores. The rates of Fe3+ reduction by the citrus seedlings followed the order: FeHEDTA >> FeDPTA > FeCDTA. No reduction occurred for FeDFOB (ferrioxamine B) and FeTAF (ferric triacetylfusagen). Low rates of Fe3+ reduction occurred for Fe2RA3 (ferric rhodotorulic acid). The levels of 55Fe taken up the citrus seedlings showed good correlations with the reduction rates. These results indicate the importance of Fe3+ reduction in the Fe uptake by citrus rootstocks. The immobility of a large percent of the 55Fe taken up by the roots is attributed to the accumulation of Fe in the root apoplasts.

Desferrioxamine, the therapeutic iron chelator, is limited in its usage by its short half-life in plasma and lack of oral activity, its side-effects and its slow penetration into cells. Several studies have emerged recently demonstrating the ability of this trihydroxamate compound to act as a radical scavenger, in addition to and independently of its iron-chelating properties. These include the interaction of desferrioxamine with the Superoxide radical and ferryl myoglobin radical, as well as its action as a chainbreaking antioxidant in peroxidizing erythrocyte membranes. We have synthesized recently a series of monohydroxamate compounds and investigated their efficacy as radical scavenging antioxidants in comparison with desferrioxamine and rhodotorulic acid, a naturally occurring dihydroxamate compound. The results show that the relative rates of reaction of these hydroxamate derivatives with ferryl myoglobin are No -methyl- N -hexanoyl hydroxylamine N -methyl- N -benzoyl hydroxylamin N -methyl- N -acetyl ydroxylamine desferrioxamine rhodotorulic acid N -methyl- N -butyryl hydroxylamine.

A new cyclic diaminodihydroxamic ligand, piperazine-1,4-bis(N-methylacetohydroxamic acid)(H2L2), presenting a reasonable analogy with the naturally occurring siderophore rhodotorulic acid (H2L1), has been prepared through an easy two-step process. This ligand, which has proved to be biologically active, forms a very stable complex [Fe2L23]. The ligand and its iron and copper complexes have been characterized by potentiometric and spectrophotometric techniques. The mechanism of electron transfer in the complexes has been studied by voltammetric methods, and the kinetics of dissociation of the iron(II) complex, which might be of crucial importance in the biological activity of this siderophore analogue, was also investigated.

To study the ecological role of siderophores in the natural environment, it is necessary to enrich the sample by several orders of magnitude. Solid phase extraction techniques are excellently suited for this type of application. Adsorption of three siderophores (Desferal, rhodotorulic acid, deferriferrichrome A) and EDTA were studied using 12 solid phase resins. These included traditional silica and C‐18 cartridges, polar (e.g., diol or amine) cartridges, non‐silica polymeric cartridges (e.g., MP‐1, MP‐2 and MP‐3) and ion exchange cartridges (e.g., SAX, CBA, SCX). Using iron‐55 labeled siderophores as probes, considerable variation was observed between the cartridge types and similar cart‐ridges from different manufacturers. For most applications, traditional or polymeric C‐18 reverse cartridges were preferred. Recoveries for EDTA and RA were low, while recoveries for DES and FCA were generally greater than 40%. C‐18 cartridges worked equally well when freshwater or seawater was the starting matr...

A variety of fungi are able to synthesize iron chelating compounds (siderophores) which are excreted into the cultivation medium for iron sequestering purposes. Most ascomycetous and basidomycetous fungi synthesize hydroxamate-type siderophores containing N5-hydroxy-N5-acyl- l -ornithine residues which constitute the iron binding ligands. After complexing of ferric ions, octahedral complexes are formed. Uptake of iron via siderophores involves the recognition of siderophores by the cognate transport systems which is dependent on the configuration of the metal centre (Λ/Δ) and its surrounding functionalities. Transport across the membrane requires metabolic energy and can be inhibited by respiratory poisons, uncouplers, low temperatures, anaerobiosis and is functioning in a pH range of pH 2–7. Four classes of hydroxamate siderophores have so far been isolated: ferrichromes, fusarinines, coprogens and rhodotorulic acid. Besides their function in solubilizing and transport of iron, hydroxamate siderophores are also involved in iron storage. Recently, a novel complexone type siderophore (rhizoferrin) has been discovered which seem to be widespread in the Mucorales. Interestingly the Mucorales are the only fungal group where ferritin has been detected so far, suggesting that the substitution of hydroxamate siderophores by complexone siderophores has led to the formation of ferritins as the eucaryotic iron storage proteins.

The role of the root apoplasm for iron acquisition was studied in wheat (Triticum aestivum L. cv Ares) grown in nutrient solution under controlled environmental conditions. To obtain different levels of Fe in the root apoplasm, plants were supplied in the dark for 5 hours (preloading period) with various (59)Fe-labeled Fe compounds [Fe(III) hydroxide; microbial siderophores: Fe rhodotorulic acid (FeRDA) and ferrioxamin (FeDesferal(3)), and synthetic Fe chelate (FeEDDHA)], each at a concentration of 5 micromolar. Large pools of apoplasmic Fe were formed after supplying Fe(III) hydroxide or FeRDA, but no such pools were observed after supplying FeDesferal or FeEDDHA. Depending on plant Fe nutritional status (preculture +/- 0.1 millimolar FeEDTA), apoplasmic Fe was used to different extent for translocation to the shoot. Under Fe deficiency, a much greater fraction of the apoplasmic Fe was utilized than in Fe-sufficient plants, as a result of the different rates of phytosiderophore release. Because of the diurnal rhythm in release of phytosiderophores in Fe-deficient plants, the utilization of the apoplasmic Fe for translocation into the shoot started 2 hours after onset of the light period and was dependent on the concentration of Fe in the apoplasm, which followed the order: Fe(III) hydroxide >> FeRDA >> FeDesferal = FeEDDHA. From these results, it can be concluded that in soil-grown plants the apoplasmic Fe pool loaded by various indigenous Fe compounds such as siderophores in the soil solution can be an important Fe source in graminaceous species, particularly during periods of limited Fe supply from the soil.