Metabolic utilization of 57Fe-labeled coprogen in Neurospora crassa. An in vivo M?ssbauer study

M?ssbauer spectra of whole cells of Neurospora crassa arg-5 ota aga (a siderophore-free mutant) show that the siderophore coprogen is accumulated inside the cell as an entity. 57Fe from 57Fe-labeled coprogen is slowly removed from the complex (45% in 27 h). The rate of removal depends on the degree of iron starvation of the cells. The distribution of 55Fe from [55Fe]coprogen in vacuoles, membranes, and cytoplasm has been also determined. From this it is clear that coprogen is accumulated in the cytoplasm. In addition to its role as a siderophore, coprogen serves as an iron-storage compound. No holoferritins could be detected. We therefore conclude that this type of iron-storage protein is lacking in N. crassa. Metabolized iron was found predominantly to exist as an envelope of Fe(II) high-spin (delta = 1.2-1.3 mm s-1; delta EQ = 3.0-3.1 mm s-1 at 4.2 K) and fast-relaxing Fe(III) high-spin species (delta approximately equal to 0.25 mm s-1 and 0.45 mm s-1; delta EQ approximately equal to 0.6 mm s-1 and 0.55 mm s-1, respectively, at 4.2 K). An assignment of these major iron metabolites is difficult. The M?ssbauer data of the Fe(II) species do not fit those reported for heme, cytochromes and ferredoxins. We therefore assume that this iron metabolite represents a novel internal iron compound. One of the Fe(III) species becomes the dominant component of the cell spectra after 65 h of metabolization and might correspond to an iron-storage compound with iron oxide cores similar to bacterioferritin. After 27 h of growth in mycelia supplied with 57Fe-labeled coprogen, the siderophore ferricrocin was observed in the cell spectra. This is unexpected, since N. crassa arg-5 ota aga is unable to synthesize ornithine. We assume that ferricrocin is synthesized by the use of coprogen degradation products.     

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.     

Synthesis and activity of p-azidobenzoyloxyferricrocin, a photoactivatable analog of ferrichrome

p-azidobenzoyloxy desferriferricrocin (AF) 2, a photoactivatable analog of ferrichrome, was prepared by selective acylation of the serine group of ferricrocin 1 in two steps: transesterification of ferricrocin followed by demetallation. A model compound, (L) 2-benzyloxycarbonylamino-3-p-azidobenzoyloxy N-isopropyl propionamide 8, was separately synthesized in order to set up optimal transesterification conditions to avoid , -elimination or epimerization of serine. Binding of iron-loaded AF (FeAF) to the FhuA outer membrane receptor protein of Escherichia coli AB2847 was demonstrated by inhibition of ferrichrome transport, interference with the infection by the bacteriophage 80 and with killing of cells by albomycin and colicin M. FeAF transported iron only weakly which indicates that the photoaffinity moiety is incompatible with transport or intracellular iron release from the siderophore.     

Iron supply of Escherichia coli with polymer-bound ferricrocin.

Uptake of ferric iron from ferricrocin was studied in Escherichia coli using a polymer-coupled ferricrocin that was unable to penetrate into the cell. Ferricrocinyl polyethylene glycol succinate (Mr 7000 -- 8500) promoted growth of E. coli K-12 AB2847 aroB under iron-limiting conditions. In iron-starved cells, uptake of 55Fe could be demonstrated; the amount of iron accumulated amounted to 10% of that observed with free ferricrocin. The iron supply by ferricrocin bound to polyethylene glycol was strictly dependent upon the functions expressed by the tonA and the tonB genes, as was the iron uptake promoted by free ferricrocin. Polymer-bound ferricrocin protected cells against colicin M and phage T5 by competition for the common tonA-coded outer membrane receptor protein. In addition, the rate of iron transport via the negatively charged ferricrocinyl succinate was as fast as via the neutral ferricrocin molecule. No ligand was found associated with the cells. Penetration of chelator beyond receptor is not necessary for siderophore-mediated iron uptake. It is concluded that sufficient amounts of iron can be released from the polymer complex to satisfy growth requirements.     

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

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

Role of porins in iron uptake by Mycobacterium smegmatis

Many bacteria rely on siderophores to extract iron from the environment. However, acquisition of iron-loaded siderophores is dependent on high-affinity uptake systems that are not produced under high-iron conditions. The fact that bacteria are able to maintain iron homeostasis in the absence of siderophores indicates that alternative iron acquisition systems exist. It has been speculated that such low-affinity uptake of iron in Gram-negative bacteria includes diffusion of iron ions or chelates across the outer membrane through porins. The outer membrane of the saprophytic Mycobacterium smegmatis contains the Msp family of porins, which enable the diffusion of small and hydrophilic solutes, such as monosaccharides, amino acids, and phosphate. However, it is unknown how cations cross the outer membrane of mycobacteria. Here, we show that the Msp porins of M. smegmatis are involved in the acquisition of soluble iron under high-iron conditions. Uptake of ferric ions by a triple porin mutant was reduced compared to wild-type (wt) M. smegmatis. An intracellular iron reporter indicated that derepression of iron-responsive genes occurs at higher iron concentrations in the porin mutant. This was consistent with the finding that the porin mutant produced more siderophores under low-iron conditions than wt M. smegmatis. In contrast, uptake of the exochelin MS, the main siderophore of M. smegmatis, was not affected by the lack of porins, indicating that a specific outer membrane siderophore receptor exists. These results provide, to our knowledge, the first experimental evidence that general porins are indeed the outer membrane conduit of low-affinity iron acquisition systems in bacteria.     

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.     

SreA-mediated iron regulation in Aspergillus fumigatus

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

Iron-activated iron uptake: A positive feedback loop mediated by iron regulatory protein 1

The love-hate relationship between iron and living matter has generated mechanisms to maintain iron concentration in a narrow range, above and below which deleterious effects occur. At the cellular level, iron homeostasis is accomplished by the activity of the IRP proteins, which, under conditions of iron depletion, up-regulate the expression of the iron acquisition proteins TfR and DMT1. It has been shown that hydrogen peroxide activates IRP1 and that this activation mediates a potentially harmful increase in cell iron uptake. Here we show that IRP1 activity is also induced by iron-mediated oxidative stress. When cells were incubated with up to 20 M of iron, a typical decrease in IRP1 and IRP2 activity was observed. Interestingly, when iron was further increased to 40 or 80 M, IRP1 was reactivated in three of the four different cell lines tested, i.e., Caco-2 cells, N2A cells and HepG2 cells. In the fourth cell line (K562) IRP1 activity did not increase, but neither did it decrease. This response to iron was largely abrogated when the antioxidant N-acetyl cysteine was added along with iron to the culture medium. Thus, the effect of iron was mediated by oxidative stress. Increases in IRP1 activity were accompanied by increases in cell iron uptake, an indication that the activated IRP1 was functional in the activation of iron uptake. Hence, this iron-induced iron uptake feedback loop results in the increase of intracellular iron and increased oxidative stress.     

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The K_m value obtained from iron uptake kinetics was 4.5 M, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.     

Iron uptake studies on erythroid cells.

Iron uptake from 55Fe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.     

Iron uptake studies on erythroid cells

Iron uptake from ⁵⁵Fe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.     

Iron uptake processes in Mycobacterium vaccae R877R, a mycobacterium lacking mycobactin

The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.     

Kinetic studies on the specificity of chelate-iron uptake in Aspergillus.

Three strains of the fungus Aspergillus, Aspergillus quadricinctus (E. Yuill), A. fumigatus (Fresenius), and A. melleus (Yukawa), each producing different iron-chelating compounds during iron-deficient cultivation, were used for 55Fe3+ uptake measurements. Iron from chelates of the ferrichrome-type family was taken up by young mycelia of all strains tested, irrespective of the ferrichrome-type compound these strains predominantly produce in low-iron cultures. Ferrichrysin-producing strains, however, seem to favor ferrichrysin iron uptake, whereas ferrichrome, ferricrocin, and even ferrirubin showed similar iron transport properties in all of these strains. Compared to iron uptake from ferrichrome-type compounds (Km approximately 4 uM) iron uptake from fusigen revealed completely different kinetic values (Km approximately 50 to 80 muM). Iron from exogenous chelates, e.g., from coprogen produced by Neurospora crassa for ferrioxamine B produced by Streptomyces pilosus, can obviously not be taken up by Aspergillus, confirming the pronounced specificity of chelate-iron transport in fungi.