Transport properties of N-acyl derivatives of the coprogen and ferrichrysin classes of siderophores inNeurospora crassa

Several derivatives of the coprogen and ferrichrysin classes of siderophores were synthesized as potential affinity labels of the iron uptake system inNeurospora crassa. While only one of these compounds has proved useful as an affinity label, all were recognized and transported byNeurospora crassa. One derivative, chloroacetyl-ferrichrysin, proved to be an unexpectedly potent reversible inhibitor (K ₁=0.4 M) of both ferrichrysin and coprogen uptake, similar to the natural siderophore, ferrirubin. The reported results provide further understanding of the steric and electronic requirements of siderophores for the iron uptake system inNeurospora crassa.Abbreviations amu atomic mass units - DMF dimethylformamide - FAB tast atom bombardment - NMR nuclear magnetic resonance - ppm parts per million - tlc thin layer chromatography     

A photoaffinity label for the siderophore-mediated iron transport system in Neurospora crassa

A photoaffinity derivative of the coprogen class of siderophores, p-azidobenzoylcoprogen B, has been synthetized. In the dark it is recognized and taken up by the iron transport system in Neurospora crassa (arg-5, ota, aga) in a concentration-dependent manner (K_m = 6 μM, V_max = 0.2 nmol·min⁻¹·mg⁻¹). It is also a competitive inhibitor of coprogen uptake, K_I ≈ 5 μM. Photolyssis of cells with near-ultraviolet light during transport in the presence of the photoaffinity label results in approx. 50% irreversible inhibition of both coprogen and ferrichrysin uptake. Uptake of p-azidobenzoylcoprogen B itself is also inhibited upon illumination. It is proposed that this affinity label be used in isolation of the iron receptor protein(s) in N. crassa.     

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.     

Chiral linear hydroxamates as biomimetic analogues of ferrioxamine and coprogen and their use in probing siderophore-receptor specificity in bacteria and fungi

Summary Linear hydroxamate derivatives, possessing chiral -amino acid moieties, were synthesized and their iron transport activities were studied in bacteria and fungi. No growth-promoting activity could be detected in the Gram-positive hydroxamate-auxotrophAureobacterium flavescens JG9. However, Gram-negative enterobacteria, such asEscherichia coli, Pantoea agglomerans andHafnia alvei were able to utilize iron from these analogues. Uptake of⁵⁵Fe-labeled analogues was inhibited by sodium azide, suggesting an active transport process. The receptors involved during uptake in enterobacteria were identified by using appropriate indicator organisms which are defective in the transport of either ferrioxamines (P. agglomerans FM13), coprogens (H. alvei), or both of these siderophore classes (E. coli fhuE). Our data suggest that the chiral hydroxamates are recognized by the ferrioxamine receptor (FoxA) and the coprogen receptor (FhuE) at a ratio which depends on the optical/ isomer fraction and the nature of side chains. Transport was also observed in the fungusNeurospora crassa, known to take up coprogen rather than ferrioxamines, suggesting that in this fungus the synthetic analogues behave like coprogen.     

Molecular recognition of siderophores in fungi: role of iron-surrounding N-acyl residues and the peptide backbone during membrane transport in Neurospora crassa.

Recognition of ferric siderophores in Neurospora crassa was found to depend on the number and kind of N-acyl residues that surrounded the iron coordination center. In the coprogen series, uptake decreased in the order of coprogen, neocoprogen I, and neocoprogen II, indicating that gradual replacement of the N-transanhydromevalonyl groups by N-acetyl groups had an adverse effect on uptake. The reverse effect was observed in the ferrichrome series, where uptake decreased in the order of ferrichrysin, asperchrome D1, asperchrome B1, and ferrirubin. Configuration of the anhydromevalonyl group (cis or trans) in ferrichromes was also an important determinant in the recognition process. On the basis of uptake and inhibition studies, it is proposed that in ferrichromes part of the molecule (iron configuration and the N-acyl groups) is responsible for binding, whereas another (cyclic peptide ring) is involved in the subsequent process of transport.     

Identification of the ferrioxamine B receptor,FoxB, inEscherichia coli K12

The photoreactivep-azidobenzoyl analog of ferrioxamine B was used to show that ferrioxamine-B-mediated iron transport is separate and distinct from coprogen-mediated iron transport inEscherichia coli. Photolysis of this analog inhibited uptake of [⁵⁹Fe]ferrioxamine B but not [⁵⁹Fe]coprogen or [⁵⁹Fe]ferrichrome. Conversely, photolysis of thep-azidobenzoyl analog of coprogen B inhibited uptake of [⁵⁹Fe]coprogen but not [⁵⁹Fe]ferrioxamine B or [⁵⁹Fe]ferrichrome. Photolabeling of outer membranes withp-azidobenzoyl-[⁵⁹Fe]ferrioxamine B resulted in the labeling of two iron-regulated peptides with molecular masses of about 66 and 26 kDa. Expression of these peptides was increased when ferrioxamine B was the sole iron source. Both peptides were present in outer membrane preparations of thefhuF mutant H1717, but the 66 kDa peptide was not inducible. These results are evidence for an outer membrane receptor inE. coli unique for linear ferrioxamines.     

Identification of siderophores and siderophore-mediated uptake of iron in Stemphylium botryosum

Under iron-deficient conditions Stemphylium botryosum f. sp. lycopersici produces three major siderophores; dimerum acid, coprogen B and an unidentified monohydroxamate siderophore designated as A. The system of siderophores mediating uptake of iron was characterized. It exhibits active transport, saturation kinetics and an optimum at pH 6 and 30°. The rate of iron uptake via dimerum acid and coprogen B was four times higher than siderophore A. S. botryosum was capable of taking up iron from hydroxamate siderophores produced by other fungi, e.g. ferrichrome, fusigen, rhodotorulic acid but not ferrioxamine B. Double labelling experiments suggest that ferric coprogen B accumulates in mycelial cells as an intact chelate.     

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.     

Enzymatic release of iron from sideramines in fungi. NADH: Sideramine oxidoreductase in Neurospora crassa

Young mycelia of the fungus Neurospora crassa contain a soluble NADH-linked sideramine reductase, which may be responsible for liberating iron in vivo from accumulated sideramines during iron-deficient cultivation. The enzymes can be assayed using a soluble supernatant fraction, EDTA, and an atmosphere of pure nitrogen. The enzyme is stable without loss of activity up to 45°C and has an optimum of activity at pH 7.0. Besides coprogen (K_m = 100 μM, V = 2.8 nmol/min. per mg protein), some other ferrichrome-type compounds are reduced. However, ferrichrome, ferrirubin, coprogen B and ferrioxamine are poor substrates. When the mucelia were grown in a medium containing 10^?5 M ferric iron, the activity of the reductase was found to be only 30% of that found under low iron conditions. The enzyme is inhibited by oxygen, SH-alkylating agents and partly by some detergents. Unlike the reductase of N. crassa, the corresponding enzyme from Aspergillus fumigatus revealed low reduction of coprogen and high reduction of ferrichrome, indicating genus-dependent specificities of sideramine reduction enzymes in fungi. The participation of acids of the citric acid cycle as natural iron acceptors during strong iron deficiency is studied and confirmed by iron uptake measurements on isolated mitochondria.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

Stoffwechselprodukte von Mikroorganismen

Zusammenfassung Bei Neurospora crassa, arg-5, ota, aga wird die Desferricoprogenbildung durch Zusatz von Fe³⁺, Ga³⁺, Al³⁺ und V³⁺ im Medium unterdrückt. Bei Anwesenheit von Cr³⁺ und Co³⁺ verhält sich die Mutante wie unter Eisenmangelbedingungen. Eine angelaufene Coprogenbiosynthese wird innerhalb von 24 h mit Fe-Coprogen (10 M), Ga-Coprogen (10 M) und Al-Coprogen (>100 M) reprimiert. Die Coprogenaufnahme entspricht einer Sättigungskinetik, die Aufnahme von Desferricoprogen und Fe³⁺ folgt dagegen einer Diffusionsgeraden. Die verschiedenen Metall-Coprogene verhalten sich bei der Aufnahme kompetitiv und werden nach folgender Affinitätsreihe angereichert: Ga>Fe>Al>V>Cr>Co. Die gleiche Reihenfolge wird eingehalten bei der Regulation der Desferricoprogenbiosynthese. Ein Modell für die Aufnahme, das auf der Stabilität der Metall-Coprogene basiert, wird vorgeschlagen.

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Metabolic products of microorganisms120. Uptake of iron by Neurospora crassa II. Regulation of the biosynthesis of sideramines and inhibition of iron transport by metal analogues of coprogen

Summary The production of desferricoprogen in Neurospora crassa, arg-5, ota, aga is suppressed by addition of Fe³⁺, Ga³⁺, Al³⁺, and V³⁺ to the medium. In the presence of Cr³⁺ and Co³⁺, the mutant behaves as under iron-deficient conditions. Once started, the biosynthesis of coprogen is suppressed within 24 h by Fe-Coprogen (10 M), Ga-Coprogen (10 M), and Al-Coprogen (>100 M). The uptake of Coprogen corresponds to a saturation kinetic, whereas the uptake of desferricoprogen and Fe³⁺ is in accordance with a diffusion line. The different metal analogues of coprogen exhibit competitive behavior during the uptake, and are concentrated by the cells in the following order of affinity: Ga>Fe>Al>V>Cr>Co, which seems to be the same sequence in the regulation of the desferricoprogen biosynthesis. A model for uptake, based on the stability of the metal coprogens, is proposed.

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119. Mitt.: Schindler, P.W., Zähner, H.: Europ. J. Biochem. (im Druck, 1973).     

Characterization of siderophore-mediated iron transport inGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum is capable of utilizing iron from hydroxamate siderophores of different structural classes. The relative rates of iron transport for ferrichrome, ferrichrysin, ferrioxamine B, fusigen, ferrichrome A, rhodotorulic acid, coprogen B, dimerium acid and ferrirhodin were 100%, 98%, 74%, 59%, 49%, 35%, 24%, 12% and 11% respectively. Ferrichrome, ferrichrysine and ferrichrome A inhibited [⁵⁹Fe]ferrioxamine-B-mediated iron transport by 71%, 68% and 28% respectively when added at equimolar concentrations to the radioactive complex. The inhibitory mechanism of [⁵⁹Fe]ferrioxamine B uptake by ferrichrome was non-competitive (K _i 2.4 M), suggesting that the two siderophores do not share a common transport system. Uptake of [⁵⁹Fe]ferrichrome, [⁵⁹Fe]rhodotorulic acid and [⁵⁹Fe]fusigen was unaffected by competition with the other two siderophores or with ferrioxamine B. Thus,G. candidum may possess independent transport systems for siderophores of different structural classes. The uptake rates of [¹⁴C]ferrioxamine B and⁶⁷Ga-desferrioxamine B were 30% and 60% respectively, as compared to [⁵⁹Fe]ferrioxamine B. The specific ferrous chelates, dipyridyl and ferrozine at 6 mM, caused 65% and 35% inhibition of [⁵⁹Fe]ferrioxamine uptake. From these results we conclude that, although about 70% of the iron is apparently removed from the complex by reduction prior to being transported across the cellular membrane, a significant portion of the chelated ligand may enter the cell intact. The former and latter mechanisms seem not to be mutually exclusive.     

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.     

Hydroxamate Recognition During Iron Transport from Hydroxamate-Iron Chelates

Kinetics of radioactive iron transport from three structurally different secondary hydroxamate-iron chelates (schizokinen-iron, produced by Bacillus megaterium ATCC 19213; Desferal-iron, produced by an actinomycete; and aerobactin-iron, produced by Aerobacter aerogenes 62-1) revealed that B. megaterium SK11 (a mutant which cannot synthesize schizokinen) has a specific transport system for utilization of ferric hydroxamates with a recognition capacity based on the chemical structure of the hydroxamate. Both Desferal and schizokinen enhanced iron uptake in this organism; however, Desferal-iron delivered only one-sixth the level of iron incorporated from the schizokinen-iron chelate. Desferal-iron did not generate the rapid rates of iron transport noted with schizokinen-iron at elevated iron concentrations. Assays containing large excesses of either iron-free Desferal or iron-free schizokinen suggested that the iron-free hydroxamate may compete with the ferric hydroxamate for acceptance by the transport system although the system has greater affinity for the iron chelate. Aerobactin-iron did not stimulate iron uptake in B. megaterium SK11 and aerobactin inhibited growth of this organism, indicating that B. megaterium SK11 cannot efficiently process the aerobactin-iron chelate.     

Ferrioxamine transport mutants and the identification of the ferrioxamine receptor protein (FoxA) inErwinia herbicola (Enterobacter agglomerans)

Summary Iron deprivation ofErwinia herbicola (Enterobacter agglomerans) induces the biosynthesis of six high-M _r outer-membrane proteins and large amounts of ferrioxamine E. Mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine and selection with ferrimycin A yielded mutants ofE. herbicola K4 (wild type), defective in the expression of a 76-kDa outer-membrane protein, as determined by SDS/polyacrylamide gel electrophoresis. While in bioassays wild-type cells showed growth promotion in the presence of ferrioxamines (B, D₁, D₂, E, G), enterobactin, citrate, ferrichrome and coprogen, these mutants failed to respond to ferrioxamines. Moreover, experiments with⁵⁵Fe-labelled siderophores confirmed that iron transport mediated by ferrioxamine E and B in the mutants was completely inhibited, whereas iron transport by other hydroxamate siderophores, such as ferrichrome and coprogen was unaffected. The results are evidence that the 76-kDa protein in the outer membrane represents the receptor protein (FoxA) for ferrioxamines inE. herbicola.     

Identification of an iron uptake system specific for coprogen and rhodotorulic acid in Escherichia coli K12

With the lac operon fusion technique, mutants were isolated in two genes that specify two outer membrane proteins designated FhuE (76 K) and Fiu (83 K). The synthesis of both proteins was increased under low iron growth conditions. The FhuE-protein was shown to be necessary for iron uptake via coprogen, an iron chelator produced by certain fungi, e.g. Neurospora crassa. In addition to fhueE the genes fhuCDB, tonB and exbB were necessary for iron coprogen uptake. The gene fhuE was mapped between kdp and gltA near 16 min on the genetic map of E. coli K12, while gene fiu was mapped near 18 min between chlA and chlE. Nor iron transport system could be assigned as yet to the Fiu protein.     

Surface iron polymers and hydroxy acids. A model of iron supply in sideramine-free fungi

Biochemical and electron microscopic evidence is presented that sideramine-free fungi form iron hydroxide polymer layers on the cell surface when grown in an iron containing medium.Iron hydroxide polymer formation on the cell surface is completely prevented in sideramine producing strains of Neurospora crassa. After feeding a sideramine-free mutant of Neurospora crassa with ornithine in order to restore the sideramine synthesis the iron hydroxide coat is gradually dissolved.The addition of excess citrate and malate to the incubation medium also prevents iron polymer adsorption, suggesting that hydroxy acids may be involved in iron supply, when sideramine-free organisms are grown in iron containing media.In order to study the interaction between iron hydroxide polymer deposition upon the cell surface and iron chelating acids in Neurospora crassa, the amount and the proportion of excreted acids was studied under various experimental conditions. Gas chromatographic analysis of the acids produced under iron deficient conditions revealed that succinate, malate and citrate were present within the cells in the early growth phase. The acids were sequentially excreted into the medium in the order succinate, malate and citrate. The amount of succinate decreased after 2 days of cultivation, whereas the amount of malate and citrate continually increased. Although citrate was present within the cells from the 1st day, excretion occurred very late, generally after the 3rd day.It is suggested that sideramine-free fungi first adsorb iron as a hydroxide polymer on the cell surface, and that it is gradually solubilized by excreted hydroxy acids such as citrate or malate. Thus high local concentrations of iron chelated by hydroxy acids provide sideramine-free fungi with a continuous iron supply.Abbreviations BSTFA N,O-Bis(trimethylsilyl)-trifluoracetamide - GC Gaschromatography - EGTA Ethylenglykol-bis(2-aminoethylether) N,N-tetraacetic acid - TMS Trimethylsilyl     

Utilization of microbial siderophores in iron acquisition by oat

Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K_m of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms.     

Excretion of pyruvate in nickel toxicity in wild type and Ni2+ resistant mutants ofNeurospora crassa

The parent wild strainNeurospora crassa Em 5297a and three Ni²⁺ resistantNeurospora crassa mutants have been shown to excrete pyruvate into the culture medium in Ni²⁺ and Co²⁺ toxicities. Ni²⁺ has a more pronounced effect in this regard. The excretion is progressive with growth inhibition and is abolished by Mg²⁺ in all strains and by Fe³⁺ partially in the Em strain but not inNeurospora crassa Ni^R1. Pyruvate, citrate and malate supplementation reverse growth inhibition caused by excess Ni²⁺, but with concomitant suppression of Ni²⁺ accumulation. It is suggested that one of the features of Ni²⁺ toxicity inNeurospora crassa is a derangement in carbohydrate metabolism at step(s) beyond pyruvate and that this is possibly due to decreased invivo activity of Mg²⁺ dependent processes