A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi. For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included. Culture filtrates of the fungi Penicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus and Neurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system. Photodiode array detection allowed the distinction between siderophores and non-siderophores. According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0-3) was possible.
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 Dl, asperchrome Bi, 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.
Uptake and competition experiments were performed with Neurospora crassa and Penicillium parvum by using 14C-labeled coprogen and 55Fe-labeled ferrichrome-type siderophores. Several siderophores of the ferrichrome family, such as ferrichrome, ferricrocin, ferrichrysin, and tetraglycyl-ferrichrome as well as the semisynthetic ferricrocin derivatives O-(phenyl-carbamoyl)-ferricrocin and O-(sulfanilyl-carbamoyl)ferricrocin were taken up by N. crassa. The ferrichrome-type siderophores used vary in the structure of the peptide backbone but possess a common A-cis configuration about the iron center and three identical ornithyl-8-N-acetyl groups as surrounding residues. This suggests that these ferrichrome-type siderophores are recognized by a common ferrichrome receptor. We also concluded that the ferrichrome receptor is X-cis specific from the inability to take up the synthetic enantiomers, enantio-ferrichrome and enantio-ferricrocin, possessing a A-cis configuration about the iron center. On the other hand, we found that coprogen, possessing a A-absolute configuration and two trans-anhydromevalonic acid residues around the metal center, was also taken up by N. crassa and was competitively inhibited by the ferrichrome-type siderophores. We therefore propose the existence of a common siderophore transport system but the presence of different siderophore receptors in N. crassa. In addition, ferrirubin, which is very slowly transported by N. crassa, inhibited both coprogen and ferrichrome-type siderophore transport. Contrary to the findings with N. crassa, transport experiments with P. parvum revealed the presence of a ferrichrome receptor but the absence of a coprogen receptor; coprogen was neither transported nor did it inhibit the ferrichrome transport.
The mechanism of siderophore transport in fungi was studied with cells of Neurospora crassa using [ 55 Fe]-ferricrocin as a siderophore. In the presence of respiratory inhibitors and uncouplers transport of siderophores is immediately inhibited. Measurements of the ATP content of the cells revealed different levels of ATP, depending on the concentration of inhibitors and the time of incubation, Thus, transport inhibition is not a result of decreased ATP level, but rather due to the concomitant membrane depolarization. To study the role of the membrane potential for siderophore iron transport, glu-II derepressed cells were used, which show membrane depolarization after 479 480 WINKELMANN AND HUSCHKA the addition of high amounts of glucose, without ATP depletion. During depolarization siderophore iron transport was inhibited for a short time, followed by a recovering phase. Inhibition of the membrane ATP-ase by N ,N'-dicyclohexyl carbodiimide (DCCD) or diethylstilbestrol (DES) during membrane depolarization prevented the regeneration of the membrane potential, resulting in a long lasting inhibition of siderophore uptake. These results strongly suggest that the membrane potential of the plasma membrane is essential for siderophore uptake in fungi and that siderophores are translocated by a proton symport.
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