Specific Metal Chelating Agents Siderophores

Despite the abundance of iron in the Earth's crust (5% by weight), in aerobic environments it exists mainly as iron oxyhydroxides (e.g., goethite) of very low solubility at neutral pH (~10~38 M), which causes a bioavailability problem for most living organisms whose metabolism depends on this element. Therefore, most plants, fungi, and bacteria produce an efficient and highly specific system for iron acquisition, comprised of siderophores (Greek "iron carriers") that act as iron scavengers or chelators of high affinity (Hider 1984; Neilands 1995; Renshaw et al. 2002; Boukhalfa and Crumbliss 2002; Kraemer 2004). However, other mechanisms such as reductive assimilation and protonation are also used by some organisms with the aim of solubilizing iron from their environments (Guerinot 1994; Kosman 2003). Although plants (grasses) produce these compounds (phyto-siderophores), the chemical structures and chelation mechanisms differ from those of fungal and bacterial origin (Sugiura and Nomoto 1984; Kraemer et al. 2006; Crowley 2006).

Siderophores are iron-chelating compounds of low molecular weight (5001,500 Da), with the ability to form stable complexes of high affinity constants (Kf > 1030) with the ferric form of iron (Fe3+), but not with the ferrous form (Fe2+). These compounds are excreted into the environment by microorganisms and after chelating the Fe3+ they return to the cell (Hider 1984; Guerinot 1994; Renshaw et al. 2002). The synthesis of siderophores and the proteins involved in their recognition and, in some cases, in their transport to the interior of the cells, is strictly regulated at the molecular level by the concentration of iron perceived by the cell, causing an induction of these systems only under iron stress. Depending on the organism, concentrations higher than 1 mM of Fe3+ can dramatically reduce or completely repress siderophore biosynthesis (van der Helm and Winkelmann 1994).

Siderophores are synthesized by cells as metal-free ligands (desferrisidero-phores), which are excreted into the extracellular environment where they solubi-lize the iron by chelation and return to the cell as ferrisiderophores, releasing the iron through different mechanisms (Hider 1984; Guerinot 1994; Neilands 1995; Renshaw et al. 2002; Kraemer 2004). At least four different mechanisms have been proposed for the transport of ferrisiderophores to the cell, mostly based on the recognition of ferric complexes by specific transport systems present in the cell membrane. Once inside the cell, the Fe3+ is reduced to Fe2+, which is released from the siderophore. A different mechanism performs the reduction of siderophore-transported Fe3+ by a reductase enzyme present in the membrane. The Fe2+ and not the siderophore is subsequently transported to the interior of the cell (van der Helm and Winkelmann 1994; Renshaw et al. 2002). Despite the existing knowledge on the ferrisiderophore transport, this has been much more studied in bacteria than in fungi. Although siderophores are produced by the majority of fungi and bacteria, there are some exceptions where the presence of these compounds has not been demonstrated. However, some microorganisms that do not produce siderophores are capable of using exogenous siderophores (xenosiderophores), produced by other species of bacteria or fungi (Guerinot 1994; Kosman 2003).

The chemical nature of siderophores varies a great deal, but it is possible to distinguish three large groups according to the functional groups involved in chelation: hydroxamates, catecholates (phenolates), and hydroxycarboxylates. In addition to these groups, characterized most frequently in microorganisms, side-rophores with other structures or a combination of the structures previously mentioned have also been described. In bacteria, the production of siderophores mainly of the catecholate and hydroxamate types has been reported. In most fungi, by contrast, it has only been possible to detect and isolate hydroxamate-type side-rophores of the ferrichrome, fusarinine, and coprogen families and, in a few cases, hydroxycarboxylates (Hider 1984; Renshaw et al. 2002; Haselwandter and Winkelmann 2007). Independent of the chemical nature, the kinetic and thermodynamically more stable complexes are obtained by the formation of hex-adentate or six-coordinate siderophores with Fe3+, which allows them to act as efficient iron scavengers from the insoluble oxyhydroxides (Hider 1984; Boukhalfa and Crumbliss 2002; Kraemer 2004). Although catechol-type siderophores can form complexes with a higher Fe3+ affinity than hydroxamates, they are very susceptible to oxidation, depending on the pH. By contrast, hydroxamates form complexes of lower affinity, but very stable over a broad pH range (Hider 1984; Boukhalfa and Crumbliss 2002).

The siderophore production by mycorrhizal fungi has been demonstrated in a limited number of investigations and for very few fungal species; however, less research has been done to illustrate the chemical nature of siderophores isolated from ECM fungi. Nevertheless, those studies demonstrate that siderophores produced by ectomycorrhizal, ericoid, orchidaceous, and ectendomycorrhizal fungi are hydroxamate ligands: mainly of the ferrichrome structural family (Haselwandter 1995; Haselwandter and Winkelmann 2007). The ferrichromes are cyclic peptides containing a tripeptide of N-acyl-N-hydroxy-ornithine and combinations of the amino acids glycine, serine, or alanine (Renshaw et al. 2002). The few studies of some species of ECM fungi (Table 15.1) have detected the presence of siderophores in axenic cultures using (a) the reagent chrome azurol S (CAS) in liquid or solid medium (Schwyn and Neilands 1987; Milagres et al. 1999), (b) the chemical assays to detect hydroxamate structures or the Csaky test (Csaky 1948), and catecholate structures or the Arnow test (Arnow 1937), or (c) through bioassays using the Aureobacterium (Arthrobacter) flavescens JG-9 strain, a hydroxamate siderophore auxotrophic soil organism (Neilands 1984). Cenococcum geophilum (only one ascomycete in Table 15.1) was the first ECM fungus described, from which side-rophores were isolated and characterized through HPLC, mass spectrometry, and NMR spectra (Haselwandter and Winkelmann 2002). This fungus produced mainly ferricrocin-type hydroxamates; other lower concentration compounds were found and although their structures were not completely identified, seemed to correspond to ferrichrome, fusarinine, fusigen, and coprogen.

On the other hand, if the studies showing the production of siderophores by ECM fungi are few, studies describing siderophore production by ECM fungi in association with roots are even fewer. Nevertheless, van Hees et al. (2006), in a careful and precise study demonstrated the simultaneous release of siderophores and organic acids by hyphae of the extraradical mycelium of Hebeloma crustuliniforme in association with Pinus sylvestris seedlings. The siderophores characterized corresponded mainly to ferricrocin; ferrichrome also appeared in the exudates, but in a much lower concentration. Oxalate in concentrations 10,000 times higher than ferricrocin was also detected in exudates. The authors suggest that the combination of hyphal exudates, siderophores and oxalic acid, can significantly alter soil conditions through mineral dissolution (van Hees et al. 2006). Using the CAS assay, the Csaky and Arnow assays, and HPLC, the presence of iron-chelating compounds and organic acids was detected in cultures of Suillus luteus, Rhizopogon luteolus and Scleroderma verrucosum, collected from pine plantations (Machuca et al. 2007).

Table 15.1 Ectomycorrhizal fungi described by production and/or characterization of siderophores

Fungal species

Fungal growth (solid, liquid,

Demonstration of siderophore

Purification/characterization of

References

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