Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.

A search for glomuferrin: a potential siderophore of arbuscular mycorrhizal fungi of the genus Glomus.

Most fungi are known to synthesize siderophores under iron limitation. However, arbuscular mycorrhizal fungi (AM fungi) have so far not been reported to produce siderophores, although their metabolism is iron-dependent. In an approach to isolate siderophores from AM fungi, we have grown plants of Tagetes patula nana in the presence of spores from AM fungi of the genus Glomus (G. etunicatum, G. mossae & unidentified Glomus sp.) symbiotically under iron limitation and sterile conditions. A siderophore was isolated from infected roots after 2-3 weeks of growth in pots containing low-iron sand with Hoagland solution. HPLC analysis of the root cell lysate revealed a peak at a retention time of 6.7 min which showed iron-binding properties in a chrome azurol S test. The compound was isolated by preparative HPLC and the structure was determined by high resolution electrospray FTICR-MS and GC/MS analysis of the hydrolysis products. From an observed absolute mass to charge ratio (m/z) of 401.11925 [M+H]+ with a relative mass error of ∆ = 0.47 ppm an elemental composition of C16H21N2O10 [M+H]+ was derived, suggesting a molecular weight of 400 Da for glomuferrin. Corresponnding ion masses of m/z 423.10 and m/z 439.06 were asigned to the Na-adduct and K-adduct respectively. A mass of 455.03836 confirmed an Fe- complex with an elemental composition of C16H19N2O10Fe (∆ = 0.15 ppm). GC/MS analysis of the HCl lysate (6 N HCL, 12 h) revealed 1,4 butanediamine. Thus the proposed structure of the isolated siderophore from Glomus species consisted of 1,4 butanediamine amidically linked to two dehydrated citrate residues, similar to the previously identified bis-amidorhizoferrin. Thus, the isolated siderophore (glomuferrin) is a member of the rhizoferrin family previously isolated from fungi of the Mucorales (Zygomycetes).

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor.

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H](+)). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C33H52N6O13Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.

Fe(III)-complexes of the tripodal trishydroxamate siderophore basidiochrome: potential biological implications.

One method of mobilization of iron by mycorrhizal organisms is through the secretion of small organic chelators called siderophores. Hydroxamate donor chelators are a common type of siderophore that is frequently used by fungal organisms. The primary siderophore that is produced by fungi from the genera Ceratobasidium and Rhizoctonia is the tripodal trishydroxamate siderophore basidiochrome. To gain some insight into the iron uptake mechanisms of these symbiotic fungi, the iron binding characteristics of basidiochrome were determined. It was found that basidiochrome exhibits a log ß(110) of 27.8±0.1 and a pFe value of 25.0. These values are similar to those of another fungal trishydroxamate siderophore, ferrichrome. The similarity in iron affinity between the two siderophores suggests that the structure of the backbone has little influence in complex formation due to the length of the pendant arms, although the identity of the terminating groups of the pendant arms is likely related to complex stability. The role of basidiochrome in the biogeochemical cycling of iron is also discussed.

Design of clinically useful macromolecular iron chelators.

OBJECTIVES: In recent years, macromolecular iron chelators have received increasing attention as human therapeutic agents. The objectives of this article are: one, to discuss the factors which should be considered when designing iron binding macromolecules as human therapeutic agents, and two, to report recent achievements in the design and synthesis of appropriate macromolecular chelators that have resulted in the production of a number of agents with therapeutic potential. KEY FINDINGS: Macromolecular drugs exhibit unique pharmaceutical properties that are fundamentally different from their traditional small-molecule counterparts. By virtue of their high-molecular-weight characteristics, many are confined to extracellular compartments, for instance, the serum and the gastrointestinal tract. In addition, they have potential for topical administration. Consequently, these macromolecular drugs are free from many of the toxic effects that are associated with their low-molecular-weight analogues. SUMMARY: The design and synthesis of macromolecular iron chelators provides a novel aspect to chelation therapy. 3-Hydroxypyridin-4-one hexadentate-based macromolecular chelators have considerable potential for the development of new treatments for iron overload and for topical treatment of infection.

Ecology of siderophores with special reference to the fungi.

Ecology of siderophores, as described in the present review, analyzes the factors that allow the production and function of siderophores under various environmental conditions. Microorganisms that excrete siderophores are able to grow in natural low-iron environments by extracting residual iron from insoluble iron hydroxides, protein-bound iron or from other iron chelates. Compared to the predominantly mobile bacteria, the fungi represent mostly immobile microorganisms that rely on local nutrient concentrations. Feeding the immobile is a general strategy of fungi and plants, which depend on the local nutrient resources. This also applies to iron nutrition, which can be improved by excretion of siderophores. Most fungi produce a variety of different siderophores, which cover a wide range of physico-chemical properties in order to overcome adverse local conditions of iron solubility. Resource zones will be temporally and spatially dynamic which eventually results in conidiospore production, transport to new places and outgrow of mycelia from conidiospores. Typically, extracellular and intracellular siderophores exist in fungi which function either in transport or storage of ferric iron. Consequently, extracellular and intracellular reduction of siderophores may occur depending on the fungal strain, although in most fungi transport of the intact siderophore iron complex has been observed. Regulation of siderophore biosynthesis is essential in fungi and allows an economic use of siderophores and metabolic resources. Finally, the chemical stability of fungal siderophores is an important aspect of microbial life in soil and in the rhizosphere. Thus, insolubility of iron in the environment is counteracted by dissolution and chelation through organic acids and siderophores by various fungi.

Environmental factors influence the production of enterobactin, salmochelin, aerobactin, and yersiniabactin in Escherichia coli strain Nissle 1917.

The probiotic Escherichia coli strain Nissle 1917 produces four siderophores: the catecholates enterobactin and salmochelin, the hydroxamate aerobactin, and the mixed-type siderophore yersiniabactin. We studied the influence of pH, temperature, and carbon source on the production of these four siderophores. Yersiniabactin and salmochelin were maximally produced under neutral to alkaline conditions (pH 7.0 and 7.6, respectively), whereas aerobactin was maximally produced at a more acidic pH (pH 5.6), which agrees with the slightly higher complex stability of hydroxamates at acidic pH values compared to the catecholates. Under nearly all conditions studied, catecholate siderophore production was higher with glycerol than with glucose as the carbon source. Yersiniabactin production was also higher with glycerol as the carbon source at pH 7.0. At 42 degrees C, strain Nissle 1917 grew poorly or not at all because of the iron-limiting conditions. In a competition experiment between wild-type strain Nissle 1917 and a mutant of this strain with a deletion in the yersiniabactin operon, the wild-type overgrew the mutant at pH 7.0 and 7.6 and not at pH 5.6. These results agree with yersiniabactin production being of greater advantage at neutral and slightly alkaline pH values. The production of four siderophores may help the probiotic E. coli Nissle 1917 to compete with other E. coli strains in the colon. The probiotic strain Nissle 1917 used in our experiments has many characteristics in common with uropathogenic E. coli and other pathogenic strains which also secrete these siderophores. Uropathogenic E. coli strains may need the multitude of siderophores to adapt to the pH of urine, which varies between pH 4.6 and 8.0.

Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization.

The siderophore salmochelin is produced under iron-poor conditions by Salmonella and many uropathogenic Escherichia coli strains. The production of salmochelin, a C-glucosylated enterobactin, is dependent on the synthesis of enterobactin and the iroBCDEN gene cluster. An E. coli IroD protein with an N-terminal His-tag cleaved cyclic salmochelin S4 to the linear trimer salmochelin S2, the dimer salmochelin S1, and the monomers dihydroxybenzoylserine and C-glucosylated dihydroxybenzoylserine (salmochelin SX, pacifarinic acid). The periplasmic IroE protein was purified as a MalE-IroE fusion protein. This enzyme degraded salmochelin S4 and ferric-salmochelin S4 to salmochelin S2 and ferric-salmochelin S2, respectively. In E. coli, uptake of ferric-salmochelin S4 was dependent on the cleavage by IroE, and independent of the FepBDGC ABC transporter in the cytoplasmic membrane. IroC, which has similarities to ABC-multidrug-resistance proteins, was necessary for the uptake of salmochelin S2 from the periplasm into the cytoplasm. IroE did not function as a classical binding protein since salmochelin S2 was taken up in the absence of a functional IroE protein. IroC mediated the uptake of iron via enterobactin in a fepB mutant. IroE was also necessary in this case for the uptake of ferric-enterobactin, which indicated that only the linear degradation products of enterobactin were taken up via IroC. PfeE, the Pseudomonas aeruginosa IroE homologue, was cloned, and its enzymic activity was shown to be very similar to that of IroE. It is suggested that homologues in other bacteria are also periplasmic IroE-type esterases of siderophores.

The detection of salmochelin and yersiniabactin in uropathogenic Escherichia coli strains by a novel hydrolysis-fluorescence-detection (HFD) method.

Escherichia coli strains produce a variety of structurally different siderophores of which enterobactin, aerobactin and yersiniabactin have been reported earlier to occur in strains of extraintestinal infections. In uropathogenic E. coli (UPEC) strains novel siderophores, named salmochelins, have recently been identified which contain C-glucosylated 2,3-dihydroxybenzoyl-L-serine (glucosyl-DHB-serine) residues connected in a linear (mono-, di- , trimeric) or cyclic form. We report here on a fast and simple hydrolysis-fluorescence-detection (HFD) method, based on identification of C-glucosylated dihydroxybenzoic acid (glucosyl-DHB). Salmochelin containing culture filtrates were bound to DEAE cellulose spin columns, hydrolyzed and the breakdown products were subsequently identified by HPLC or thin layer chromatography (TLC). The hydrolysis products can be easily detected by their fluorescence, either during HPLC separation connected to a fluorescence detector or after TLC on cellulose plates viewed under a UV254 or UV365 lamp. While DHB originates from the hydrolysis of enterobactin and salmochelin, glucosyl-DHB is only found as a characteristic hydrolysis product of salmochelins (S1, S2, S4). The HFD method allows detection of salmochelin in the presence of other siderophores, such as enterobactin, aerobactin and yersiniabactin. Several clinical UPEC isolates containing the iroN gene cluster were analyzed by this procedure, showing that all isolates were glucosyl-DHB positive indicating salmochelin production, while a collection of other pathogenic E. coli strains (EHEC, EIEC, ETEC, EAggEC and EPEC) were glucosyl-DHB negative. In addition, the HFD method allowed the identification of yersiniabactin due to a fluorescent salicylate-containing degradation product.

The determination of ferric iron in plants by HPLC using the microbial iron chelator desferrioxamine E.

Common methods for plant iron determination are based on atomic absorption spectroscopy, radioactive measurements or extraction with subsequent spectrophotometry. However, accuracy is often a problem due to background, contamination and interfering compounds. We here describe a novel method for the easy determination of ferric iron in plants by chelation with a highly effective microbial siderophore and separation by high performance liquid chromatography (HPLC). After addition of colourless desferrioxamine E (DFE) to plant fluids, the soluble iron is trapped as a brown-red ferrioxamine E (FoxE) complex which is subsequently separated by HPLC on a reversed phase column. The formed FoxE complex can be identified due to its ligand-to-metal charge transfer band at 435 nm. Alternatively, elution of both, DFE and FoxE can be followed as separate peaks at 220 nm wavelength with characteristic retention times. The extraordinarily high stability constant of DFE with ferric iron of K = 10(32) enables extraction of iron from a variety of ferrous and ferric iron compounds and allows quantitation after separation by HPLC without interference by coloured by-products. Thus, iron bound to protein, amino acids, citrate and other organic acid ligands and even insoluble ferric hydroxides and phosphates can be solubilized in the presence desferrioxamine E. The "Ferrioxamine E method" can be applied to all kinds of plant fluids (apoplasmic, xylem, phloem, intracellular) either at physiological pH or even at acid pH values. The FoxE complex is stable down to pH 1 allowing protein removal by perchloric acid treatment and HPLC separation in the presence of trifluoroacetic acid containing eluents.

The configuration of the chiral carbon atoms in staphyloferrin A and analysis of the transport properties in Staphylococcus aureus.

Staphyloferrin A, the iron-transporting siderophore of Staphylococci, contains two citric acid residues linked to a D-ornithine backbone, having thus three chiral centers. While the chirality of the backbone can be determined after hydrolysis, the chirality of the two citryl residues can only be determined from the intact staphyloferrin A molecule by circular dichroism spectra. The chirality of the quarternary carbon atoms of citryl residues in fungal rhizoferrin and bacterial enantio-rhizoferrin have been determined previously to be R,R and S,S respectively. The present investigation shows that of the three chiral centers in staphyloferrin A, the citryl residues can be assigned an S,S-configuration by comparison with synthetic analogs, confirming a common chirality among the bacterial enantio-rhizoferrin and staphyloferrin A. This suggests that the bacterial carboxylates originate from a common biosynthetic pathway leading to an S,S-configuration, while the fungal rhizoferrin possessing an R,R-configuration must have a different biosynthetic origin. Growth promotion tests with staphylococci revealed that the S,S-configuration of staphyloferrin A and enantio-rhizoferrin enabled iron uptake, while the fungal rhizoferrin with R,R-configuration was not utilized.

The structure of salmochelins: C-glucosylated enterobactins of Salmonella enterica.

Salmochelins represent novel carbohydrate containing catecholate siderophores, which are excreted by Salmonella enterica and uropathogenic Escherichia coli strains under low-iron stress. While previous analytical data showed salmochelins to contain 2,3-dihydroxybenzoyl-L-serine and glucose, the molecular structure remained elusive. Structure elucidation with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), GC-MS and 2D-NMR now revealed that salmochelins are enterobactin-related compounds, which are beta-C-glucosylated at the 5-position of a 2,3-dihydroxybenzoyl residue. The key compound salmochelin S4 is a twofold beta-C-glucosylated enterobactin analogue. Comparison of partial structures of salmochelin with a C-glycosylated compound previously characterized by another group strongly suggest that salmochelins represent the long sought compounds termed Salmonella resistance factors (SRF) or pacifarins. Transformation of iro-genes into enterobactin-producing E. coli K12 confers the ability to produce salmochelins. A detailed analysis proved iroB to be the sole gene with glycosyltransferase activity necessary for salmochelin production. Salmochelins compared to enterobactin are the better siderophores in the presence of serum albumin. This may indicate for salmochelins a considerably more important role for pathogenic processes in certain Escherichia coli and Salmonella infections than formerly assigned to enterobactin. This conclusion is supported by the location of the iro genes on pathogenicity islands of uropathogenic E. coli strains.

Differential gene expression in auristatin PHE-treated Cryptococcus neoformans.

The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans. To gain a broader understanding of the cellular response of C. neoformans to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 micro M) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1 and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune 14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-caju was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.

Ferrichrome in Schizosaccharomyces pombe--an iron transport and iron storage compound.

Schizosaccharomyces pombe has been assumed not to produce siderophores. Nevertheless, the genomic sequence of this fission yeast revealed the presence of siderophore biosynthetic genes for hydroxamates. Applying a bioassay based on an Aspergillus nidulans strain deficient in siderophore biosynthesis, and using reversed-phase HPLC and mass spectrometry analysis, we demonstrate that S. pombe excretes and accumulates intracellularly the hydroxamate-type siderophore ferrichrome. Under iron-limiting conditions, the cellular ferrichrome pool was present in the desferri-form, while under iron-richconditions, in the ferri-form. In contrast to S. pombe, hydroxamate-type siderophores could not be detected intwo other yeast species, Saccharomyces cerevisiae and Candida albicans.

Characterization of the Aspergillus nidulans transporters for the siderophores enterobactin and triacetylfusarinine C.

The filamentous ascomycete Aspergillus nidulans produces three major siderophores: fusigen, triacetylfusarinine C, and ferricrocin. Biosynthesis and uptake of iron from these siderophores, as well as from various heterologous siderophores, is repressed by iron and this regulation is mediated in part by the transcriptional repressor SREA. Recently we have characterized a putative siderophore-transporter-encoding gene ( mirA ). Here we present the characterization of two further SREA- and iron-regulated paralogues (mirB and mirC ), including the chromosomal localization and the complete exon/intron structure. Expression of mirA and mirB in a Saccharomyces cerevisiae strain, which lacks high affinity iron transport systems, showed that MIRA transports specifically the heterologous siderophore enterobactin and that MIRB transports exclusively the native siderophore triacetylfusarinine C. Construction and analysis of an A. nidulans mirA deletion mutant confirmed the substrate specificity of MIRA. Phylogenetic analysis of the available sequences suggests that the split of the species A. nidulans and S. cerevisiae predates the divergence of the paralogous Aspergillus siderophore transporters.

Effect of auristatin PHE on microtubule integrity and nuclear localization in Cryptococcus neoformans.

The mechanism of action of the fungicidal peptide auristatin PHE was investigated in Cryptococcus neoformans. Since auristatin PHE causes budding arrest in C. neoformans (T. Woyke, G. R. Pettit, G. Winkelmann, and R. K. Pettit, Antimicrob. Agents Chemother. 45:3580-3584, 2001), microtubule integrity and nuclear localization in auristatin PHE-treated cells were examined. Iterative deconvolution in conjunction with an optimized C. neoformans microtubule immunolabeling procedure enabled detailed visualization of the microtubule cytoskeleton in auristatin PHE-treated C. neoformans. The effect of auristatin PHE on C. neoformans microtubule organization was compared with that of the tubulin-binding agent nocodazole. Both drugs produced complete disruption first of cytoplasmic and then of spindle microtubules in a time- and concentration-dependent manner. Sub-MICs of auristatin PHE caused complete microtubule disruption within 4.5 h, while 1.5 times the nocodazole MIC was required for the same effect. For both drugs, disruption of microtubules was accompanied by blockage of nuclear migration and of nuclear and cellular division, resulting in cells arrested in a uninucleate, large-budded stage. Nocodazole and the linear peptide auristatin PHE are remarkably different in structure and spectrum of activity, yet on the cellular level, they have similar effects.

The siderophore iron transporter of Candida albicans (Sit1p/Arn1p) mediates uptake of ferrichrome-type siderophores and is required for epithelial invasion.

The human fungal pathogen Candida albicans contains a close homologue of yeast siderophore transporters, designated Sit1p/Arn1p. We have characterized the function of SIT1 in C. albicans by constructing sit1 deletion strains and testing their virulence and ability to utilize a range of siderophores and other iron complexes. sit1 mutant strains are defective in the uptake of ferrichrome-type siderophores including ferricrocin, ferrichrysin, ferrirubin, coprogen, and triacetylfusarinine C. A mutation of FTR1 did not impair the use of these siderophores but did affect the uptake of ferrioxamines E and B, as well as of ferric citrate, indicating that their utilization was independent of Sit1p. Hemin was a source of iron for both sit1 and ftr1 mutants, suggesting a pathway of hemin uptake distinct from that of siderophores and iron salts. Heterologous expression of SIT1 in the yeast Saccharomyces cerevisiae confirmed the function of Sit1p as a transporter for ferrichrome-type siderophores. The sit1 mutant was defective in infection of a reconstituted human epithelium as a model for human oral mucosa, while the SIT1 strain was invasive. In contrast, both sit1 and SIT1 strains were equally virulent in the mouse model of systemic infection. These results suggest that siderophore uptake by Sit1p/Arn1p is required in a specific process of C. albicans infection, namely epithelial invasion and penetration, while in the blood or within organs other sources of iron, including heme, may be used.

Bisucaberin--a dihydroxamate siderophore isolated from Vibrio salmonicida, an important pathogen of farmed Atlantic salmon (Salmo salar).

A siderophore of the bacterial fish pathogen, Vibrio salmonicida, was isolated from low-iron culture supernatant and structurally characterized as bisucaberin by FTICR- and FAB-MS, NMR and GC-MS analysis of the hydrolysis products. Although the cyclic dihydroxamate bisucaberin has previously been isolated from a marine bacterium, Alteromonas haloplanktis, its involvement in cold-water vibriosis of Atlantic salmon (Salmon salar) is novel. Bisucaberin production in iron-limited media was highest at temperatures around and below 10 degrees C, correlating well with temperatures at which outbreaks of cold-water vibriosis occur. Due to the very high stability constant of K = 32.2, bisucaberin is a most efficient iron scavenger which may contribute to the virulence of V. salmonicida in Atlantic salmon.

X-ray crystallographic structures of the Escherichia coli periplasmic protein FhuD bound to hydroxamate-type siderophores and the antibiotic albomycin.

Siderophore-binding proteins play an essential role in the uptake of iron in many Gram-positive and Gram-negative bacteria. FhuD is an ATP-binding cassette-type (ABC-type) binding protein involved in the uptake of hydroxamate-type siderophores in Escherichia coli. Structures of FhuD complexed with the antibiotic albomycin, the fungal siderophore coprogen and the drug Desferal have been determined at high resolution by x-ray crystallography. FhuD has an unusual bilobal structure for a periplasmic ligand binding protein, with two mixed beta/alpha domains connected by a long alpha-helix. The binding site for hydroxamate-type ligands is composed of a shallow pocket that lies between these two domains. Recognition of siderophores primarily occurs through interactions between the iron-hydroxamate centers of each siderophore and the side chains of several key residues in the binding pocket. Rearrangements of side chains within the binding pocket accommodate the unique structural features of each siderophore. The backbones of the siderophores are not involved in any direct interactions with the protein, demonstrating how siderophores with considerable chemical and structural diversity can be bound by FhuD. For albomycin, which consists of an antibiotic group attached to a hydroxamate siderophore, electron density for the antibiotic portion was not observed. Therefore, this study provides a basis for the rational design of novel bacteriostatic agents, in the form of siderophore-antibiotic conjugates that can act as "Trojan horses," using the hydroxamate-type siderophore uptake system to actively deliver antibiotics directly into targeted pathogens.

Coordination Chemistry of the Carboxylate Type Siderophore Rhizoferrin: The Iron(III) Complex and Its Metal Analogs.

Rhizoferrin is a member of a new class of siderophores (microbial iron transport compounds) based on carboxylate and hydroxy donor groups rather than the commonly encountered hydroxamates and catecholates. We have studied the coordination chemistry of rhizoferrin (Rf), as a representative of this group, with Fe(3+), Rh(3+), Cr(3+), Al(3+), Ga(3+), VO(2+), and Cu(2+). The metal complexes have been studied by UV-vis, CD, NMR, and EPR spectroscopies and mass spectrometry. The formation constants for the iron complex have also been measured and yield a log K(LFe) of 25.3. The Rh and Cr rhizoferrin complexes are unusual in that they appear to adopt a chirality about the metal center that is the opposite of the native iron analog. Several of the alternative metal ion complexes are found to have biological activity toward Morganella morganii in a plate type assay.