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1. The effect of iron chelators on iron uptake, ferritin and total protein synthesis was studied in cultured Chang cells. Desferrioxamine depressed ferritin synthesis and completely inhibited iron uptake by ferritin protein. Rhodotorulic acid reduced iron uptake by the cells but had little effect on ferritin synthesis. Diethylenetriamine pentaacetic acid produced complete inhibition of iron uptake and all protein synthesis. 2,3-Dihydroxybenzoic acid (2,3-DHB) had no effect in this system. 2. When 2,3-DHB was incubated with a liver homogenate, its subsequent addition to a Chang cell culture resulted in depression of ferritin synthesis, iron uptake into the protein and some depression of total protein synthesis. Pretreatment of rhodotorulic acid did not affect its properties. 3. Non-ferritin iron in the Chang cell cytosol was dialysable, available for binding to transferrin and formed chelates which appeared, on gel chromatography, to be of low molecular weight. Gel chromatography of cytosol after incubation of the cells with chelating agents showed non-ferritin iron to be in a similar form. 4. Loss of non-ferritin iron from the cells occurred only when the transferrin in the medium was unsaturated. In the presence of chelating agents non-ferritin iron was lost from the cells even when transferrin was 100% saturated. 5. The results confirm the presence of an intracellular labile iron pool which is available for chelation, and demonstrate that different iron chelators have different metabolic effects.

A Puerto Rican isolate of the colorless dinoflagellate Crypthecodinium cohnii was grown in a defined marine medium. Fe was added as Fe(NH4)2(SO3)2 - 6H2O (2.0 mg%), FeCl3 - 6H2O (1.0 mg%) or a particulate slurry prepared from FeCl3 + KOH, along with varying concentrations of several chelators. Heavy growth at pH 7.5-7.7 occurred with salicylhydroxamic acid, aurintricarboxylic acid, EDTA, NTA, and humic acid; and at pH 7.9-8.1 with SHAM and ATA. Moderate growth occurred at pH 7.5-7.7 with sulfosalicylic acid, dipicolinic acid, pyrocatecholdisulfonic acid, hexanohydroxamic acid, L-histidine, and at pH 7.9-8.1 with 1-naphthohydroxamic acid, EDTA, NTA. Slight growth occurred at pH 7.5-7.7 with benzohydroxamic acid, 1-naphohydroxamic acid, 2.6-dipicolinic acid N-oxide, salicylic acid, rhodotorulic acid, Na oxalate, EDDHA, sorbohydroxamic acid, gamma-pyrone-2, 6-decarboxylic acid, and at pH 7.9-8.1 with hexanohydroxamic acid, benzohydroxamic acid. Some ecological and physiological implications are discussed.

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.

Siderophores are iron transport compounds produced by numerous microorganisms and which strongly chelate Fe(III), but not Fe(II). Other trivalent metals, such as Al(III), Cr(III), or Ga(III), are not capable of significantly displacing iron from siderophores. However, I demonstrate here that Ga(III) can effectively displace iron under reducing conditions. With ascorbate as reductant and ferrozine as Fe(II) trapping agent, the kinetics of reductive displacement of iron by Ga(III) were followed spectroscopically by the increase of absorbance at 562 nm due to formation of the Fe(II)-ferrozine complex. No significant reduction of siderophore occurred in the absence of Ga(III). With excess Ga(III), the displacement was quantitative and very rapid. The rate of metal exchange was pseudo first order with respect to Ga(III) concentration and highly pH dependent, suggesting that siderophore ligands are displaced from the iron in a concerted mechanism by Ga(III) and protonation to expose the Fe(III) to reduction by ascorbate. Reaction rates were dependent upon the structure of the siderophore, being greatest for ferric rhodotorulic acid and slowest for ferrichrome A at pH 5.4. The pH profile for ferric rhodotorulic acid was unusual in that it showed a maximum at pH 6.5, while all other siderophores examined showed an increase in rate as pH was lowered from 7.0. The physiological significance of this reaction to the clinical use of gallium is discussed.

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.

The mechanism by which iron uptake is facilitated by the siderophore rhodotorulic acid (RA) in the yeast Rhodotorula pilimanae was investigated with radioactively labeled Fe and RA and kinetically inert, chromic-substituted RA complexes. The weight of the evidence supports a model in which RA mediates iron transport to the cell but does not actually transport iron into the cell. It is proposed that RA exchanges the ferric ion at the cell surface with a membrane-bound chelating agent that completes the active transport of iron into the cell. Uptake of 55Fe in ferric rhodotorulate was much more rapid than uptake of RA itself. Two exchange-inert chromic complexes of RA showed no uptake.

The cyclic trihydroxamic acid, N,N',N''-triacetylfusarinine C, produced by Mycelia sterilia EP-76, was shown to be a ferric ionophore for this organism. The logarithm of the association constant k for the ferric triacetylfusarinine C chelate was determined to be 31.8. Other iron-chelating agents, such as rhodotorulic acid, citric acid, and the monomeric subunit of triacetylfusarinine C, N-acetylfusarinine, delivered iron to the cells by an indirect mechanism involving iron exchange into triacetylfusarinine C. In vitro ferric ion exchange was found to be rapid with triacetylfusarinine C. Gallium uptake rates comparable to those of iron were observed with the chelating agents that transport iron into the cell. Ferrichrome, but not ferrichrome A, was also capable of delivering iron and gallium to this organism, but not by an exchange mechanism. Unlike triacetylfusarinine C, the 14C-ligand of ferrichrome was retained by the cell. A midpoint potential of -690 mV with respect to the saturated silver chloride electrode was obtained for the ferric triacetylfusarinine C complex, indicating that an unfavorable reduction potential was not the reason for the use of a hydrolytic mechanism of intracellular iron release from the ferric triacetylfusarinine C chelate.

Each of the four hydroxamate compounds, ferrichrome, aerobactin, rhodotorulic acid, and coprogen, known to transport ferric iron into Escherichia coli requires a specific outer membrane receptor protein. In addition, common transport functions for all four ferric hydroxamate compounds have been identified in the 3.5-min region of the linkage map and designated fhu. The fhu region was cloned into pBR322. By restriction analysis, Tn5 insertion mutations, and complementation studies between plasmid fragments and chromosomal mutants at least four loci in the order fhuA fhuC fhuD fhuB were found. The genetic products were determined in maxicells and minicells. fhuA codes for the known 78,000-dalton receptor protein and the 81,000-dalton precursor in the outer membrane, fhuC codes for a 30,000-dalton protein, and fhuD encodes a 26,000-dalton protein in the cytoplasmic membrane. No protein(s) could be assigned to the fhuB region. Truncated proteins derived from partial fhuA genes (68,000, 42,000, and 39,000 daltons) and a partial fhuD gene (24,000 daltons) and the strong polar effect on the expression of the genes indicated the direction of transcription to be from fhuA to fhuD.

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.

A number of hydroxamic acid derivatives, including salicyl-, octano-, decano- and dodecanohydroxamic acid and rhodotorulic acid, inhibited 3H-thymidine incorporation into DNA, lowered the concentration of dATP and raised the concentration of dTTP in human PHA-stimulated lymphocytes. These effects suggest that these compounds, like desferrioxamine, inhibit ribonucleotide reductase, an iron-requiring enzyme. On the other hand, benzoic acid derivatives did not inhibit ribonucleotide reductase assessed by the dATP and dTTP pool changes, although some did inhibit DNA synthesis judged by 3H-thymidine incorporation into DNA. A number of other compounds known to chelate iron also inhibited DNA synthesis without inhibiting ribonucleotide reductase. These results suggest that different iron binding compounds affect different iron pools in the cell and that some of them may have use as cytotoxic agents.

The production of siderochromes in 46 strains of Heterobasidiomycetes with ontogenetic yeast phases and some related species was investigated. The strains, representing 24 species of 13 genera, were cultivated in a modified Sundstroem medium under low-iron conditions. The metabolites were isolated from the medium by chromatography on Amberlite XAD-2. The identification of the metabolic products was carried out by thin layer chromatography and cellogel electrophoresis by comparison with authentic samples. Of the investigated strains only one species failed to grow; furthermore, three species did not produce siderochromes under the conditions used. The remaining strains produced siderochromes. Besides the well known sideramines ferrichrome, ferrichrome A, and rhodotorulic acid, a new metabolite was isolated from cultures of Ustilago major, U. violacea , and U. violacea var. stellariae. The results of the antagonism test with this metabolite suggest a ferrichrome or ferrioxamine type of compound. Additionally, the iron auxotrophic fungus Pilobolus kleinii shows similar growth rates on media containing either ferrichrome or the new metabolite. The taxonomic value of the production of sideramines by the species investigated is discussed. KEYWORDS Ferrichrome;, ferrichrome A;, rhodotorulic acid;, new iron binding compound;, Heterobasidiomycetes;, taxonomy.

Synthese en particulier de N 5 -benzyloxycarbonyl-, N 5 -acetyl- et N 5 -[trichloro-2 ethoxycarbonyl]- Nα-t-butoxycarbonyl N 5 -benzyloxyornithines

Tris[(acetylhydroxyamino)alkyl] isocyanurates 2a-c were synthesized from alpha, omega-dibromoalkanes 5 in four steps. The alkylation of the bromides 5a-c with O-benzyl-N-[(trichloroethoxy)carbonyl]hydroxylamine in the presence of DBU gave N-alkylation products 7a-c. The (trichloroethoxy)cabronyl protecting group of 7a-c was easily removed by Zn dust in acetic acid. When the reaction was performed with acetic anhydride, the desired N-acetylated materials 10a-c were obtained. The alkylation of cyanuric acid with 12 in the presence of base provided the N-alkylated materials 13, which were hydrogenated to provide 2a-c. In order to determine the affect of structural modifications on biological activity, various chain lengths of the side arms were utilized and the retroanalogue 3 was prepared. Most of the compounds examined acted as ferrichrome in supporting the iron nutrition of Escherichia coli. However, tris[(acetylhydroxyamino)butyl] isocyanurate 2b, and to some extent its pentyl analogue, 2c, displayed the unique and remarkable property of supporting growth of fhuB mutants, the latter unresponsive to the other analogues and to all natural siderophores tested.

Three human isolates of Campylobacter jejuni were grown in a biphasic culture medium with and without the addition of a synthetic chelator to induce iron limitation. Cells grown in low-iron medium exhibited slower growth rates and altered cellular morphology. Increased numbers of longer, more filamentous forms were seen in Gram-stained smears. Three proteins, with apparent Mrs of 82,000, 76,000, and 74,000, were consistently present in the outer membrane of cells grown in low-iron medium. At least one of these proteins (76,000 to 74,000) was exposed on the cell surface. A bioassay was used to look for the production of siderophores by these and other strains of C. jejuni. Seven of 26 strains tested produced detectable amounts of siderophores. Growing strains at 42 degrees C failed to suppress siderophore synthesis or to alter the outer membrane protein profiles of iron-starved cells. The ability of three strains to utilize exogenously supplied siderophores for growth in low-iron medium was also examined. All three strains were able to utilize enterochelin and ferrichrome, but none utilized aerobactin, rhodotorulic acid, or desferrioxamine B. The effect of iron on the virulence of C. jejuni for 11-day-old chicken embryos inoculated via the chorioallantoic membrane was also determined.

Cells of Escherichia coli can derive iron from a variety of chelators (siderophores) in addition to enterochelin, the catechol derivative excreted by many enteric bacteria. The genetic control of hydroxamate siderophore utilization was investigated in mutants of E. coli K-12 selected for resistance to lethal agents which adsorb to the tonA protein of the outer membrane (albomycin, colicin M, and phages T5 and φ80). Many of the mutants were unable to utilize hydroxamate siderophores as an iron source. This phenotype was termed Fhu, for ferric hydroxamate uptake. Mutants carrying lesions in the tonA region of the chromosome were studied and fell into several types. Members of one class had lost some or all of the tonA receptor protein's functions in that they were resistant to the lethal agents and unable to utilize ferrichrome and its analogs, although able to respond to the hydroxamate rhodotorulic acid (FhuA phenotype). Other mutants were unable to utilize any of the hydroxamate siderophores tested and were resistant to albomycin, althogh many were sensitive to the other lethal agents (FhuB phenotype). Members of these classes lacked ferrichrome-mediated iron uptake. Strains carrying transposon Tn 10 insertions in fhuA (previously termed tonA ) lacked the 78,000-molecular-weight outer membrane protein previously described; insertions in fhuB retained this protein and had no detectable change in outer membrane composition. Three-point transduction crosses revealed the gene order to be pan-fhuA-fhuB-metD in the min 3.5 region of the chromosome map. Complementation analyses with F′ merodiploid strains showed that fhuA and fhuB comprise separate transcription units which are both required for utilization of ferrichrome. Response of diploid strains to rhodotorulic acid suggested the existence of a third gene, fhuC , required for utilization of this siderophore, but not ferrichrome. It is suggested that fhuB encodes a transport component in the cytoplasmic membrane that is necessary for the uptake of all hydroxamate siderophores following their receptor-mediated passage across the outer membrane.

A new hydroxamate-based siderophore analogue, 1,4,8,11-tetraazacyclotetradecane-12,14-dioxo-4,8-bis(N-methylacetohydroxamicacid) (DOCYDMAHA) was synthesized and characterized in terms of its acid-base behaviour. It is shown that this ligand forms a quite stable ferric complex (Fe2L3) and its coordination properties were studied by potentiometric and spectrophotometric techniques. To further characterize this dimeric complex, electron paramagnetic resonance spectra and magnetic moments were measured in aqueous solution. The mechanism of electron transfer of the ferric complex, thought to be important in the biological activity of this siderophore analogue, was also studied by cyclic voltammetry. Theoretical simulation studies were performed using molecular mechanics methods to find the most probable structure of the ferric complex. The properties of this ferric complex are compared with those of rhodotorulic acid, a naturally occurring dihydroxamate siderophore.

Summary The production of siderophores was investigated in 25 isolates of smut fungi which form their sori in the flowers of members of the Polygonaceae. The parasites originated from 6 species of host plants which are classified within the genera Bilderdykia, Oxyria and Polygonum. Ferrichrome and rhodotorulic acid were isolated from Ustilago inflorescentiae. The remaining species of the genera Sphacelotheca and Ustilago are characterized by the formation of rhodotorulic acid only. On the basis of these results Ustilago inflorescentiae does not belong to the inflorescentic smuts of Polygonaceae. The teliospore ornamentation of this species is similar to that of those smuts that parasitizes on the leaves of Polygonaceae.

Although iron is abundant in soils (1–6%), it is often unavailable to plants because its solubility is dependent on pH and controlled by the low solubility of ferric oxides. Iron availability to plant roots may thus depend on organic chelators which Lindsay reports would maintain an adequate iron supply by diffusion and mass flow at concentrations as low as 10−8 M1,2. Hydroxamate siderophores (HS), microbially produced, ferric-specific, iron transport molecules with stability constants3 as high as 1032, may represent the chelators which maintain these soil iron concentrations. Such peptide derivatives were shown to control iron availability in aquatic ecosystems4, but little is known about their soil role beyond their function as microbial growth factors5,6. We report here the occurrence of HS in aqueous extracts of a variety of soils in concentrations (10−7 M–10−8 M after correction to 10% soil moisture) sufficient to affect plant nutrition1,2.

The iron complex of rhodotorulic acid (RA) at neutral pH has been found to be dimeric with the formulation Fe2(RA)3, where both iron atoms have the Δ-cis configuration; it is this dimeric complex that functions as an iron transport agent in yeast.