Iron uptake by the yeast Saccharomyces cerevisiae: involvement of a reduction step

Among several parameters affecting the rate and amount of iron uptake by Saccharomyces cerevisiae, the oxidation state of iron appeared to be determinant. Iron presented as Fe(II) was taken up faster than Fe(III) and the kinetic parameters were different. Iron was taken up by the cells from different ferric chelates, at rates that did not depend on their stability constants, and uptake was strongly inhibited by an iron(II)-trapping reagent like ferrozine. Iron was physiologically reduced by a transplasmamembrane redox system, which was induced in iron-deficient conditions. We propose that iron must be reduced to be taken up by the cells in the same way as other divalent cations.     

Reductive and non-reductive mechanisms of iron assimilation by the yeast Saccharomyces cerevisiae

Iron reduction and uptake was studied in wild-type and haem-deficient strains of Saccharomyces cerevisiae. Haem-deficient strains lacked inducible ferri-reductase activity and were unable to take up iron from different ferric chelates such as Fe(III)-citrate or rhodoturulic acid. In contrast, ferrioxamine B was taken up actively by the mutants as well as by the wild-type strains. At a low extracellular concentration, uptake was insensitive to ferrozine and competitively inhibited by Ga(III)-desferrioxamine B. Extracellular reductive dissociation of the siderophore occurred at higher extracellular concentrations. Two mechanisms appear to contribute to the uptake of ferrioxamine B by S. cerevisiae: one with high affinity, by which the siderophore is internalized as such and another with lower affinity by which iron is dissociated from the ligand prior to uptake.     

Iron uptake from ferrichrome A and iron citrate in Ustilago sphaerogena.

Double radioactive label transport assays with iron, chromium, and gallium chelates were used to investigate the mechanism of iron uptake by Ustilago sphaerogena. In iron-deficient cells, ferrichrome A iron was taken up without appreciable uptake of the ligand. Iron-sufficient cells partially accumulated the ligand with the metal. The chromium- and gallium-containing analogs of ferrichrome A were transported as intact chelates. Ferrichrome A iron uptake was inhibited by dipyridyl. The data suggest that the intact ferrichrome A chelate binds to a specific receptor, the iron is then separated from the ligand at the membrane by reduction, and the metal is released to the inside of the cell while the ligand is released to the exterior. The reduction step is not transport rate limiting. Iron chelated to citrate was taken up by an energy-dependent process. The citrate ligand was not taken up with the metal. Uptake was sensitive to dipyridyl and ferrozine. Chromic ion chelated to citrate was not transported, suggesting that the iron, rather than the chelate, is recognized by the receptor or that reduction of the metal is required for transport.     

Evidence for a common siderophore transport system but different siderophore receptors in Neurospora crassa.

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 lambda-cis configuration about the iron center and three identical ornithyl-delta-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 lambda-cis specific from the inability to take up the synthetic enantiomers, enantio-ferrichrome and enantio-ferricrocin, possessing a delta-cis configuration about the iron center. On the other hand, we found that coprogen, possessing a delta-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.     

Siderophore-mediated iron (III) transport in the mycelia of the cultivated fungus, Agaricus bisporus.

Three structurally diverse iron (III) sequestering compounds (siderophores) were isolated from the supernatants of early stationary phase iron-deficient cultures of vegetative mycelia of the cultivated mushroom, Agaricus bisporus (ATCC 36416). The compounds were purified as their ferric chelates to homogeneity by gel permeation, cation exchange, and low-pressure reversed phase C18 chromatographies, and characterized as trihydroxamic acids. The chelates were identified as ferrichrome, ferric fusarinine C, and an unusual compound, des (diserylglycyl) ferrirhodin (DDF) by HPTLC cochromatography and electrophoresis against authentic samples, hydrolysis and amino acid analysis, and FAB-MS and 1H NMR spectroscopy. The iron transport activities of the three compounds (and of some structurally similar exogenous compounds) in young mycelial cells were determined by time- and concentration-dependent kinetic assays and inhibition experiments (CN-, N3-) using 55Fe(3+)-labeled chelates. 55Iron (III) uptake mediated by all three compounds was found to be via high affinity, energy-dependent processes; transport effectiveness was in the order: ferrichrome > DDF > ferric fusarinine C. The relative uptake of iron by lambda-cis ferrichromes was: ferrichrome > ferrirhodin > ferrichrome A; transport activity by the delta-cis fusarinines was: ferric fusarinine C > tris cis-(and trans-) fusarinine iron (III) > ferric N1-triacetylfusarinine C.     

Membrane transport of non-transferrin-bound iron by reticulocytes

The transport of non-transferrin-bound iron into rabbit reticulocytes was investigated by incubating the cells in 0.27 M sucrose with iron labelled with 59Fe. In most experiments the iron was maintained in the reduced state, Fe(II), with mercaptoethanol. The iron was taken up by cytosolic, haem and stromal fractions of the cells in greater amounts than transferrin-iron. The uptake was saturable, with a Km value of approx. 0.2 microM and was competitively inhibited by Co2+, Mn2+, Ni2+ and Zn2+. It ceased when the reticulocytes matured into erythrocytes. The uptake was pH and temperature sensitive, the pH optimum being 6.5 and the activation energy for iron transport into the cytosol being approx. 80 kJ/mol. Ferric iron and Fe(II) prepared in the absence of reducing agents could also be transported into the cytosol. Sodium chloride inhibited Fe(II) uptake in a non-competitive manner. Similar degrees of inhibition was found with other salts, suggesting that this effect was due to the ionic strength of the solution. Iron chelators inhibited Fe(II) uptake by the reticulocytes, but varied in their ability to release 59Fe from the cells after it had been taken up. Several lines of evidence showed that the uptake of Fe(II) was not being mediated by transferrin. It is concluded that the reticulocyte can transport non-transferrin-bound iron into the cytosol by a carrier-mediated process and the question is raised whether the same carrier is utilized by transferrin-iron after its release from the protein.     

Effect of metabolic inhibitors on uptake of non-transferrin-bound iron by reticulocytes

The relationship between transferrin-free iron uptake and cellular metabolism was investigated using rabbit reticulocytes in which energy metabolism was altered by incubation with metabolic inhibitors (antimycin A, 2,4-dinitrophenol, NaCN, NaN3 and rotenone) or substrates. Measurements were made of cellular ATP concentration and the rate of uptake of Fe(II) from a sucrose solution buffered at pH 6.5. There was a highly significant correlation between the rate of iron uptake into cytosolic and stromal fractions of the cells and ATP levels. Iron transport into the cytosol showed saturation kinetics. The metabolic inhibitors all reduced the Vmax but had no effect on the Km values for this process. It is concluded that the uptake of transferrin-free iron by reticulocytes is dependent on the cellular concentration of ATP and that it crosses the cell membrane by an active, carrier-mediated transport process. Additional studies were performed using transferrin-bound iron. The metabolic inhibitors also reduced the uptake of this form of iron but the inhibition could be accounted for entirely by reduction in the rate of transferrin endocytosis.     

Distinction of emigration by telotroch formation and death by predation in peritrich ciliates: SEM observations on the remaining stalk ends

Iron uptake mechanisms were investigated in different species of Salmonella isolated from environmental waters. All strains examined were able to grow in the presence of high concentrations (10 mM) of the iron chelator EDDA. All strains excreted phenolate and hydroxamate siderophores, as assessed by bioassays and chemical tests. Bioassays with different indicator strains showed that all Salmonella strains can cross-feed other Enterobacteria, as well as mutants of Salmonella typhimurium deficient in the Enterobactin system, suggesting that this siderophore may be produced by the environmental Salmonella strains. The siderophore aerobactin may also be produced by one of the strains, according to the bioassays results. The same pattern of outer membrane proteins are synthesized under iron-limiting conditions in all species tested, which suggests a similarity of iron uptake systems in many species of Salmonella. This system could be also of great importance in the survival of these bacteria in natural waters, as well as in possible pathogenic mechanisms.     

BIOLOGICAL AVAILABILITY OF IRON TO THE FRESHWATER CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition from various ferric chelates and colloids was studied using iron‐limited cells of Anabaena flos‐aquae (Lyng.) Brèb UTEX 1444, a cyanobacterial strain that produces high levels of siderophores under iron limitation. Various chelators of greatly varying affinity for Fe³⁺ (HEDTA, EDDHA, desferrioxamine mesylate, HBED, 8‐hydroxyquinoline) were assayed for the degree of iron acquisition by iron‐limited cyanobacterial cells. Iron uptake rates (measured by graphite furnace atomic absorption spectrometry) varied approximately inversely with calculated [Fe³⁺] (calculated as pFe) and decreased with increasing chelator‐to‐iron ratio. No iron uptake was observed when Fe³⁺ was chelated with HBED, the strongest of the tested chelators. Iron‐limited Anabaena cells were able to take up iron from 8‐hydroxyquinoline (oxine or 8HQ), a compound sometimes used to quantify aquatic iron bioavailability. Iron bound to purified humic acid was poorly available but did support some growth at high humic acid concentrations. These results suggest that for cyanobacteria, even tightly bound iron is biologically available, including to a limited extent iron bound to humic acids. However, iron bound to some extremely strong chelators (e.g. HBED) is likely to be biologically unavailable.     

Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus

A system was designed to investigate ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus. It involved the incubation of the organisms with labeled ferrous iron in the Norris medium at pH 5, in which the bacteria had grown. Iron uptakes were similar under aerobic and anaerobic conditions. Ferrous but not ferric iron was taken up by the organisms. Iron uptake showed saturation kinetics and a marked temperature dependence. 2,4-Dinitrophenol and thenoltrifluoroacetate but not azide or trypsin treatment inhibited iron uptake. Zinc inhibited iron uptake competitively. Iron uptake from used medium was much greater than that from fresh medium at the same pH. It is concluded that ferrous iron uptake by the microorganisms is a carrier-mediated active phenomenon, inhibited by zinc, which may involve a substance elaborated into the medium by the organism.     

Iron transporters in marine prokaryotic genomes and metagenomes

In the pelagic environment, iron is a scarce but essential micronutrient. The iron acquisition capabilities of selected marine bacteria have been investigated, but the recent proliferation of marine prokaryotic genomes and metagenomes offers a more comprehensive picture of microbial iron uptake pathways in the ocean. Searching these data sets, we were able to identify uptake mechanisms for Fe(3+), Fe(2+) and iron chelates (e.g. siderophore and haem iron complexes). Transport of iron chelates is accomplished by TonB-dependent transporters (TBDTs). After clustering the TBDTs from marine prokaryotic genomes, we identified TBDT clusters for the transport of hydroxamate and catecholate siderophore iron complexes and haem using gene neighbourhood analysis and co-clustering of TBDTs of known function. The genomes also contained two classes of siderophore biosynthesis genes: NRPS (non-ribosomal peptide synthase) genes and NIS (NRPS Independent Siderophore) genes. The most common iron transporters, in both the genomes and metagenomes, were Fe(3+) ABC transporters. Iron uptake-related TBDTs and siderophore biosynthesis genes were less common in pelagic marine metagenomes relative to the genomic data set, in part because Pelagibacter ubique and Prochlorococcus species, which almost entirely lacked these Fe uptake systems, dominate the metagenomes. Our results are largely consistent with current knowledge of iron speciation in the ocean, but suggest that in certain niches the ability to acquire siderophores and/or haem iron chelates is beneficial.     

Role of two siderophores in Ustilago sphaerogena. Regulation of biosynthesis and uptake mechanisms

Under iron-deficient conditions the smut fungus Ustilago sphaerogena produces two kinds of siderophores, ferrichrome and ferrichrome A. Regulation of ligand biosyntheses and uptake mechanisms of the iron chelates were studied to determine the role of each chelate in U. sphaerogena. The biosynthesis of each ligand was differentially regulated. Ferrichrome A, the more effective chelate, was preferentially synthesized under more extreme conditions of iron stress, but completely repressed when the cell was supplied with sufficient iron. In contrast, biosynthesis of ferrichrome was strongly but not completely repressed by iron. The mechanism of repression was examined using a newly developed in vivo synthesis assay. Chromium and gallium-containing siderophore analogs had no effect on siderophore ligand biosynthesis. Iron, added as siderophores, resulted in increased oxygen uptake and amino acid transport, which was soon followed by decreased ligand biosynthesis, suggesting that regulation may be indirect and related to oxidative metabolism. Uptake experiments were used to rule out a ligand-exchange mechanism for ferrichrome A-iron transport. The data suggest that ferrichrome A-iron is taken up at a specific site that results in a rapid distribution of iron inside the cell.     

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.     

Role of two siderophores in Ustilago sphaerogena: Regulation of biosynthesis and uptake mechanisms

Under iron-deficient conditions the smut fungus Ustilago sphaerogena produces two kinds of siderophores, ferrichrome and ferrichrome A. Regulation of ligand biosyntheses and uptake mechanisms of the iron chelates were studied to determine the role of each chelate in U. sphaerogena. The biosynthesis of each ligand was differentially regulated. Ferrichrome A, the more effective chelate, was preferentially synthesized under more extreme conditions of iron stress, but completely repressed when the cell was supplied with sufficient iron. In contrast, biosynthesis of ferrichrome was strongly but not completely repressed by iron. The mechanism of repression was examined using a newly developed in vivo synthesis assay. Chromium and gallium-containing siderophore analogs had no effect on siderophore ligand biosynthesis. Iron, added as siderophores, resulted in increased oxygen uptake and amino acid transport, which was soon followed by decreased ligand biosynthesis, suggesting that regulation may be indirect and related to oxidative metabolism. Uptake experiments were used to rule out a ligand-exchange mechanism for ferrichrome A-iron transport. The data suggest that ferrichrome A-iron is taken up at a specific site that results in a rapid distribution of iron inside the cell.     

Ferrous iron uptake by a magnesium transport system is toxic for Escherichia coli and Salmonella typhimurium.

At low magnesium concentrations, Escherichia coli and Salmonella typhimurium LT2 accumulate ferrous iron independent of the ferrous iron transport system feo. Mutant strains with mutations in the magnesium transport gene corA accumulated less ferrous iron than the parent strains. corA+ and corA strains also differed in their sensitivity to ferrous iron under oxic conditions. corA mutants were more resistant to ferrous iron than their parent corA+ strains. Part of the ferrous iron accumulated can be chased by the addition of magnesium. Much less iron was chased when ferric iron was taken up by the siderophore ferrichrome. These results may indicate that the intracellular metabolism of the iron taken up by these systems differs and that it depends on the uptake route of the iron.     

Hexose transport in L6 rat myoblasts. I. Rate-limiting step, kinetic properties, and evidence for two systems

The hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2-Deoxy-D-glucose (2-DOG) and 2-deoxy-2-fluoro-D-glucose (2FG) were used as analogues to investigate the rate-limiting step of hexose uptake into the cell. Virtually all of the 2-DOG or 2FG taken up into the cell was found to be in the phosphorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate-limiting step. The inhibitory effect of various glucose analogues on 2-DOG and 3-O-methyl-D-glucose (3-OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2-DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3-OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.     

Influence of chelating ligands on bioavailability and mobility of iron in plant growth media and their effect on radish growth

In this study, the effects of chelating ligands on iron movement in growth medium, iron bioavailability, and growth of radish sprouts (Raphanus sativus) were investigated. Iron is an important nutrient for plant growth, yet the insoluble state of iron hydroxides in alkaline conditions decreases its bioavailability. Iron chelates increase iron uptake and have been used in agriculture to correct iron chlorosis. While previous studies have reported the effects of chelating ligands on iron solubility and bioavailability, the present study elucidates the pattern of iron movement by chelating ligands in plant growth medium. The apparent mobility of iron in growth medium was calculated using a ‘4-box’ model. Ethylenediaminedisuccinic acid (EDDS) and hydroxy-iminodisuccinic acid (HIDS) produced the highest apparent mobility of iron from the bottom layer of the medium (initially 10⁻⁴ M Fe(III)) to the upper layer (no iron), followed by glutamatediacetic acid (GLDA), ethylenediaminetetraacetic acid (EDTA), methylglycinediacetic acid (MGDA), and iminodisuccinic acid (IDS). Iron movement in the growth medium was influenced by the chelating ligand species, pH, and ligand exposure time. The iron uptake and growth of radish sprouts were related to the iron mobility produced by the chelating ligands. These results suggest that, in alkaline media, chelating ligands dissolve the hardly soluble iron hydroxide species, thus increasing iron mobility, iron uptake, and plant growth. HIDS, which is biodegradable, was one of the most effective ligands studied; therefore, this compound would be a good alternative to other environmentally persistent chelating ligands.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Siderophore-mediated iron uptake in two clades of Marinobacter spp. associated with phytoplankton: the role of light

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). We have previously shown that algal-associated heterotrophic bacteria belonging to the γ-proteobacterial Marinobacter genus release the siderophore vibrioferrin (VF). The iron-VF complex was shown to be both far more photolabile than all previously examined photolabile siderophores and to generate a photoproduct incapable of re-chelating the released iron. Thus, the photo-generated iron was shown to be highly bioavailable both to the producing bacterium and its algal partner. In exchange, we proposed that algal cells produced dissolved organic matter that helped support bacterial growth and ultimately fueled the biosynthesis of VF through a light-dependent “carbon for iron mutualism”. While our knowledge of the importance of light to phototrophs is vast, there are almost no studies that examine the effects of light on microbial heterotrophs. Here, we characterize iron uptake mechanisms in “algal-associated” VF-producers. Fe uptake by a VF knock-out mutant mimics the wild-type strain and demonstrates the versatility of iron uptake mechanisms in Marinobacter VF-producers. We also show that VF-producers selectively regulate a subset of their siderophore-dependent iron uptake genes in response to light exposure. The regulation of iron uptake and transport genes by light is consistent with the light driven algal–bacterial “carbon for iron mutualism” hypothesis in the marine environment.     

Siderophore iron transport followed by electron paramagnetic resonance spectroscopy

Siderophore iron transport was followed in Ustilago sphaerogena using isotope transport assays coupled with EPR spectroscopy. EPR spectroscopy was used as a quantitative tool to follow the rate of reduction of siderophore iron(III) to iron(II) in the cell suspension by following the disappearance of the signal at g = 4.3. This rate was compared with the rate of iron transport, measured by the disappearance of radioactively labeled iron from the medium. The transport of three iron chelates was examined: the ferric siderophores ferrichrome and ferichrome A, and iron(III) chelated to excess citrate. For the transport of ferrichrome, an iron(III) ionophore, the rate of reduction of iron(III) to iron(II) was significantly lower than the rate of uptake of isotope from the medium supernatant, which is consistent with the established mechanism of uptake of the entire complex followed by intracellular reduction to remove the iron from the ligand. However, the rate of reduction of ferrichrome A, a non-ionophore, was identical with the rate of transport of iron into the cell. Iron(III) citrate was reduced at a rate slightly lower than the rate of transport. These data suggest that reduction of iron(III) is involved in the transport of iron from ferichrome A and possibly from iron(III) citrate.