Iron nutrition and physiological responses to iron stress in Nitrosomonas europaea

Nitrosomonas europaea, as an ammonia-oxidizing bacterium, has a high Fe requirement and has 90 genes dedicated to Fe acquisition. Under Fe-limiting conditions (0.2 μM Fe), N. europaea was able to assimilate up to 70% of the available Fe in the medium even though it is unable to produce siderophores. Addition of exogenous siderophores to Fe-limited medium increased growth (final cell mass). Fe-limited cells had lower heme and cellular Fe contents, reduced membrane layers, and lower NH₃- and NH₂OH-dependent O₂ consumption activities than Fe-replete cells. Fe acquisition-related proteins, such as a number of TonB-dependent Fe-siderophore receptors for ferrichrome and enterobactin and diffusion protein OmpC, were expressed to higher levels under Fe limitation, providing biochemical evidence for adaptation of N. europaea to Fe-limited conditions.     

Impact of bimetallic combinations of Cu,Ni and Fe on growth rate,uptake of nitrate and ammonium,14C02 fixation,nitrate reductase and urease activity ofChlorella vulgaris

Summary The toxicity of Cu, Ni and Fe individually, as well as in combination (Cu + Ni, Cu + Fe, Ni + Fe), on growth-rate depression, uptake of NO₃ ^– and NH₄ ⁺, photosynthesis, nitrate reductase and urease activity ofChlorella vulgaris has been studied. All the test metals when used individually showed pronounced toxicity on all the parameters studied. However, their interactive effect was mostly antagonistic except for Cu + Ni (synergism). Pre-addition of Fe offered more protection to the cells against copper and nickel toxicity. The data of statistical analysis reconfirmed that¹⁴C0₂ uptake is the most sensitive parameter (significant atP<0.005, both for time and treatment) than others in metal toxicity assessment. However, these results suggest further that exposure time and sequence of metal addition are very important in biomonitoring of heavy metal toxicity.     

Reduction of Fe(III), Mn(III), and Cu(II) chelates by roots of pea (Pisum sativum L.) or soybean (Glycine max)

Neither the reduction of Fe(III) to Fe(II) by roots nor its induction by Fe-deficiency are unique characteristics of the reductive activities of roots. We show that chelated Mn(III) or chelated Cu(II), as well as chelated Fe(III), may be reduced by Fe-stressed roots of pea (Pisum sativum L.). Deficiency of Fe stimulated the reduction of Fe(III)EDTA about 20-fold, the reduction of Mn(III)CDTA about 11-fold, the reduction of Cu(II)(BPDS)₂ about 5-fold, and the reduction of Fe(III)(CN)₆ by only about 50%. Not only are metals other than Fe reduced as part of the Fe-stress response, but deficiencies of metals other than Fe stimulate the reductive activity of roots. We show that depriving peas or soybeans (Glycine max) of Cu or Zn stimulates the reduction of Fe(III).     

Characterization and in vitro selection for iron efficiency inPyrus andCydonia

Summary Responses to low Fe were characterized in tissue cultures ofPyrus amygdaliformis andCydonia oblonga (quince), two species used as rootstocks for pear. Cultured shoots and plantlets ofP. amygdaliformis had a higher chlorophyll concentration and Fe²⁺/total Fe ratio than those ofC. oblonga when grown under low Fe conditions. This tolerance to low Fe was correlated with high Fe³⁺-reducing ability and medium acidification. The adaptive responses were manifested in roots of plantlets, shoot bases, root cultures, and cell suspension cultures. Shoots were regenerated from leaves of quince and subjected to Fe-deficient conditions. Two somaclonal variants (IE-1 and IE-2) were recovered; each displayed higher ability to reduce Fe³⁺ and acidify the medium. These variants may be useful as rootstocks for regions with calcareous soils, which limit Fe availability.     

Siderophore production byBradyrhizobium spp. strains nodulating groundnut

EighteenBradyrhizobium spp. strains, fourRhizobium spp. strains and oneAzorhizobium caulinodans strain were grown under Fe limitation and assayed for siderophore production. It was further assessed if Fe accumulation in two groundnut cultivars was influenced by inoculant strain or nitrate fertilisation. Growth ofBradyrhizobium spp. strains nodulating groundnut was slow with mean generation times from 11–24 h. All strains, except MAR 967, showed a reduced growth rate when deprived of Fe; none of the strains showed starvation at 1 M Fe. In the CAS (chrome azurol S)-agar assay, all strains, which formed colonies, produced siderophores as visualised by orange halos around the colonies on blue plates.Bradyrhizobium strains produced much smaller halos than the referenceRhizobium meliloti strain. In the CAS-supernatant assay, all strains, except MAR 967, gave positive responses (measured as absorbance at 630 nm) when supernatants of Fe-depleted cultures were assayed with CAS-indicator complex in comparison with Fe-supplemented cultures. Responses of all fourRhizobium spp. strains were large, while responses of allBradyrhizobium strains, exceptB. japonicum MAR 1491 (USDA 110), were small and mostly insignificant. A small response, i.e. a low Fe-scavenging ability, implies either the production of small quantities of siderophores or the production of low affinity siderophores. Among theBradyrhizobium strains, MAR 1574 and MAR 1587 gave the largest responses taken over the two assays. Fe accumulation in groundnut cultivar Falcon was seven times larger than in cultivar Natal Common. No correlation was found between the quantity of nodule tissue and Fe accumulation, making it unlikely that bacteroids are involved in Fe acquisition by groundnuts. Nitrate-fertilised plants accumulated significantly more Fe, suggesting involvement of nitrate reductase in Fe assimilation in groundnut. The two most successful Fe-scavengingBradyrhizobium spp. strains were also the most effective in nodulating groundnut, the reverse also being true. Strain MAR 967, with the lowest Fe requirement, produced the largest nodule dry weight. These data indicate that improved Fe scavenging properties and/or reduced Fe requirement improve rhizospheric growth and with that nodulation effectiveness.     

拟南芥bHLH Ib转录因子调控FIT的转录

FIT是调控拟南芥铁稳态的一个关键调控因子，它在转录水平上受到缺铁诱导，但其背后的调控机制还不甚清楚。该研究以拟南芥bHLH38和FIT的单、双过表达植物及bHLH Ib四突变体植物为材料，采用缺铁(-Fe)处理实验和定量RT-PCR的方法从RNA角度分析了FIT转录水平的变化。结果表明：(1)在铁充足时，bHLH38过表达植物中FIT的转录水平显著高于其在野生型中的水平。(2)在bHLH Ib四突变体植物中FIT的转录水平不受缺铁诱导。(3)FIT单过表达不能激活内源FIT的转录，而在加铁(+Fe)条件下bHLH38和FIT的双过表达则可以激活内源FIT的转录。(4)在缺铁条件下，所有植物中FIT的转录水平均与野生型中的FIT水平无明显差异。基于以上结果认为，bHLH Ib转录因子是缺铁诱导FIT转录的必要条件，而非充分条件。该研究结果为深入了解植物通过多种途径共同维持铁稳态提供了新的见解。     

Growth,N2 fixation and photosynthesis in a cyanobacterium,Trichodesmium sp., under Fe stress

Trichodesmium sp., isolated from the Great Barrier Reef lagoon, was cultured in artificial seawater media containing a range of Fe concentration. Fe additions stimulated growth, N₂ fixation, cellular chlorophyll a content, light-saturated chlorophyll a-specific gross photosynthetic capacity (P_m ^chla) and the dark respiration rate (R_d ^chla). Cell yields only doubled for 9 nM Fe relative to zero added Fe, whereas N₂ fixation increased 11-fold considerably for 450 nM Fe. The results suggest that N₂ fixation of Trichodesmium is more sensitive to Fe limitation than are the cell yields.     

Ammonia switch-off of nitrogenase from Rhodobacter sphaeroides and Methylosinus trichosporium: no evidence for Fe protein modification

In vivo switch-off of nitrogenase activity by NH ₄ ⁺ is a reversible process in Rhodobacter sphaeroides and Methylosinus trichosporium OB3b. The same pattern of switch-off in Rhodospirillum rubrum is explained by ADP-ribosylation of one of the Fe protein subunits, however, no evidence of covalent modification could be found in the subunits from either R. sphaeroides or M. trichosporium. Fe protein subunits from these organisms showed no variant behaviour on SDS-PAGE, nor were they ³²P-labeled following switch-off. These observations suggest either that the attachment of the modifying group to the Fe protein in these organisms is quite labile and does not survive in vitro manipulation, or that the mechanism of switch-off is different than that seen in Rhodospirillum.     

Differences between Bradyrhizobium strains NC92 and TAL1000 in their nodulation and nitrogen fixation with peanut in iron deficient soil

The effects of Bradyrhizobium (strains NC92 and TAL1000) and Fe supply on nodulation and nitrogen fixation of two peanut (Arachis hypogaea L.) cultivars (cv. Tainan 9 (Fe inefficient) and cv. 71-234 (Fe efficient)) grown under Fe deficient conditions (imposed by adding 40% CaCO₃ to a ferruginous soil) were examined in a glasshouse experiment. When inoculated with TAL1000 without Fe, both cultivars had low shoot N concentration, very low nodule numbers and weight and no measurable acetylene reduction activity per plant. Inoculation with NC92 without Fe increased all these parameters substantially; addition of Fe with NC92 had no further effect on N concentration but doubled nodule number, weight and acetylene reduction activity per plant. Addition of Fe with TAL1000 increased all parameters to the same level as Fe+NC92, indicating that the poorer nodulation and N₂ fixation of TAL1000 in the absence of Fe, resulted from a poorer ability in getting its Fe supply from the alkaline soil. The nodules from all treatments with measurable activity had the same specific acetylene reduction activity suggesting that Fe deficiency limited nodule development.The results support previous suggestions that Bradyrhizobium strains differ greatly in their ability to obtain Fe from soils and that selection of Fe efficient strains could complement plant breeding in the selection of legume crops for Fe deficient soils.     

Ferric iron reduction by Desulfovibrio vulgaris Hildenborough wild type and energy metabolism mutants

Desulfovibrio vulgaris Hildenborough wild type and its hyn1, hyd and hmc mutants, lacking genes for periplasmic [NiFe] hydrogenase-1, periplasmic [FeFe] hydrogenase or the transmembrane high molecular weight cytochrome (Hmc) complex, respectively, were able to reduce Fe(III) chelated with nitrilotriacetic acid (NTA), but not insoluble ferric oxide, with lactate as the electron donor. The rate and extent of Fe(III)-NTA reduction followed the order hyn = WT > hmc >> hyd, suggesting that reduction of soluble Fe(III) is a periplasmic process that requires the presence of periplasmic [FeFe] hydrogenase. Reduction of Fe(III)-NTA was not coupled to cell growth. In fact cell concentrations declined when D. vulgaris was incubated with Fe(III)-NTA as the only electron acceptor. Wild type and mutant cells reducing a limiting concentration of sulfate (2 mM), reduced Fe(III)-NTA with similar rates. However, these were similarly incapable of catalyzing subsequent lactate-dependent reduction of Fe(III)-NTA to completion. Periplasmic reduction of Fe(III)-NTA appeared to inhibit the productive, sulfate-reducing metabolism of D. vulgaris, possibly because it prevents the cycling of reducing equivalents needed to achieve a net bioenergetic benefit.     

Reduction of Fe(II)EDTA-NO by a newly isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain DN-2 in NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> scrubber solution

Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N₂ is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO _x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW⁻¹ h⁻¹. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO _x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.     

Response ofAnabaena doliolum to bimetallic combinations of Cu,Ni and Fe with special reference to sequential addition

This study reports physiological features of a N₂-fixing cyanobacteriumAnabaena doliolum in response to metal mixtures. Exposure of the cyanobacterium to Cu, Ni and Fe individually, as well as in combinations (Cu + Ni, Cu + Fe, Ni + Fe), showed marked differences in growth inhibition, nutrient uptake (NH₄ ⁺ and NO₃ ⁻), photosynthesis, ATP content, nitrate reductase, glutamine synthetase and urease activities. The response to metal combinations was also dependent upon the order in which the metals were added. The Cu-Ni combination resulted in synergistic interaction, in contrast to the antagonism of Cu-Fe and Ni-Fe. Pre-addition of Fe protected the cyanobacterium against Cu and Ni toxicity. Statistically significant (P < 0.005) inhibition of all the processes following metal supplementation was observed. This study suggests that carbon fixation is the most suitable variable for assessing heavy metal toxicity.     

Isocyanides inhibit [Fe]-hydrogenase with very high affinity

[Fe]-Hydrogenase catalyzes the reversible activation of H₂. CO and CN⁻ inhibit this enzyme with low affinity (K_i ≅ 0.1 mM) by binding to the iron site of the bound iron-guanyrylpyridinol cofactor. We report here that isocyanides, which are formally isoelectronic with CO and CN⁻, strongly inhibit [Fe]-hydrogenase (K_i as low as 1 nM). The [NiFe]- and [FeFe]-hydrogenases tested were not inhibited by isocyanides. UV–Vis and infrared spectra revealed that the isocyanides bind to the iron center of [Fe]-hydrogenase. The inhibition kinetics are in agreement with the proposed catalytic mechanism, including the open/closed conformational change of the enzyme.     

Iron nitrosyl complexes as models for biological nitric oxide transfer reagents

Owing to the indiscriminate reactivity of the free NO radical, intricate control mechanisms are required for storage, transport and transfer of NO to its various biological targets. Among the proposed storage components are protein-bound thionitrosyls (R_protein–SNO) and protein-bound dinitrosyl iron complexes. Current knowledge suggests the latter are derived from iron–sulfur cluster degradation in the presence of excess NO. Mobilization of protein-bound NO could involve NO or Fe(NO)₂ unit transfer to small serum molecules such as glutathione, free cysteine, or iron-porphyrins. The study reported is of a reaction model which addresses the key steps in NO transfer from a prototypal iron dinitrosyl complex. While the N,N′-bis(2-mercaptoethyl)-N,N′-diazacyclooctane (bme-daco) ligand typically binds in square-planar N₂S₂ coordination, it also serves as a bidentate dithiolate donor for tetrahedral structures in the preparation of the (H⁺bme-daco)Fe(NO)₂ derivative (Chiang et al., J. Am. Chem. Soc. 126:10867–10874, 2004). The removal of one NO produces the mononitrosyl complex, (bme-daco)Fe(NO), and simplifies studies of NO release mechanisms. We have used heme-type model complexes, Fe or Co porphyrins as NO acceptors, yielding (porphyrin)M(NO), where M is Fe or Co, and monitored reactions by ν(NO) Fourier transform IR spectroscopy. Reaction products were verified by electrospray ionization mass spectrometry. Rudimentary mechanistic studies suggest a role for HNO in the NO release from the dinitrosyl; the mononitrosyl benefits as well from acid catalysis. Other NO uptake complexes such as [(N₂S₂)Fe]₂ [N₂S₂ is bme-daco or N,N’-bis(2-mercapto-2-methylpropyl)-daco] are shown to form Fe(NO) mononitrosyls with stability and spectroscopic signatures similar to those of the porphyrins.     

The respiratory inhibitor antimycin A specifically binds Fe(III) ions and mediates utilization of iron by the halotolerant alga Dunaliella salina (Chlorophyta)

It is demonstrated that Antimycin A (AA), a respiratory inhibitor produced by Streptomyces bacteria, forms lipophylic complexes with Fe(III) ions. Spectroscopic titration indicates that Fe(III) ions interact with 2AA molecules. At growth-limiting Fe concentrations, AA mediates Fe uptake and promotes growth and chlorophyll synthesis better than other Fe chelators in the halotolerant alga Dunaliella salina. It is proposed that AA enhances Fe bioavailability in hypersaline solutions by formation of lipophylic Fe-AA complexes which are taken-up and utilized by the algae. The results suggest that the respiratory inhibitor AA can affect Fe metabolism in microorganisms.     

External Iron Regulates Polyphosphate Content in the Acidophilic, Thermophilic Alga Cyanidium caldarium

Transmission electron microscopy revealed the presence of electron-dense bodies (EDB) in the cytosol of the acidophilic, thermophilic red alga Cyanidium caldarium. These bodies contain almost exclusively Fe, P, and O and can play a role in Fe storage. ³¹P-nuclear magnetic resonance analysis identified a sharp signal at −23.3 ppm, which was attributed to the phosphate groups of the inner portions of polyphosphate chains. From this evidence, as well as that of a previous ESR study (Nagasaka et al., BioMetals 16:465–470, 2003), it can be concluded that polyphosphates are the major anionic constituents of the EDB. Omission of Fe from the culture medium resulted in substantially decreased polyphosphate levels, demonstrating the control of cellular polyphosphate content by the Fe status of the culture medium.     

Self-hydroxylation of taurine/α-ketoglutarate dioxygenase: evidence for more than one oxygen activation mechanism

2-Aminoethanesulfonic acid (taurine)/α-ketoglutarate (αKG) dioxygenase (TauD) is a mononuclear non-heme iron enzyme that catalyzes the hydroxylation of taurine to generate sulfite and aminoacetaldehyde in the presence of O₂, αKG, and Fe(II). Fe(II)TauD complexed with αKG or succinate, the decarboxylated product of αKG, reacts with O₂ in the absence of prime substrate to generate 550- and 720-nm chromophores, respectively, that are interconvertible by the addition or removal of bound bicarbonate and have resonance Raman features characteristic of an Fe(III)–catecholate complex. Mutagenesis studies suggest that both reactions result in the self-hydroxylation of the active-site residue Tyr73, and liquid chromatography nano-spray mass spectrometry/mass spectrometry evidence corroborates this result for the succinate reaction. Furthermore, isotope-labeling resonance Raman studies demonstrate that the oxygen atom incorporated into the tyrosyl residue derives from H₂ ¹⁸O and ¹⁸O₂ for the αKG and succinate reactions, respectively, suggesting distinct mechanistic pathways. Whereas the αKG-dependent hydroxylation likely proceeds via an Fe(IV)=O intermediate that is known to be generated during substrate hydroxylation, we propose Fe(III)–OOH (or Fe(V)=O) as the oxygenating species in the succinate-dependent reaction. These results demonstrate the two oxygenating mechanisms available to enzymes with a 2-His-1-carboxylate triad, depending on whether the electron source donates one or two electrons.     

Root exudation and Fe uptake and transport in wheat genotypes differing in tolerance to Zn deficiency

Tolerance to Zn deficiency in wheat germplasm may be inversely related to uptake and transport of Fe to shoots. The present study examined eight bread (Triticum aestivum) and two durum (T. turgidum L. conv. durum) wheat genotypes for their capacity to take up and transport Fe when grown under either Fe or Zn deficiency. Bread wheat genotypes Aroona, Excalibur and Stilleto showed tolerance to Zn and Fe deficiency, while durum wheat genotypes are clearly less tolerant to either deficiency. Roots of bread wheats tolerant to Zn deficiency exuded more phytosiderophores than sensitive bread and durum genotypes. Greater amounts of phytosideophores were exuded by roots grown under Fe than Zn deficiency. A relatively poor relationship existed between phytosiderophore exudation or the Fe uptake rate and relative shoot growth under Fe deficiency. At advanced stages of Zn deficiency, genotypes tolerant to Zn deficiency (Aroona and Stilleto) had a greater rate of Fe uptake than other genotypes. Zinc deficiency depressed the rate of Fe transport to shoots in all genotypes in early stages, while advanced Zn deficiency had the opposite effect. Compared with Zn-sufficient plants, 17-day-old Zn-deficient plants of genotypes tolerant to Zn deficiency had a lower rate of Fe transport to shoots, while genotypes sensitive to Zn deficiency (Durati, Yallaroi) had the Fe transport rate increased by Zn deficiency. A proportion of total amount of Fe taken up that was transported to shoots increased with duration of either Fe or Zn deficiency. It is concluded that greater tolerance to Zn deficiency among wheat genotypes is associated with the increased exudation of phytosiderophores, an increased Fe uptake rate and decreased transport of Fe to shoots. This revised version was published online in June 2006 with corrections to the Cover Date.