High-affinity Iron Transport Research Articles

Chemical genomic analysis reveals the interplay between iron chelation, zinc homeostasis, and retromer function in the bioactivity of an ethanol adduct of the feijoa fruit-derived ellagitannin vescalagin.

Nature has been a rich source of pharmaceutical compounds, producing 80% of our currently prescribed drugs. The feijoa plant, Acca sellowiana, is classified in the family Myrtaceae, native to South America, and currently grown worldwide to produce feijoa fruit. Feijoa is a rich source of bioactive compounds with anticancer, anti-inflammatory, antibacterial, and antifungal activities; however, the mechanism of action of these compounds is largely not known. Here, we used chemical genetic analyses in the model organism Saccharomyces cerevisiae to investigate the mechanism of action of a feijoa-derived ethanol adduct of vescalagin (EtOH-vescalagin). Genome-wide barcode sequencing analysis revealed yeast strains lacking genes in iron metabolism, zinc metabolism, retromer function, or mitochondrial function were hypersensitive to 0.3 µM EtOH-vescalagin. This treatment increased expression of iron uptake proteins at the plasma membrane, which was a compensatory response to reduced intracellular iron. Likewise, EtOH-vescalagin increased expression of the Cot1 protein in the vacuolar membrane that transports zinc into the vacuole to prevent cytoplasmic accumulation of zinc. Each individual subunit in the retromer complex was required for the iron homeostatic mechanism of EtOH-vescalagin, while only the cargo recognition component in the retromer complex was required for the zinc homeostatic mechanism. Overexpression of either retromer subunits or high-affinity iron transporters suppressed EtOH-vescalagin bioactivity in a zinc-replete condition, while overexpression of only retromer subunits increased EtOH-vescalagin bioactivity in a zinc-deficient condition. Together, these results indicate that EtOH-vescalagin bioactivity begins with extracellular iron chelation and proceeds with intracellular transport of zinc via the retromer complex. More broadly, this is the first report of a bioactive compound to further characterize the poorly understood interaction between zinc metabolism and retromer function.

A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

Phosphatidylinositol 3-phosphate regulates iron transport via PI3P-binding CgPil1 protein

Iron homeostasis, which is pivotal to virulence, is regulated by the phosphatidylinositol 3-kinase CgVps34 inthe human fungal pathogen Candida glabrata. Here, we identify CgPil1 as a phosphatidylinositol 3-phosphate (PI3P)-binding protein and unveil its role in retaining the high-affinity iron transporter CgFtr1 at the plasma membrane (PM), with PI3P negatively regulating CgFtr1-CgPil1 interaction. PI3P production and its PM localization are elevated in the high-iron environment. Surplus iron also leads to intracellular distribution and vacuolar delivery of CgPil1 and CgFtr1, respectively, from the PM. Loss of CgPil1 or CgFtr1 ubiquitination at lysines 391 and 401 results in CgFtr1 trafficking to the endoplasmic reticulum and a decrease in vacuole-localized CgFtr1. The E3-ubiquitin ligase CgRsp5 interacts with CgFtr1 and forms distinct CgRsp5-CgFtr1 puncta at the PM, with high iron resulting in their internalization. Finally, PI3P controls retrograde transport of many PM proteins. Altogether, we establish PI3P as a key regulator of membrane transport in C.glabrata.

Identification and analysis of iron transporters from the fission yeast Schizosaccharomyces pombe.

Iron is an essential trace metal ion required for all living organisms, and is taken up by iron transporters. Here, we identified and characterized three-candidate high-affinity (Fio1, Frp1 and Frp2) and two-candidate low-affinity iron transporters (Fet4 and Pdt1) from the fission yeast Schizosaccharomyces pombe. Protein sequence analyses revealed that Fio1 is a multicopper oxidase that contains three cupredoxin domains with eleven candidate iron-binding ligands, whereas Frp1 harbors a ferric reductase domain with three-candidate heme-binding ligands. Protein sequence analyses also revealed that Fet4 and Pdt1 are integral membrane proteins with 10 and 11 transmembrane regions, respectively. Deletion of fio1 and, to a lesser extent, frp1 impaired growth under iron-depleted conditions, whereas deletion of frp1 and, to a lesser extent, frp2 inhibited growth under iron-replete conditions. Deletion of fet4 and pdt1 did not affect the growth of cells under iron-depleted and iron-replete conditions. Deletion of fio1 or frp1 also increased the sensitivity of cells to other transition metals. The copper sensitivity of Δfio1 cells could be rescued by iron, suggesting that the addition of iron mightdecrease the uptake of potentially toxic copper in Δfio1 cells. The copper sensitivity of Δfio1 cells could also be rescued by deletion of frp1, suggesting that Fio1 and Frp1 may function together in iron and copper uptakes in S. pombe. Our results revealed that iron and copper uptake systems may be partially overlapped in S. pombe.

High-affinity iron and calcium transport pathways are involved in U(VI) uptake in the budding yeast Saccharomyces cerevisiae

Uranium (U) is a naturally-occurring radionuclide that is toxic for all living organisms. To date, the mechanisms of U uptake are far from being understood. Here we provide a direct characterization of the transport machineries capable of transporting U, using the yeast Saccharomyces cerevisiae as a unicellular eukaryote model. First, we evidenced a metabolism-dependent U transport in yeast. Then, competition experiments with essential metals allowed us to identify calcium, iron and copper entry pathways as potential routes for U uptake. The analysis of various metal transport mutants revealed that mutant affected in calcium (mid1Δ and cch1Δ) and Fe(III) (ftr1Δ) transport, exhibited highly reduced U uptake rates and accumulation, demonstrating the implication of the calcium channel Mid1/Cch1 and the iron permease Ftr1 in U uptake. Finally, expression of the Mid1 gene into the mid1Δ mutant restored U uptake levels of the wild type strain, underscoring the central role of the Mid1/Cch1 calcium channel in U absorption process in yeast. Our results also open up the opportunity for rapid screening of U-transporter candidates by functional expression in yeast, before their validation in more complex higher eukaryote model systems.

Quantification and comparison of gene expression associated with iron regulation and metabolism in a virulent and attenuated strain of Flavobacterium psychrophilum.

Iron is essential for growth and virulence in most pathogenic bacterial strains. In some cases, the hosts for these pathogenic bacteria develop specialized strategies to sequester iron and limit infectivity. This in turn may result in the invading pathogens utilizing high-affinity iron transport mechanisms, such as the use of iron-chelating siderophores, to extend beyond the host defences. Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) in salmonids, relies on iron metabolism for infectivity, and the genome of the model CSF-259-93 strain has recently been made available. Further, this strain serves as a parent strain for a live-attenuated vaccine strain, B.17, which has been shown to provide rainbow trout with protection against BCWD. To elucidate specific gene expression responses to iron metabolism and compare strain differences, both F.psychrophilum strains were grown under iron-limiting conditions and 26 genes related to iron metabolism were mapped for 96hr in culture via qPCR analyses. Results indicate increased production of the ferrous iron transport protein B (FITB; p=.008), and ferric receptor CfrA (FR 1; p=.012) in the wild-type CSF-259-93 strain at 72hr and 96hr post-exposure to iron-limiting media. In the B.17 vaccine strain, siderophore synthase (SS) expression was found to be downregulated at 72hr, in comparison with 0h (p=.018). When strains were compared, FITB (p=.021), FR1 (p=.009) and SS (p=.016) were also elevated in B.17 at 0hr and TonB outer protein membrane receptor 1 (TBomr1; p=.005) had a lower expression at 96hr. Overall, this study demonstrated strain-related gene expression changes in only a fraction of the iron metabolism genes tested; however, results provide insight on potential virulence mechanisms and clarification on iron-related gene expression for F.psychrophilum.

Novel 2-Substituted 3-Hydroxy-1,6-dimethylpyridin-4(1H)-ones as Dual-Acting Biofilm Inhibitors of Pseudomonas aeruginosa.

2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS), a compound from P. aeruginosa, functions as both a quorum sensing (QS) regulator and a potent iron chelator to induce expression of pyoverdine and pyochelin which are involved in high-affinity iron transport systems. A potential dual-acting antibiofilm strategy requires molecules designed to interfere with iron uptake and the QS system of P. aeruginosa. A series of 2-substituted 3-hydroxy-1,6-dimethylpyridin-4-ones have been designed, synthesized, and tested as biofilm inhibitors of P. aeruginosa. One compound, N-((1,3,6-trimethyl-4-oxo-1,4-dihydropyridin-2-yl)methyl)hexanamide (10d), exhibits 68.67% biofilm inhibitory activity at 20 μM. Further mechanistic studies have confirmed that this compound not only inhibits the QS systems of P. aeruginosa but also acts as an iron chelator to compete strongly with pyoverdine, causing iron deficiency in bacteria. The pyoverdine receptor FpvA was revealed as the target of 10d by the Pvds mutant strain, fpvA-overexpressed strain, and in silico studies.

IroT/MavN Is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by in Vitro Real-Time Transport.

In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, a putative transmembrane protein inserted on the surface of the host pathogen-containing vacuole, IroT/MavN, facilitates intravacuolar iron acquisition from the host by an unknown mechanism, bypassing the problem of Fe(III) insolubility and mobilization. We developed a platform for purification and reconstitution of IroT in artificial lipid bilayer vesicles (proteoliposomes). By encapsulating the fluorescent reporter probe Fluozin-3, we reveal, by real-time metal transport assays, that IroT is a high-affinity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains, and loops important for substrate recognition and translocation. The work establishes the substrate transport properties in a novel transporter family important for iron acquisition at the host-pathogen intravacuolar interface and provides chemical tools for a comparative investigation of the translocation properties in other iron transporter families.

Universal fluorescent sensors of high-affinity iron transport, applied to ESKAPE pathogens

Sensitive assays of biochemical specificity, affinity, and capacity are valuable both for basic research and drug discovery. We created fluorescent sensors that monitor high-affinity binding reactions and used them to study iron acquisition by ESKAPE bacteria, which are frequently responsible for antibiotic-resistant infections. By introducing site-directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells or purified proteins that bind but do not transport target compounds. These constructs sensitively detected ligand concentrations in solution, enabling accurate, real-time spectroscopic analysis of membrane transport by other cells. We assessed the efficacy of these "fluorescent decoy" (FD) sensors by characterizing active iron transport in the ESKAPE bacteria. The FD sensors monitored uptake of both ferric siderophores and hemin by the pathogens. An FD sensor for a particular ligand was universally effective in observing the uptake of that compound by all organisms we tested. We adapted the FD sensors to microtiter format, where they allow high-throughput screens for chemicals that block iron uptake, without genetic manipulations of the virulent target organisms. Hence, screening assays with FD sensors facilitate studies of mechanistic biochemistry, as well as discovery of chemicals that inhibit prokaryotic membrane transport. With appropriate design, FD sensors are potentially applicable to any pro- or eukaryotic high-affinity ligand transport process.

Candida albicans possess a highly versatile and dynamic high-affinity iron transport system important for its commensal-pathogenic lifestyle.

Iron is an essential nutrient for nearly all organisms, but iron overdose is toxic. The human commensal-pathogenic fungus Candida albicans traverses host niches with markedly different iron availability. During systemic infection, C. albicans must activate the high-affinity iron permease Ftr1 to acquire iron sequestered by the host's iron-withholding defense and suppresses iron uptake while residing in the iron-rich gut to avoid toxicity. Ftr1 associates with a ferroxidase to form an iron transporter. C. albicans contains four permeases and five ferroxidase homologs, suggesting 20 possible subunit combinations. Here, we investigated the iron-dependent expression, cellular localization and interacting partners of all permeases and ferroxidases and the significance of each subunit for gastrointestinal colonization and systemic infection in mice. We uncovered three distinct patterns of iron-dependent expression and highly flexible ferroxidase-permease partnerships, which underlie a dynamic iron transport system that can be deftly tuned according to iron availability. We found functional differentiation as well as redundancy among the ferroxidases and permeases during both gastrointestinal colonization and bloodstream infection. We propose that C. albicans possesses a sophisticated iron acquisition and utilization system befitting its commensal-pathogenic lifestyle. Our findings reveal new possibilities for medical intervention of C. albicans infection.

Identification of Ftr1 and Zrt1 as iron and zinc micronutrient transceptors for activation of the PKA pathway in Saccharomyces cerevisiae

Multiple types of nutrient transceptors, membrane proteins that combine a transporter and receptor function, have now been established in a variety of organisms. However, so far all established transceptors utilize one of the macronutrients, glucose, amino acids, ammonium, nitrate, phosphate or sulfate, as substrate. This is also true for the Saccharomyces cerevisiae transceptors mediating activation of the PKA pathway upon re-addition of a macronutrient to glucose-repressed cells starved for that nutrient, re-establishing a fermentable growth medium. We now show that the yeast high-affinity iron transporter Ftr1 and high-affinity zinc transporter Zrt1 function as transceptors for the micronutrients iron and zinc. We show that replenishment of iron to iron-starved cells or zinc to zinc-starved cells triggers within 1-2 minutes a rapid surge in trehalase activity, a well-established PKA target. The activation with iron is dependent on Ftr1 and with zinc on Zrt1, and we show that it is independent of intracellular iron and zinc levels. Similar to the transceptors for macronutrients, Ftr1 and Zrt1 are strongly induced upon iron and zinc starvation, respectively, and they are rapidly downregulated by substrate-induced endocytosis. Our results suggest that transceptor-mediated signaling to the PKA pathway may occur in all cases where glucose-repressed yeast cells have been starved first for an essential nutrient, causing arrest of growth and low activity of the PKA pathway, and subsequently replenished with the lacking nutrient to re-establish a fermentable growth medium. The broadness of the phenomenon also makes it likely that nutrient transceptors use a common mechanism for signaling to the PKA pathway.

Combined genomic and structural analyses of a cultured magnetotactic bacterium reveals its niche adaptation to a dynamic environment.

BackgroundMagnetotactic bacteria (MTB) are a unique group of prokaryotes that have a potentially high impact on global geochemical cycling of significant primary elements because of their metabolic plasticity and the ability to biomineralize iron-rich magnetic particles called magnetosomes. Understanding the genetic composition of the few cultivated MTB along with the unique morphological features of this group of bacteria may provide an important framework for discerning their potential biogeochemical roles in natural environments.ResultsGenomic and ultrastructural analyses were combined to characterize the cultivated magnetotactic coccus Magnetofaba australis strain IT-1. Cells of this species synthesize a single chain of elongated, cuboctahedral magnetite (Fe3O4) magnetosomes that cause them to align along magnetic field lines while they swim being propelled by two bundles of flagella at velocities up to 300 μm s−1. High-speed microscopy imaging showed the cells move in a straight line rather than in the helical trajectory described for other magnetotactic cocci. Specific genes within the genome of Mf. australis strain IT-1 suggest the strain is capable of nitrogen fixation, sulfur reduction and oxidation, synthesis of intracellular polyphosphate granules and transporting iron with low and high affinity. Mf. australis strain IT-1 and Magnetococcus marinus strain MC-1 are closely related phylogenetically although similarity values between their homologous proteins are not very high.Conclusion Mf. australis strain IT-1 inhabits a constantly changing environment and its complete genome sequence reveals a great metabolic plasticity to deal with these changes. Aside from its chemoautotrophic and chemoheterotrophic metabolism, genomic data indicate the cells are capable of nitrogen fixation, possess high and low affinity iron transporters, and might be capable of reducing and oxidizing a number of sulfur compounds. The relatively large number of genes encoding transporters as well as chemotaxis receptors in the genome of Mf. australis strain IT-1 combined with its rapid swimming velocities, indicate that cells respond rapidly to environmental changes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3064-9) contains supplementary material, which is available to authorized users.

Transcriptomic response of yeast cells to ATX1 deletion under different copper levels

BackgroundIron and copper homeostatic pathways are tightly linked since copper is required as a cofactor for high affinity iron transport. Atx1p plays an important role in the intracellular copper transport as a copper chaperone transferring copper from the transporters to Ccc2p for its subsequent insertion into Fet3p, which is required for high affinity iron transport.ResultsIn this study, genome-wide transcriptional landscape of ATX1 deletants grown in media either lacking copper or having excess copper was investigated. ATX1 deletants were allowed to recover full respiratory capacity in the presence of excess copper in growth environment. The present study revealed that iron ion homeostasis was not significantly affected by the absence of ATX1 either at the transcriptional or metabolic levels, suggesting other possible roles for Atx1p in addition to its function as a chaperone in copper-dependent iron absorption. The analysis of the transcriptomic response of atx1∆/atx1∆ and its integration with the genetic interaction network highlighted for the first time, the possible role of ATX1 in cell cycle regulation, likewise its mammalian counterpart ATOX1, which was reported to play an important role in the copper-stimulated proliferation of non-small lung cancer cells.ConclusionsThe present finding revealed the dispensability of Atx1p for the transfer of copper ions to Ccc2p and highlighted its possible role in the cell cycle regulation. The results also showed the potential of Saccharomyces cerevisiae as a model organism in studying the capacity of ATOX1 as a therapeutic target for lung cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2771-6) contains supplementary material, which is available to authorized users.

Mechanistic insights into the mode of action of anticandidal sesamol

Previously we have deciphered the antifungal effect of sesamol (Ses), a phenolic compound obtained from sesame oil, against human fungal pathogen Candida albicans. To gain deeper insights into the possible mechanisms involved, transcription profiling was done in presence of Ses which revealed various targets through which Ses was barricading the growth of C. albicans. We observed that Ses perturbs membrane integrity confirming our previous observations and displayed disrupted plasma membrane ATPase activity. We further investigated that Ses leads to inhibited morphological transition, biofilm formation and epithelial cell adhesion which are significant virulence attributes required for pathogenesis. Interestingly, Ses also causes amendment in iron homeostasis as revealed by hypersensitivity under iron deprivation, ferroxidase assay to estimate iron levels and concomitant upregulation of FTR2, a high affinity iron transporter. Finally we assessed that Ses causes defect in mitochondrial functioning and DNA repair mechanism. Together, being source of consumable natural product, further studies on Ses are warranted so that it can be exploited as effective antifungal agent.

A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata.

Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

The impact of iron limitation on the physiology of the Antarctic diatom Chaetoceros simplex.

Iron availability strongly governs the growth of Southern Ocean phytoplankton. To investigate how iron limitation affects photosynthesis as well as the uptake of carbon and iron in the Antarctic diatom Chaetoceros simplex, a combination of chlorophyll a fluorescence measurements and radiotracer incubations in the presence and absence of chemical inhibitors was conducted. Iron limitation in C. simplex led to a decline in growth rates, photochemical efficiency and structural changes in photosystem II (PSII), including a reorganisation of photosynthetic units in PSII and an increase in size of the functional absorption cross section of PSII. Iron-limited cells further exhibited a reduced plastoquinone pool and decreased photosynthetic electron transport rate, while non-photochemical quenching and relative xanthophyll pigment content were strongly increased, suggesting a photoprotective response. Additionally, iron limitation resulted in a strong decline in carbon fixation and thus the particulate organic carbon quotas. Inhibitor studies demonstrated that, independent of the iron supply, carbon fixation was dependent on internal, but not on extracellular carbonic anhydrase activity. Orthovanadate more strongly inhibited iron uptake in iron-limited cells, indicating that P-type ATPase transporters are involved in iron uptake. The stronger reduction in iron uptake by ascorbate in iron-limited cells suggests that the re-oxidation of iron is required before it can be taken up and further supports the presence of a high-affinity iron transport pathway. The measured changes to photosystem architecture and shifts in carbon and iron uptake strategies in C. simplex as a result of iron limitation provide evidence for a complex interaction of these processes to balance the iron requirements for photosynthesis and carbon demand for sustained growth in iron-limited waters.

A novel siderophore‐independent strategy of iron uptake in the genus Burkholderia

Like many other bacteria, Burkholderia sp. take up iron in its ferric form via siderophore-dependent transporters. We observed that mutant strains of B. cenocepacia H111 unable to synthesize siderophores did not exhibit any growth defect under iron limited conditions. This finding suggested that this opportunistic pathogen can adopt an alternative iron uptake strategy to compensate for the loss of siderophores. We identified a putative iron uptake locus, ftrBcc ABCD, in the genome of B. cenocepacia H111, which is also conserved in other members of the genus Burkholderia. Mutants deficient in both siderophore-dependent and FtrBcc ABCD systems failed to grow under iron-limited conditions and radiolabelled iron transport assays showed that these mutants were impaired in iron uptake. In addition, expression of ftrBcc ABCD can restore growth of an E. coli strain lacking all known high-affinity iron transport systems under iron-limited conditions. We show that all four proteins encoded by ftrBcc ABCD are essential for iron uptake. Furthermore, our results indicate that the expression of ftrBcc ABCD is regulated at the transcriptional level by iron concentration. This study provides evidence of an alternative, siderophore-independent, iron uptake system in Burkholderia species.

Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria.

Iron bioavailability limits biological activity in many aquatic and terrestrial environments. Broad scale genomic meta-analyses indicated that within a single organism, multiple iron transporters may contribute to iron acquisition. Here, we present a functional characterization of a cyanobacterial iron transport pathway that utilizes concerted transporter activities. Cyanobacteria are significant contributors to global primary productivity with high iron demands. Certain cyanobacterial species employ a siderophore-mediated uptake strategy; however, many strains possess neither siderophore biosynthesis nor siderophore transport genes. The unicellular, planktonic, freshwater cyanobacterium, Synechocystis sp. PCC 6803, employs an alternative to siderophore-based uptake-reduction of Fe(III) species before transport through the plasma membrane. In this study, we combine short-term radioactive iron uptake and reduction assays with a range of disruption mutants to generate a working model for iron reduction and uptake in Synechocystis sp. PCC 6803. We found that the Fe(II) transporter, FeoB, is the major iron transporter in this organism. In addition, we uncovered a link between a respiratory terminal oxidase (Alternate Respiratory Terminal Oxidase) and iron reduction - suggesting a coupling between these two electron transfer reactions. Furthermore, quantitative RNA transcript analysis identified a function for subunits of the Fe(III) transporter, FutABC, in modulating reductive iron uptake. Collectively, our results provide a molecular basis for a tightly coordinated, high-affinity iron transport system.

Structures of Streptococcus pneumoniae PiaA and Its Complex with Ferrichrome Reveal Insights into the Substrate Binding and Release of High Affinity Iron Transporters

Iron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at 2.7 and 2.1 Å resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an α-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues: Glu119 and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome.

Requirement and Functional Redundancy of Ib Subgroup bHLH Proteins for Iron Deficiency Responses and Uptake in Arabidopsis thaliana

The Ib subgroup of the bHLH gene family in Arabidopsis contains four members (AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101). AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deficiency induced transcription factor (FIT), directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1. In this work, we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake. Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT. Dual expression of either AtbHLH100 or AtbHLH101 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1. The plants overexpressing FIT together with AtbHLH101 showed constitutive expression of FRO2 and IRT1 in roots, and accumulated more iron in shoots. Further, the single, double, and triple knockout mutants of AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 were generated and characterized. The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39, AtbHLH100, and AtbHLH101 than that of the other available double and triple mutants of the four genes. Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100, AtbHLH38, AtbHLH101, and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake. Taken all together, we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.