Low-iron Conditions Research Articles

DSF inactivator RpfB homologous FadD upregulated in Bradyrhizobium japonicum under iron limiting conditions

Phytopathogenic bacteria Xanthomonas campestris pv. campestris (Xcc) causes black rot and other plant diseases. Xcc senses diffusible signal factor (DSF) as a quorum-sensing (QS) signal that mediates mainly iron uptake and virulence. RpfB deactivates DSF in this DSF–QS circuit. We examined differential gene expression profiles of Bradyrhizobium japonicum under low versus high iron conditions and found that fadD and irr were upregulated under low iron (log2 fold change 0.825 and 1.716, respectively). In addition to having similar protein folding patterns and functional domain similarities, FadD shared 58% sequence similarity with RpfB of Xcc. The RpfB–DSF and FadD–DSF complexes had SWISSDock molecular docking scores of − 8.88 kcal/mol and − 9.85 kcal/mol, respectively, and the 100 ns molecular dynamics simulation results were in accord with the docking results. However, significant differences were found between the binding energies of FadD–DSF and RpfB–DSF, indicating possible FadD-dependent DSF turnover. The protein–protein interaction network showed that FadD connected indirectly with ABC transporter permease (ABCtp), which was also upregulated (log2 fold change 5.485). We speculate that the low iron condition may be a mimetic environmental stimulus for fadD upregulation in B. japonicum to deactivate DSF, inhibit iron uptake and virulence of DSF-producing neighbors. This finding provides a new option of using B. japonicum or a genetically improved B. japonicum as a potential biocontrol agent against Xcc, with the added benefit of plant growth-promoting properties.

The small RNA PrrH of Pseudomonas aeruginosa regulates hemolysis and oxidative resistance in bloodstream infection

Small regulatory RNAs (sRNAs) regulate multiple physiological functions in bacteria, and sRNA PrrH can regulate iron homeostasis and virulence. However, the function of PrrH in Pseudomonas aeruginosa (P. aeruginosa) bloodstream infection (BSI) is largely unknown. The aim of this study was to investigate the role of PrrH in P. aeruginosa BSI model. First, P. aeruginosa PAO1 was co-cultured with peripheral blood cells for 6 h. qRT-PCR results showed a transient up-regulation of PrrH expression at 1 h. Simultaneously, the expression of iron uptake genes fpvA, pvdS and phuR were upregulated. In addition, the use of iron chelator 2,2′-dipyridyl to create low-iron conditions caused up-regulation of PrrH expression, a result similar to the BSI model. Furthermore, the addition of FeCl3 was found to decrease PrrH expression. These results support the hypothesis that the expression of PrrH is regulated by iron in BSI model. Then, to clarify the effect of PrrH on major cells in the blood, we used PrrH mutant, overexpressing and wild-type strains to act separately on erythrocytes and neutrophils. On one hand, the hemolysis assay revealed that PrrH contributes to the hemolytic activity of PAO1, and its effect was dependent on the T3SS system master regulator gene exsA, yet had no association with the hemolytic phospholipase C (plcH), pldA, and lasB elastase genes. On the other hand, PrrH mutant enhanced the oxidative resistance of PAO1 in the neutrophils co-culture assay, H2O2-treated growth curve and conventional plate spotting assays. Furthermore, the katA was predicted to be a target gene of PrrH by bioinformatics software, and then verified by qRT-PCR and GFP reporter system. In summary, dynamic changes in the expression of prrH are iron-regulated during PAO1 bloodstream infection. In addition, PrrH promotes the hemolytic activity of P. aeruginosa in an exsA-dependent manner and negatively regulates katA to reduce the oxidative tolerance of P. aeruginosa.

Overexpression of quorum sensing genes in Acidithiobacillus ferrooxidans enhances cell attachment and covellite bioleaching

Cell adhesion is generally a prerequisite to the microbial bioleaching of sulfide minerals, and surface biofilm formation is modulated via quorum sensing (QS) communication. We explored the impact of the overexpression of endogenous QS machinery on the covellite bioleaching capabilities of Acidithiobacillus ferrooxidans, a representative acidophilic chemolithoautotrophic bacterium. Cells were engineered to overexpress the endogenous qs-I operon or just the afeI gene under control of the tac promoter. Both strains exhibited increased transcriptional gene expression of afeI and improved cell adhesion to covellite, including increased production of extracellular polymeric substances and increased biofilm formation. Under low iron conditions, the improved bioleaching of covellite was more evident when afeI was overexpressed alone as compared to the native operon. These observations demonstrate the potential for the genetic modulation of QS as a mechanism for increasing the bioleaching efficiency of covellite, and potentially other copper sulfide minerals.

Changes of biomarkers for erythropoiesis, iron metabolism, and FGF23 by supplementation with roxadustat in patients on hemodialysis

This study aimed to confirm changes in biomarkers of erythropoiesis and iron metabolism and serum fibroblast growth factor 23 (FGF-23) during darbepoetin-α treatment and then switching to the hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat. A total of 28 patients on hemodialysis who received weekly doses of darbepoetin-α were switched to roxadustat. Biomarkers for erythropoiesis and iron metabolism and intact and C-terminal FGF-23 were measured in blood samples collected before the HD session on days − 7 (darbepoetin-α injection), − 4, and − 2, and days 0 (switch to roxadustat treatment, three times weekly), 3, 5, 7, 14, 21, and 28. Erythropoietin and erythroferrone levels were elevated on day − 4 by darbepoetin-α injection and decreased to baseline levels at day 0. Levels of erythropoietin were not significantly increased by roxadustat supplementation, but erythroferrone levels were continuously elevated, similar to darbepoetin-α treatment. Hepcidin-25 and total iron binding capacity were significantly decreased or increased in patients treated with roxadustat compared with darbepoetin-α. Changes of intact and C-terminal FGF-23 levels were parallel to changes of phosphate levels during roxadustat treatment. However, the actual and percentage changes of intact FGF-23 and C-terminal FGF-23 in patients with low ferritin levels were greater than those in patients with high ferritin levels. Roxadustat might stimulate erythropoiesis by increasing iron usage through hepcidin-25, which was suppressed by erythroferrone in the physiological erythropoietin condition. Changes of intact FGF-23 and C-terminal FGF-23 levels might be affected by roxadustat in patients on hemodialysis, especially those with a low-iron condition.

Liver sinusoidal endothelial cells induce BMP6 expression in response to non–transferrin-bound iron

AbstractHomeostatic adaptation to systemic iron overload involves transcriptional induction of bone morphogenetic protein 6 (BMP6) in liver sinusoidal endothelial cells (LSECs). BMP6 is then secreted to activate signaling of the iron hormone hepcidin (HAMP) in neighboring hepatocytes. To explore the mechanism of iron sensing by LSECs, we generated TfrcTek-Cre mice with endothelial cell–specific ablation of transferrin receptor 1 (Tfr1). We also used control Tfrcfl/fl mice to characterize the LSEC-specific molecular responses to iron using single-cell transcriptomics. TfrcTek-Cre animals tended to have modestly increased liver iron content (LIC) compared with Tfrcfl/fl controls but expressed physiological Bmp6 and Hamp messenger RNA (mRNA). Despite a transient inability to upregulate Bmp6, they eventually respond to iron challenges with Bmp6 and Hamp induction, yet occasionally to levels slightly lower relative to LIC. High dietary iron intake triggered the accumulation of serum nontransferrin bound iron (NTBI), which significantly correlated with liver Bmp6 and Hamp mRNA levels and elicited more profound alterations in the LSEC transcriptome than holo-transferrin injection. This culminated in the robust induction of Bmp6 and other nuclear factor erythroid 2–related factor 2 (Nrf2) target genes, as well as Myc target genes involved in ribosomal biogenesis and protein synthesis. LSECs and midzonal hepatocytes were the most responsive liver cells to iron challenges and exhibited the highest expression of Bmp6 and Hamp mRNAs, respectively. Our data suggest that during systemic iron overload, LSECs internalize NTBI, which promotes oxidative stress and thereby transcriptionally induces Bmp6 via Nrf2. Tfr1 appears to contribute to iron sensing by LSECs, mostly under low iron conditions.

Differential metal sensing and metal-dependent degradation of the broad spectrum root metal transporter IRT1.

Iron is an essential micronutrient for plant growth and development. Under low iron conditions, Arabidopsis plants take up soil iron using the root iron transporter IRT1. In addition to iron, IRT1 also transports others divalent metals, including cadmium, which consequently accumulates into plant tissues and enters the food chain. IRT1 expression was shown to be regulated at the transcriptional and post-translational levels by its essential metal substrates to maximize iron uptake while limiting the accumulation of zinc, manganese, or cobalt. Here, we characterized the regulation of IRT1 by cadmium. A short-term exposure to cadmium decreased the cell surface levels of IRT1 through endocytosis and degradation, but with a lower efficiency than observed for other IRT1 metal substrates. We demonstrated that IRT1 endocytosis in response to cadmium is mediated through the direct binding of cadmium to histidine residues within the regulatory loop of IRT1. However, we revealed that the affinity of the metal sensing motif is much lower for cadmium compared to other metal substrates of IRT1. Finally, we proved that cadmium-induced IRT1 degradation takes place through ubiquitin-mediated endocytosis driven by the UBC35/36 E2 ubiquitin-conjugating enzymes and the IDF1 E3 ubiquitin ligase. Altogether, this work sheds light on the mechanisms of cadmium-mediated downregulation of IRT1 and provides an additional molecular basis for cadmium accumulation and toxicity in plants.

Roles of Ferredoxin-NADP+ Oxidoreductase and Flavodoxin in NAD(P)H-Dependent Electron Transfer Systems.

Distinct isoforms of FAD-containing ferredoxin-NADP+ oxidoreductase (FNR) and ferredoxin (Fd) are involved in photosynthetic and non-photosynthetic electron transfer systems. The FNR (FAD)-Fd [2Fe-2S] redox pair complex switches between one- and two-electron transfer reactions in steps involving FAD semiquinone intermediates. In cyanobacteria and some algae, one-electron carrier Fd serves as a substitute for low-potential FMN-containing flavodoxin (Fld) during growth under low-iron conditions. This complex evolves into the covalent FNR (FAD)-Fld (FMN) pair, which participates in a wide variety of NAD(P)H-dependent metabolic pathways as an electron donor, including bacterial sulfite reductase, cytochrome P450 BM3, plant or mammalian cytochrome P450 reductase and nitric oxide synthase isoforms. These electron transfer systems share the conserved Ser-Glu/Asp pair in the active site of the FAD module. In addition to physiological electron acceptors, the NAD(P)H-dependent diflavin reductase family catalyzes a one-electron reduction of artificial electron acceptors such as quinone-containing anticancer drugs. Conversely, NAD(P)H: quinone oxidoreductase (NQO1), which shares a Fld-like active site, functions as a typical two-electron transfer antioxidant enzyme, and the NQO1 and UDP-glucuronosyltransfease/sulfotransferase pairs function as an antioxidant detoxification system. In this review, the roles of the plant FNR-Fd and FNR-Fld complex pairs were compared to those of the diflavin reductase (FAD-FMN) family. In the final section, evolutionary aspects of NAD(P)H-dependent multi-domain electron transfer systems are discussed.

Iron and light limitation of phytoplankton growth off East Antarctica

Phytoplankton production controls the transfer of carbon dioxide from the atmosphere into the ocean through the biological carbon pump, facilitating the sequestration of carbon dioxide and thus, contributing to the regulation of global climate. In the Southern Ocean, a high-nutrient low-chlorophyll region, productivity is limited by micronutrients including iron. The limiting role of light is also important, especially in the context of Southern Ocean phytoplankton adaptation to the low-iron conditions. To investigate the relative dominance of iron and light limitation on phytoplankton growth, we conducted a series of incubation experiments during a 2019 summer research voyage off East Antarctica. Our results show that, while light was the primary limiting factor of phytoplankton growth, iron addition became favourable for growth under high light conditions. This supports similar findings from laboratory (Strzepek et al., 2019) and field experiments in the Ross Sea (Alderkamp et al., 2019) and provides a new insight from a different region of the Southern Ocean, at a later stage of the austral summer. Similar results have previously been documented in larger diatoms, and here we provide evidence that it is also true for smaller cells. Our findings add to the body of knowledge regarding iron and light limitation in the Southern Ocean, in a poorly sampled area, leading to a better understanding of how primary production will change in a future warming ocean.

Contrasting Roles of DOP as a Source of Phosphorus and Energy for Marine Diazotrophs

The oceanic dissolved organic phosphorus (DOP) pool is mainly composed of P-esters and, to a lesser extent, equally abundant phosphonate and P-anhydride molecules. In phosphate-limited ocean regions, diazotrophs are thought to rely on DOP compounds as an alternative source of phosphorus (P). While both P-esters and phosphonates effectively promote dinitrogen (N2) fixation, the role of P-anhydrides for diazotrophs is unknown. Here we explore the effect of P-anhydrides on N2 fixation at two stations with contrasting biogeochemical conditions: one located in the Tonga trench volcanic arc region (“volcano,” with low phosphate and high iron concentrations), and the other in the South Pacific Gyre (“gyre,” with moderate phosphate and low iron). We incubated surface seawater with AMP (P-ester), ATP (P-ester and P-anhydride), or 3polyP (P-anhydride) and determined cell-specific N2 fixation rates, nifH gene abundance, and transcription in Crocosphaera and Trichodesmium. Trichodesmium did not respond to any DOP compounds added, suggesting that they were not P-limited at the volcano station and were outcompeted by the low iron conditions at the gyre station. Conversely, Crocosphaera were numerous at both stations and their specific N2 fixation rates were stimulated by AMP at the volcano station and slightly by 3polyP at both stations. Heterotrophic bacteria responded to ATP and 3polyP additions similarly at both stations, despite the contrasting phosphate and iron availability. The use of 3polyP by Crocosphaera and heterotrophic bacteria at both low and moderate phosphate concentrations suggests that this compound, in addition to being a source of P, can be used to acquire energy for which both groups compete. P-anhydrides may thus leverage energy restrictions to diazotrophs in the future stratified and nutrient-impoverished ocean.

Overexpression of MxbHLH18 Increased Iron and High Salinity Stress Tolerance in Arabidopsis thaliana.

In the life cycle of apple, it will suffer a variety of abiotic stresses, such as iron stress and salt stress. bHLH transcription factors (TFs) play an indispensable role in the response of plants to stress. In this study, a new bHLH gene named MxbHLH18 was separated from Malus xiaojinensis. According to the results of subcellular localization, MxbHLH18 was localized in the nucleus. Salt stress and iron stress affected the expression of MxbHLH18 in Malus xiaojinensis seedlings to a large extent. Due to the introduction of MxbHLH18, the resistance of Arabidopsis thaliana to salt, high iron and low iron was significantly enhanced. Under the environmental conditions of high iron and low iron, the overexpression of MxbHLH18 increased many physiological indexes of transgenic Arabidopsis compared to wild type (WT), such as root length, fresh weight and iron content. The high level expression of MxbHLH18 in transformed Arabidopsis thaliana can not only increased the content of chlorophyll and proline, as well as increasing the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); it also reduced the content of malondialdehyde (MDA), which was more obvious under high salt conditions. In addition, the relative conductivity, H2O2 content and O2− content in transgenic Arabidopsis decreased under salt stress. Meanwhile, MxbHLH18 can also regulate the expression of downstream genes associated with salt stress (AtCBF1/2/3, AtKIN1 and AtCOR15a/b) and iron stress (AtIRT1, AtFRO2, AtNAS2, ATACT2, AtZIF1 and AtOPT3). Therefore, MxbHLH18 can actively promote the adaptability of plants to the growth environment of salt and low and/or iron.

Sib1, Sib2, and Sib3 proteins are required for ferrichrome-mediated cross-feeding interaction between Schizosaccharomyces pombe and Saccharomyces cerevisiae.

Although Saccharomyces cerevisiae is unable to produce siderophores, this fungal organism can assimilate iron bound to the hydroxamate-type siderophore ferrichrome (Fc) produced and secreted by other microbes. Fc can enter S. cerevisiae cells via Arn1. Unlike S. cerevisiae, Schizosaccharomyces pombe synthesizes and secretes Fc. The sib1+ and sib2+ genes encode, respectively, a Fc synthetase and an ornithine-N5-oxygenase, which are required for Fc production. When both genes were expressed in S. pombe, cross-feeding experiments revealed that S. cerevisiae fet3Δ arn1-4Δ cells expressing Arn1 could grow in the vicinity of S. pombe under low-iron conditions. In contrast, deletion of sib1+ and sib2+ produced a defect in the ability of S. pombe to keep S. cerevisiae cells alive when Fc is used as the sole source of iron. Further analysis identified a gene designated sib3+ that encodes an N5-transacetylase required for Fc production in S. pombe. The sib3Δ mutant strain exhibited a severe growth defect in iron-poor media, and it was unable to promote Fc-dependent growth of S. cerevisiae cells. Microscopic analyses of S. pombe cells expressing a functional Sib3-GFP protein revealed that Sib3 was localized throughout the cells, with a proportion of Sib3 being colocalized with Sib1 and Sib2 within the cytosol. Collectively, these results describe the first example of a one-way cross-feeding interaction, with S. pombe providing Fc that enables S. cerevisiae to grow when Fc is used as the sole source of iron.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A.

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore–iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species.

Comparative Thermophysiology of Marine Synechococcus CRD1 Strains Isolated From Different Thermal Niches in Iron-Depleted Areas.

Marine Synechococcus cyanobacteria are ubiquitous in the ocean, a feature likely related to their extensive genetic diversity. Amongst the major lineages, clades I and IV preferentially thrive in temperate and cold, nutrient-rich waters, whilst clades II and III prefer warm, nitrogen or phosphorus-depleted waters. The existence of such cold (I/IV) and warm (II/III) thermotypes is corroborated by physiological characterization of representative strains. A fifth clade, CRD1, was recently shown to dominate the Synechococcus community in iron-depleted areas of the world ocean and to encompass three distinct ecologically significant taxonomic units (ESTUs CRD1A-C) occupying different thermal niches, suggesting that distinct thermotypes could also occur within this clade. Here, using comparative thermophysiology of strains representative of these three CRD1 ESTUs we show that the CRD1A strain MITS9220 is a warm thermotype, the CRD1B strain BIOS-U3-1 a cold temperate thermotype, and the CRD1C strain BIOS-E4-1 a warm temperate stenotherm. Curiously, the CRD1B thermotype lacks traits and/or genomic features typical of cold thermotypes. In contrast, we found specific physiological traits of the CRD1 strains compared to their clade I, II, III, and IV counterparts, including a lower growth rate and photosystem II maximal quantum yield at most temperatures and a higher turnover rate of the D1 protein. Together, our data suggests that the CRD1 clade prioritizes adaptation to low-iron conditions over temperature adaptation, even though the occurrence of several CRD1 thermotypes likely explains why the CRD1 clade as a whole occupies most iron-limited waters.

Impact of Lhcx2 on Acclimation to Low Iron Conditions in the Diatom Phaeodactylum tricornutum.

Iron is a cofactor of photosystems and electron carriers in the photosynthetic electron transport chain. Low concentrations of dissolved iron are, therefore, the predominant factor that limits the growth of phototrophs in large parts of the open sea like the Southern Ocean and the North Pacific, resulting in “high nutrient–low chlorophyll” (HNLC) areas. Diatoms are among the most abundant microalgae in HNLC zones. Besides efficient iron uptake mechanisms, efficient photoprotection might be one of the key traits enabling them to outcompete other algae in HNLC regions. In diatoms, Lhcx proteins play a crucial role in one of the main photoprotective mechanisms, the energy-dependent fluorescence quenching (qE). The expression of Lhcx proteins is strongly influenced by various environmental triggers. We show that Lhcx2 responds specifically and in a very sensitive manner to iron limitation in the diatom Phaeodactylum tricornutum on the same timescale as the known iron-regulated genes ISIP1 and CCHH11. By comparing Lhcx2 knockout lines with wild type cells, we reveal that a strongly increased qE under iron limitation is based on the upregulation of Lhcx2. Other observed iron acclimation phenotypes in P. tricornutum include a massively reduced chlorophyll a content/cell, a changed ratio of light harvesting and photoprotective pigments per chlorophyll a, a decreased amount of photosystem II and photosystem I cores, an increased functional photosystem II absorption cross section, and decoupled antenna complexes. H2O2 formation at photosystem I induced by high light is lowered in iron-limited cells, while the amount of total reactive oxygen species is rather increased. Our data indicate a possible reduction in singlet oxygen by Lhcx2-based qE, while the other iron acclimation phenotype parameters monitored are not affected by the amount of Lhcx2 and qE.

Aspergillus Utilizes Extracellular Heme as an Iron Source During Invasive Pneumonia, Driving Infection Severity.

Depriving microbes of iron is critical to host defense. Hemeproteins, the largest source of iron within vertebrates, are abundant in infected tissues in aspergillosis due to hemorrhage, but Aspergillus species have been thought to lack heme import mechanisms. We hypothesized that heme provides iron to Aspergillus during invasive pneumonia, thereby worsening the outcomes of the infection. We assessed the effect of heme on fungal phenotype in various in vitro conditions and in a neutropenic mouse model of invasive pulmonary aspergillosis. In mice with neutropenic invasive aspergillosis, we found a progressive and compartmentalized increase in lung heme iron. Fungal cells cultured under low iron conditions took up heme, resulting in increased fungal iron content, resolution of iron starvation, increased conidiation, and enhanced resistance to oxidative stress. Intrapulmonary administration of heme to mice with neutropenic invasive aspergillosis resulted in markedly increased lung fungal burden, lung injury, and mortality, whereas administration of heme analogs or heme with killed Aspergillus did not. Finally, infection caused by fungal germlings cultured in the presence of heme resulted in a more severe infection. Invasive aspergillosis induces local hemolysis in infected tissues, thereby supplying heme iron to the fungus, leading to lethal infection.

Changes in mRNA stability play an important role in the adaptation of yeast cells to iron deprivation

Eukaryotic cells rely on iron as an indispensable cofactor for multiple biological functions including mitochondrial respiration and protein synthesis. The budding yeast Saccharomyces cerevisiae utilizes both transcriptional and posttranscriptional mechanisms to couple mRNA levels to the requirements of iron deprivation. Thus, in response to iron deficiency, transcription factors Aft1 and Aft2 activate the expression of genes implicated in iron acquisition and mobilization, whereas two mRNA-binding proteins, Cth1 and Cth2, posttranscriptionally control iron metabolism. By using a genome-wide approach, we describe here a global stabilization of mRNAs, including transcripts encoding ribosomal proteins (RPs), when iron bioavailability diminishes. mRNA decay assays indicate that the mRNA-binding protein Pub1 contributes to RP transcript stabilization during adaptation to iron limitation. In fact, Pub1 becomes critical for growth and translational repression in low-iron conditions. Remarkably, we observe that pub1Δ cells also exhibit an increase in the transcription of RP genes that evidences the crosstalk between transcription and degradation mechanisms to maintain the appropriate mRNA balance under iron deficiency conditions.

Genome-wide analysis of the NAAT, DMAS, TOM, and ENA gene families in maize suggests their roles in mediating iron homeostasis

BackgroundNicotianamine (NA), 2′-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2′-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.).ResultsHere, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles.ConclusionsOur results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

Iron-Utilization System in Vibrio vulnificus M2799.

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

NCOA4 Links Iron Bioavailability to DNA Metabolism

Iron (Fe) is a key element of many proteins involved in primary cellular processes such as energy production, DNA synthesis and repair (Fuss et al. 2015). Unbalanced iron levels are poorly tolerated thus a tight control over iron trafficking and storage is necessary in order to ensure cell growth and genome stability (Zhang et al. 2014). Indeed, under low iron condition, cells arrest in G1 phase and restrain DNA synthesis till normal iron levels are restored (Lui et al 2015). The Nuclear Receptor CoActivator 4 (NCOA4) has been recently involved in cellular and systemic iron homeostasis as a selective cargo receptor of ferritin, the iron storage macromolecule (Mancias et al. 2014). Under iron deficient condition, NCOA4 promotes the autophagic degradation of ferritin (ferritinophagy) restoring appropriate iron levels. Conversely, excess iron induces the proteosomal degradation of NCOA4 by HERC2 ubiquitin ligase allowing iron storage into ferritin (Mancias et al. 2015). Lack of ferritinophagy in NCOA4-deficient mice leads to tissue iron overload as well as anaemia because of inability to mobilize iron from deposits (Bellelli et al. 2016). In addition to this iron-related function, we uncovered that nuclear NCOA4 binds to MCM7, a member of the core engine of the replicative DNA Helicase and blocks DNA replication origin activation (Bellelli et al. 2014). Since low iron levels impair cell proliferation and block DNA replication, we hypothesized that cytosolic and nuclear NCOA4 functions (eg ferritinophagy and DNA replication origin control, respectively) were linked in order to couple iron availability to DNA metabolism. We first evaluated the effects of iron chelation on NCOA4 levels and found that Deferoxamine (DFO) treatment of HeLa cell lines increased both cytosolic and nuclear NCOA4 fraction. In particular, under low iron conditions, we found that NCOA4 not only induced ferritin degradation but also increased its interaction with MCM7 at canonical DNA origins in order to block DNA replication. Consistently, upon iron chelation, NCOA4-/- HeLa cells failed to block DNA synthesis, and underwent replication stress with a robust activation of the DNA damage response (DDR) and a permanent cell cycle arrest, as confirmed by low colony-forming ability of NCOA4 deficient cells. Then, we asked whether the dual function of NCOA4 was relevant during DNA Damage, when replication is blocked and many iron-requiring enzymes work to repair DNA. Interestingly, although DNA damage did not substantially reduce labile iron pool, as suggested by no change of IRP2 stability, treatment with genotoxic agents, such as Hydroxyurea (HU), decreased NCOA4 binding to HERC2. The HU-dependent NCOA4 stabilization promoted block of DNA replication origin activation and, as iron depletion, increased ferritin turnover. Collectively, our data identify NCOA4 functions as two faces of a common response, which aims at coordinating cellular iron availability to DNA metabolism. In this light, NCOA4 by controlling activation of DNA origins reduces replication stress and by regulating cellular iron levels sustains DNA iron-requiring enzymatic activities in order to maintain genome stability. DisclosuresCamaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.