Copper binding in IscA inhibits iron‐sulphur cluster assembly in Escherichia coli

Among the iron‐sulphur cluster assembly proteins encoded by gene cluster iscSUA‐hscBA‐fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron‐sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe‐4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron‐sulphur cluster biogenesis. Here we report that among the iron‐sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA‐mediated [4Fe‐4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe‐4S] clusters in dehydratases, but also block the [4Fe‐4S] cluster assembly in proteins by targeting IscA in cells.     

Glutathione plays an essential role in nitric oxide‐mediated iron‐deficiency signaling and iron‐deficiency tolerance in Arabidopsis

Iron (Fe) deficiency is a common agricultural problem that affects both the productivity and nutritional quality of plants. Thus, identifying the key factors involved in the tolerance of Fe deficiency is important. In the present study, the zir1 mutant, which is glutathione deficient, was found to be more sensitive to Fe deficiency than the wild type, and grew poorly in alkaline soil. Other glutathione‐deficient mutants also showed various degrees of sensitivity to Fe‐limited conditions. Interestingly, we found that the glutathione level was increased under Fe deficiency in the wild type. By contrast, blocking glutathione biosynthesis led to increased physiological sensitivity to Fe deficiency. On the other hand, overexpressing glutathione enhanced the tolerance to Fe deficiency. Under Fe‐limited conditions, glutathione‐deficient mutants, zir1, pad2 and cad2 accumulated lower levels of Fe than the wild type. The key genes involved in Fe uptake, including IRT1, FRO2 and FIT, are expressed at low levels in zir1; however, a split‐root experiment suggested that the systemic signals that govern the expression of Fe uptake‐related genes are still active in zir1. Furthermore, we found that zir1 had a lower accumulation of nitric oxide (NO) and NO reservoir S‐nitrosoglutathione (GSNO). Although NO is a signaling molecule involved in the induction of Fe uptake‐related genes during Fe deficiency, the NO‐mediated induction of Fe‐uptake genes is dependent on glutathione supply in the zir1 mutant. These results provide direct evidence that glutathione plays an essential role in Fe‐deficiency tolerance and NO‐mediated Fe‐deficiency signaling in Arabidopsis.     

Validation of reference genes for quantitative real‐time polymerase chain reaction in Drosophila melanogaster exposed to two chemicals

Drosophila melanogaster is attracted to chemicals produced by fermentation and it is abundantly found in rotten fruits. Considering its habitat, the fruit fly is reported to be tolerant to environmental chemicals. Quantitative real‐time polymerase chain reaction was employed to investigate the expression pattern and physiological function of genes putatively involved in chemical detoxification. In quantitative real‐time polymerase chain reaction assays, normalization of target gene expression with internal reference genes is required. These reference genes should be stably expressed during chemical exposure and in chemical‐free conditions. In this study, therefore, we used two programs (geNorm and BestKeeper) to evaluate the expression stability of five reference genes (nd, rpL18, ef1β, hsp22 and tbp) in female adult flies exposed to various concentrations of methanol and ethyl acetate. Four genes (nd, rpL18, ef1β and tbp) were found to be suitable for use as reference genes in methanol‐treated flies and three genes (ef1β, nd, tbp) were found to be suitable for use as reference genes in ethyl acetate‐treated flies. These results suggested that a combination of two genes among these stably expressed genes can be used for accurate normalization of target gene expression in quantitative real‐time polymerase chain reaction‐based determination of gene expression profiles in D. melanogaster treated with both chemicals.     

Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis

Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The Isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.     

Different physiological responses of cyanobacteria to ultraviolet‐B radiation under iron‐replete and iron‐deficient conditions: Implications for underestimating the negative effects of UV‐B radiation

Iron deficiency has been considered one of the main limiting factors of phytoplankton productivity in some aquatic systems including oceans and lakes. Concomitantly, solar ultraviolet‐B radiation has been shown to have both deleterious and positive impacts on phytoplankton productivity. However, how iron‐deficient cyanobacteria respond to UV‐B radiation has been largely overlooked in aquatic systems. In this study, physiological responses of four cyanobacterial strains (Microcystis and Synechococcus), which are widely distributed in freshwater or marine systems, were investigated under different UV‐B irradiances and iron conditions. The growth, photosynthetic pigment composition, photosynthetic activity, and nonphotochemical quenching of the different cyanobacterial strains were drastically altered by enhanced UV‐B radiation under iron‐deficient conditions, but were less affected under iron‐replete conditions. Intracellular reactive oxygen species (ROS) and iron content increased and decreased, respectively, with increased UV‐B radiation under iron‐deficient conditions for both Microcystis aeruginosa FACHB 912 and Synechococcus sp. WH8102. On the contrary, intracellular ROS and iron content of these two strains remained constant and increased, respectively, with increased UV‐B radiation under iron‐replete conditions. These results indicate that iron‐deficient cyanobacteria are more susceptible to enhanced UV‐B radiation. Therefore, UV‐B radiation probably plays an important role in influencing primary productivity in iron‐deficient aquatic systems, suggesting that its effects on the phytoplankton productivity may be underestimated in iron‐deficient regions around the world.     

Bacterial cyclic β‐(1,2)‐glucans sequester iron to protect against iron‐induced toxicity

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron‐restricted conditions while also limiting the potential for the production of iron‐induced reactive oxygen species (ROS). Here, we report that membrane‐derived oligosaccharide (mdo) glucan, an intrinsic component of Gram‐negative bacteria, sequesters the ferrous form of iron. Iron‐binding, uptake, and localization experiments indicated that both secreted and periplasmic β‐(1,2) ‐ glucans bind iron specifically and promote growth under iron‐restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β‐(1,2) ‐ glucan accumulate low amounts of intracellular iron under iron‐restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron‐replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram‐negative bacteria.     

A novel iron‐ and copper‐binding protein in the Lyme disease spirochaete

Iron and copper are transition metals that can be toxic to cells due to their abilities to react with peroxide to generate hydroxyl radical. Ferritins and metallothioneins are known to sequester intracellular iron and copper respectively. The Lyme disease pathogen Borrelia burgdorferi does not require iron, but its genome encodes a ferritin‐like Dps (D NA‐binding p rotein from s tarved bacteria) molecule, which has been shown to be important for the spirochaete's persistence in the tick and subsequent transmission to a new host. Here, we show that the c arboxyl‐terminal c ysteine‐r ich (CCR) domain of this protein functions as a copper‐binding metallothionein. This novel fusion between Dps and metallothionein is unique to and conserved in all Borrelia species. We term this molecule BicA for B orrelia i ron‐ and c opper‐binding protein A . An isogenic mutant lacking BicA had significantly reduced levels of iron and copper and was more sensitive to iron and copper toxicity than its parental strain. Supplementation of the medium with iron or copper rendered the spirochaete more susceptible to peroxide killing. These data suggest that an important function of BicA is to detoxify excess iron and copper the spirochaete may encounter during its natural life cycle through a tick vector and a vertebrate host.     

Molecular characterization,spatial‐temporal expression and magnetic response patterns of iron‐sulfur cluster assembly1 (IscA1) in the rice planthopper,Nilaparvata lugens

The mechanisms of magnetoreception have been proposed as the magnetitebased, the chemical radical-pair and biocompass model, in which magnetite particles, the cryptochrome (Cry) or iron-sulfur cluster assembly 1 (IscA1) may be involved. However, little is known about the association among the molecules. Here we investigated the molecular characterization and the mRNA expression of IscA1 in different developmental stages, tissues and magnetic fields in the migratory brown planthopper (BPH), Nilaparvata lugens. NlIscA1 contains an open reading frame of 390 bp, encoding amino acids of 129, with the predicted molecular weight of 14.0 kDa and the isoelectric point of 9.10. Well-conserved Fe-S cluster binding sites were observed in the predicted protein. Phylogenetic analysis demonstrated NlIscA1 to be clustered into the insect's IscA1. NlIscA1 showed up-regulated mRNA expression during the period of migration. The mRNA expression of NlIscA1 could be detected in all the three tissues of head, thorax and abdomen, with the highest expression level in the abdomen. For the macropterous migratory Nilaparvata lugens, mRNA expression of NlIscA1 and N. lugens cryptochromel (Nlcry1) were up-regulated under the magnetic fields of 5 Gauss and 10 Gauss in strength (vs. local geomagnetic field), while N. lugens cryptochrome 2 (Nlcry2) remained stable. For the brachyterous non-migratory Nilaparvata lugens, no significant changes were found in mRNA expression of NlIscA1, Nlcry1 and Nlcry2 among different magnetic fields. These findings preliminarily reveal that the expression of NlIscA1 and Nlcry1 exhibited coordinated responses to the magnetic field. It suggests some potential associations among the putative magneto-sensitive molecules of cryptochrome and iron-sulfur cluster assembly.     

TCA cycle activity in Staphylococcus aureus is essential for iron‐regulated synthesis of staphyloferrin A,but not staphyloferrin B: the benefit of a second citrate synthase

Staphylococcus aureus elaborates two citrate‐containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron‐restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron‐regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S. aureus in iron‐restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB‐deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron‐restricted media, and is significantly more impaired for virulence than either of the single‐deletion mutants. We propose that SB is the more important of the two siderophores for S. aureus insofar as it is synthesized, and supports iron‐restricted growth, without need of TCA cycle activity.     

Tim‐2 is the receptor for H‐ferritin on oligodendrocytes

Oligodendrocytes stain more strongly for iron than any other cell in the CNS, and they require iron for the production of myelin. For most cell types transferrin is the major iron delivery protein, yet neither transferrin receptor protein nor mRNA are detectable in mature oligodendrocytes. Thus an alternative iron delivery mechanism must exist. Given the significant long term consequences of developmental iron deficiency and the iron requirements for normal myelination, identification of the iron delivery mechanism for oligodendrocytes is important. Previously we have reported that oligodendrocytes bind H‐ferritin and that H‐ferritin binds to white matter tracts in vivo. Recently, T cell immunoglobulin and mucin domain‐containing protein‐2 (Tim‐2) was shown to bind and internalize H‐ferritin. In the present study we show that Tim‐2 is expressed on oligodendrocytes both in vivo and in vitro. Further, the onset of saturable H‐ferritin binding in CG4 oligodendrocyte cell line is accompanied by Tim‐2 expression. Application of a blocking antibody to the extracellular domain of Tim‐2 significantly reduces H‐ferritin binding to the differentiated CG4 cells and primary oligodendrocytes. Tim‐2 expression on CG4 cells is responsive to iron; decreasing with iron loading and increasing with iron chelation. Taken together, these data provide compelling evidence that Tim‐2 is the H‐ferritin receptor on oligodendrocytes suggesting it is the primary mechanism for iron acquisition by these cells.