Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA

Recent studies on metalloregulatory proteins suggest that coordination number/geometry and metal ion availability in a host cytosol are key determinants for biological specificity. Here, we investigate the contribution that individual metal ligands of the alpha5 sensing site of Staphylococcus aureus CzrA (Asp84, His86, His97', and His100') make to in vitro metal ion binding affinity, coordination geometry, and allosteric negative regulation of DNA operator/promoter region binding. All ligand substitution mutants exhibit significantly reduced metal ion binding affinity (K(Me)) by > or =10(3) M(-1). Substitutions of Asp84 and His97 give rise to non-native coordination geometries upon metal binding and are non-functional in allosteric coupling of metal and DNA binding (DeltaG(coupling) approximately 0 kcal mol(-1)). In contrast, His86 and His100 could be readily substituted with potentially liganding (Asp, Glu) and poorly liganding (Asn, Gln) residues with significant native-like tetrahedral metal coordination geometry retained in these mutants, leading to strong functional coupling (DeltaG(coupling) > or = +3.0 kcal mol(-1)). 1H-(15)N heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4 degrees structural switching on binding Zn(II), while D84N, H97N and H97D CzrAs do not. Thus, only those variant CzrAs that retain some tetrahedral coordination geometry characteristic of wild-type CzrA upon metal binding are capable of driving 4 degrees structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of K(Me).     

The Role of Copper and Zinc Toxicity in Innate Immune Defense against Bacterial Pathogens

Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function.     

Ceramide Synthase Schlank Is a Transcriptional Regulator Adapting Gene Expression to Energy Requirements

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Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.     

Structural Analysis of Metal Sites in Proteins: Non-heme Iron Sites as a Case Study

In metalloproteins, the protein environment modulates metal properties to achieve the required goal, which can be protein stabilization or function. The analysis of metal sites at the atomic level of detail provided by protein structures can thus be of benefit in functional and evolutionary studies of proteins. In this work, we propose a structural bioinformatics approach to the study of metalloproteins based on structural templates of metal sites that include the PDB coordinates of protein residues forming the first and the second coordination sphere of the metal. We have applied this approach to non-heme iron sites, which have been analyzed at various levels. Templates of sites located in different protein domains have been compared, showing that similar sites can be found in unrelated proteins as the result of convergent evolution. Templates of sites located in proteins of a large superfamily have been compared, showing possible mechanisms of divergent evolution of proteins to achieve different functions. Furthermore, template comparisons have been used to predict the function of uncharacterized proteins, showing that similarity searches focused on metal sites can be advantageously combined with typical whole-domain comparisons. Structural templates of metal sites, finally, may constitute the basis for a systematic classification of metalloproteins in databases.     

Strength of Cu-efflux response in Escherichia coli coordinates metal resistance in Caenorhabditis elegans and contributes to the severity of environmental toxicity

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host–microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C. elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host’s response to chronic Cu stress.     

利用黑腹果蝇（Drosophila melanogaster）研究微量金属元素代谢

微量金属参与了生物体许多化学反应过程,同时也可作为蛋白质的辅基或辅因子起作用,对机体生长发育以及正常生物功能的维持具有重要作用;微量金属元素的代谢失衡与生物体许多疾病密切相关,如威尔森氏病、门克斯病、铁色素沉积、肠变性皮炎以及一些神经退行性疾病。黑腹果蝇（Drosophila melanogaster）是遗传背景清楚、生活周期短、操作方便的模式生物,利用果蝇研究金属离子代谢以及金属离子代谢与疾病的联系具有独特的优势,近年来,随着果蝇基因组测序的完成以及许多转基因果蝇株的建立,果蝇也越来越多的用于金属离子代谢的研究。介绍了近年来果蝇在金属离子代谢研究领域的进展,以及其与神经退行性疾病关系研究上的一些应用。     

Integrated tolerance mechanisms: constitutive and adaptive plant responses to elevated metal concentrations in the environment

Isolation and study of metal tolerant and hypersensitive strains of higher plant (and yeast) species has greatly increased our knowledge of the individual pathways that are involved in tolerance. Plants have both constitutive (present in most phenotypes) and adaptive (present only in tolerant phenotypes) mechanisms for coping with elevated metal concentrations. Where studies on the mechanisms of tolerance fall down is in their failure to integrate tolerance mechanisms within cell or whole-plant function by not relating adaptive mechanisms to constitutive mechanisms. This failure often distorts the relative importance of a proposed tolerance mechanism, and indeed has confused the search for adaptive mechanisms. The fundamental goal of both constitutive and adaptive mechanisms is to limit the perturbation of cell homeostasis after exposure to metals so that normal or near-normal physiological function may take place. Consideration of the response to metals at a cellular rather than a biochemical level will lead to a greater understanding of mechanisms to withstand elevated levels of metals in both contaminated and uncontaminated environments. Recent advances in the study of Al, As, Cd, and Cu tolerance and hypersensitivity are reported with respect to the cellular response to toxic metals. The role of genetics in unravelling tolerance mechanisms is also considered.     

Fe and Cu stable isotopes in archeological human bones and their relationship to sex

Accurate sex assignment of ancient human remains usually relies on the availability of coxal bones or well-preserved DNA. Iron (Fe) and copper (Cu) stable isotope compositions ((56)Fe/(54)Fe and (65)Cu/(63)Cu, respectively) were recently measured in modern human blood, and an unexpected result was the discovery of a (56)Fe-depletion and a (65)Cu-enrichment in men's blood compared to women's blood. Bones, being pervasively irrigated by blood, are expected to retain the (56)Fe/(54)Fe and (65)Cu/(63)Cu signature of blood, which in turn is useful for determining the sex of ancient bones. Here, we report the (56)Fe/(54)Fe, (65)Cu/(63)Cu, and (66)Zn/(64)Zn ratios from a suite of well-preserved phalanxes (n = 43) belonging to individuals buried in the 17th and 18th centuries at the necropolis of Saint-Laurent de Grenoble, France, and for which the sex was independently estimated from pelvic bone morphology. The metals were purified from the bone matrix by liquid chromatography on ion exchange resin and the isotope compositions were measured by multiple-collector inductively coupled plasma mass spectrometry. The results show that, as expected from literature data on blood, male bone iron is depleted in (56)Fe and enriched in (65)Cu relative to female. No sex difference is found in the (66)Zn/(64)Zn ratios of bone. The concentration and isotopic data show no evidence of soil contamination. Four samples of five (77%) can be assigned their correct sex, a result comparable to sex assignment using Fe and Cu isotopes in blood (81%). Isotopic analysis of metals may therefore represent a valid method of sex assignment applicable to incomplete human remains.     

Siderophores,NTA, and Citrate: Potential Soil Amendments to Enhance Heavy Metal Mobility in Phytoremediation

Phytoremediation of heavy metal-contaminated sites is often limited by the low bioavailability of the contaminants. Complexing agents can help to improve this technique by enhancing heavy metal solubility. We investigated the effect of three organic chelating agents, that is, the siderophore desferrioxamine B (DFOB), nitrilotriacetate (NTA), and citrate on binding of Cu, Zn, and Cd by either Namontmorillonite, kaolinite, or goethite. The different effects of the complexing agents on metal sorption can mainly be explained by the differences in stability constants and surface charge characteristics. In the presence of clay minerals, NTA was the most efficient ligand with respect to mobilization of heavy metals. In goethite suspensions, the effect of DFOB was more pronounced. In all systems, Cu proved to be the most affected element by the presence of the ligands. In batch experiments with heavy metal-contaminated soils from field sites, NTA was the most efficient metal mobilizer.     

Assessment of Metal Availability in Soils from a Pb-Zn Mine Site of South-Central Spain

Heavy metal pollution of the soils around an abandoned Pb-Zn mine site located in the Alcudia Valley (South Central Spain) have been characterized by analysis of extractable and total metal concentrations in 60 samples of arable, pasture, and mine lands. The samples showed a broad range of size-particle distribution, cation exchange capacity, and pH values as well as high levels of total metal concentrations (up to 98510 mg kg^?1 of Pb, up to 20912 mg kg^?1 of Zn, and up to 61 mg kg^?1 of Cd). In order to assess the potential availability of metals the metal partitioning in two different soil size fractions (<2 mm and <63 μm) was determined using EDTA and CaCl₂ as sequestering reagents. The average contents of Pb, Zn, and Cd in the <63 μm particle size fraction for both extractions were higher than those of the <2 mm fraction due to the high metal adsorption capacity of the fine soil particles. Concentrations of heavy metals extracted by CaCl₂ were up to three orders of magnitude lower than those extracted by EDTA, because CaCl₂ only extracts the easily mobile fraction. Metal concentrations extracted by both procedures in the two granulometric fractions increased with total metal concentrations, thus increasing the potential environmental risk associated to heavy metal pollution.     

Biogeochemistry and effects of copper,manganese and zinc added to enclosures in Island Billabong,Magela Creek,northern Australia

Three large plastic enclosures (5 m diam, volume 40 m³) were used to study the effects of copper, manganese and zinc, on the phytoplankton community in Island billabong, a floodplain billabong (waterhole) situated in the Magela Creek in tropical northern Australia. Copper was added to one enclosure, and manganese and zinc to another, to give initial concentrations around ten times the normal wet season values. The enclosures and the billabong were monitored over a ten week period towards the end of the dry season, with the enclosures allowed to stabilise for four weeks before the metals were added.The control enclosure adequately simulated the temperature and pH changes in the billabong. The trends in conductivity, dissolved oxygen and major ion concentrations were similar in the enclosure and the billabong, with the minor differences observed attributed to either epiphytic growth on the enclosure walls (influenced dissolved oxygen, pH and bicarbonate concentration) or ingress of sulphate-rich groundwater into the billabong (influenced sulphate concentration and conductivity). Major differences in both the composition of species and the size of the phytoplankton populations were observed between the three enclosures and between the control enclosure and the billabong. This variability reflects the great natural variability in the phytoplankton communities in tropical lentic systems, and means that enclosures are unlikely to adequately simulate the biological communities in the billabongs.The control enclosure appeared to simulate quite well the longer term changes in total concentration and speciation of the three metals (copper, manganese & zinc) in the billabong. The mean concentrations of copper and zinc were similar in the two systems, although the mean concentration of manganese in the billabong was almost double that in the enclosure, possibly due to ingress of manganese-enriched groundwater. Particulate forms dominated the speciation of copper and manganese. There was considerable short term variation in both total metal concentration and speciation in both the enclosure and the billabong. This variability appears to be a feature of these small tropical waterbodies.The added heavy metals were found to have minimal detrimental effect on the phytoplankton community in each metal-loaded enclosure. The high natural variability in the phytoplankton community in these tropical systems will make it difficult to separate natural changes from those caused by low level contamination from mining operations should this occur.All three metals were rapidly removed from the water column, so that by the end of the six week period, only ca. 5% of each added metal remained in the water column. Association with the particulate matter (phytoplankton, abiotic particulate matter and MnO_x in enclosure 2) followed by sedimentation was the major removal pathway. Epiphytes growing on the enclosure walls appeared to have a minor influence (<10% of the total amount of metal added) on the removal of the added metals. For copper, uptake by phytoplankton followed by sedimentation was the major (65%) removal process. Manganese and zinc, added together, were found to influence each other. The major manganese removal process (60%) was rapid (ca. 3 days) involving bacterial oxidation and sedimentation of the MnO_x formed. This material appeared to have little influence on the behaviour of zinc, possibly because other particulate matter competed more effectively for the zinc. A further 30% of the added manganese was removed via initial adsorption to other particulate matter, possibly phytoplankton. Approximately one third of this adsorbed manganese (10% of the total added) appeared to undergo delayed oxidation some 8 days after the initial additions, and the heavier particles settled out more rapidly. This path was responsible for removing the major amount (ca. 60%) of the added zinc. We hypothesis that the sorbed zinc inhibited the bacterial oxidation of the manganese. A further 25% of the zinc was removed in association with a burst of phytoplankton activity. The occurrence of bursts in the phytoplankton activity, when populations can increase very substantially and then decrease again, all within the space of a day, appears to be an important mechanism for removing copper and zinc from the water column in these tropical water bodies     

Toward a property/function relationship for metallothioneins: histidine coordination and unusual cluster composition in a zinc-metallothionein from plants

Early cysteine labeled (E(C)) proteins are plant metallothioneins, which were first identified in wheat embryos and are thought to be seed-specific. An exhaustive analysis of expressed sequence tag (EST) entries reveals that homologs are expressed in embryos of both classes of flowering plants (monocotyledons and dicotyledons), but also occur in conifers (gymnosperms) and seed-free spike moss (lycophyta). Mass spectrometric and elemental analysis results indicate that, contrary to the widely propagated number of five, E(C) binds predominantly six zinc ions in at least two zinc-thiolate clusters. 1H and 111Cd NMR experiments suggest that, in contrast to the majority of previously characterized metallothioneins, two conserved histidine residues participate in metal binding. The collected data is consistent with the presence of clusters unprecedented in metallothioneins so far. This novel cluster composition is accompanied by metal-binding properties that are substantially different from other metallothioneins; thus wheat E(C) binds zinc less strongly than either mammalian or cyanobacterial MTs. The unique biochemical properties of wheat E(C) render it ideally suited for a role in zinc donation to nascent proteins during seed development, a role that has been suggested based on the fact that E(C) is induced by the plant hormone abscisic acid, but not by heavy metals. Our results provide a step further toward developing a property/function relationship for metallothioneins.     

OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds

Plants and seeds are the main dietary sources of zinc, iron, manganese, and copper, but are also the main entry point for toxic elements such as cadmium into the food chain. We report here that an Arabidopsis oligopeptide transporter mutant, opt3-2, over-accumulates cadmium （Cd） in seeds and roots but, unexpectedly, under-accumulates Cd in leaves. The cadmium distribution in opt3-2 differs from iron, zinc, and manganese, suggesting a metal-specific mechanism for metal partitioning within the plant. The opt3-2 mutant constitutively up-regulates the Fe/Zn/Cd transporter IRT1 and FRO2 in roots, indicative of an iron-deficiency response. No genetic mutants that impair the shoot-to-root signaling of iron status in leaves have been identified. Interestingly, shoot-specific expression of OPT3 rescues the Cd sensitivity and complements the aberrant expression of IRT1 in opt3-2 roots, suggesting that OPT3 is required to relay the iron status from leaves to roots. OPT3 expression was found in the vasculature with preferential expression in the phloem at the plasma membrane. Using radioisotope experiments, we found that mobilization of Fe from leaves is severely affected in opt3-2, suggesting that Fe mobilization out of leaves is required for proper trace-metal homeostasis. When expressed in yeast, OPT3 does not localize to the plasma membrane, precluding the identification of the OPT3 substrate. Our in planta results show that OPT3 is important for leaf phloem-loading of iron and plays a key role regulating Fe, Zn, and Cd distribution within the plant. Furthermore, ferric chelate reductase activity analyses provide evidence that iron is not the sole signal transferred from leaves to roots in leaf iron status signaling.