Copper Availability Research Articles

The Meter Reader: Early magnetic studies in the Lake Superior region

In the mid-19th century, the quality of life in the United States was improving rapidly with widespread electrification of the nation and growth in the industrial revolution. These advances were dependent on the increasing availability of copper for instruments and electric wires and iron ore for the production of iron and steel. Accordingly, reconnaissance geologic investigations in the Lake Superior region of North America that discovered copper and iron-rich rocks in the 1840s rapidly attracted the attention of prospectors and mining companies. However, ore deposits were a challenge to find in this region because of superimposed geologic events and limited rock exposures due to the widespread cover of unconsolidated sediment deposited from Pleistocene glaciation, the abundant lakes, and, locally, the cover of Phanerozoic sedimentary rocks. Limitations to surface geologic mapping were evidenced by the few outcrops of iron formation in the iron ranges and the even fewer ore exposures. As a result, geologic mapping was restricted to locating regions for constructing test shafts and pits and drilling that would lead to identifying favorable mining sites.

Multifactor transcriptional control of alternative oxidase induction integrates diverse environmental inputs to enable fungal virulence

Metabolic flexibility enables fungi to invade challenging host environments. In Candida albicans, a common cause of life-threatening infections in humans, an important contributor to flexibility is alternative oxidase (Aox) activity. Dramatic induction of this activity occurs under respiratory-stress conditions, which impair the classical electron transport chain (ETC). Here, we show that deletion of the inducible AOX2 gene cripples C. albicans virulence in mice by increasing immune recognition. To investigate further, we examined transcriptional regulation of AOX2 in molecular detail under host-relevant, ETC-inhibitory conditions. We found that multiple transcription factors, including Rtg1/Rtg3, Cwt1/Zcf11, and Zcf2, bind and regulate the AOX2 promoter, conferring thousand-fold levels of inducibility to AOX2 in response to distinct environmental stressors. Further dissection of this complex promoter revealed how integration of stimuli ranging from reactive species of oxygen, nitrogen, and sulfur to reduced copper availability is achieved at the transcriptional level to regulate AOX2 induction and enable pathogenesis.

Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energise their transport into cells and catalyse their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

The critical role of copper homeostasis during the immune response

Abstract Copper homeostasis is tightly regulated in living organisms, as copper is essential to numerous cellular processes but toxic in excess. Dysregulated copper homeostasis is associated with numerous human diseases, including genetic diseases of copper transport, cancer, autoimmune disease, and infection. Although patients with mutations in copper transporters have an increased susceptibility to infection, little is known about the changes in immune function in these diseases. Further, altering copper availability has shown promise in treating either autoimmunity or infection, but the mechanisms underlying this effect are unresolved. We examined serum copper levels in human samples during acute influenza or SARS-CoV-2 virus infections, revealing unique virus-dependent copper dynamics. As copper is required for important immune cell processes such as metabolism, signaling and maintenance of reactive oxygen species, we next investigated the effect of modulating copper levels on T cell activation. We found that copper chelation impaired T cell proliferation, reduced expression of T cell activation markers, and altered gene expression following stimulation. To determine the impact of copper levels during the immune response in vivo, we then treated mice with copper chelation prior to and during an influenza A virus infection. Notably, mice treated with copper chelation prior to infection had altered serum copper dynamics, greater weight loss, and more flu-specific T cells compared to untreated mice. Overall, our results demonstrate the importance of copper homeostasis during the immune response. Ongoing experiments will investigate the mechanisms underlying this role in immune function during disease. Supported by ALSAC and NIH (5T32AI106700).

Organic fertilization and mycorrhization increase copper phytoremediation by Canavalia ensiformis in a sandy soil

Organic fertilization and mycorrhization can increase the phytoremediation of copper-contaminated soils. The time of vermicomposting alters the properties of vermicompost, which can affect copper's availability and uptake. Therefore, this study sought to evaluate the effect of different organic fertilizers and mycorrhization on copper-contaminated soil phytoremediation. The soil was contaminated with 100 mg Cu kg-1 dry soil and received mineral fertilizer (MIN), bovine manure (CM), and vermicompost produced in 45 days (V45) or 120 days (V120), all in doses equivalent to 40 mg kg-1 dry soil of phosphorus. Half of the jack bean (Canavalia ensiformis) plants were inoculated with the arbuscular mycorrhizal fungus Rhizophagus clarus. At plant flowering, the dry mass and concentrations of Cu, Zn, Mn, Ca, Mg, P, and K in the soil, solution, and plant tissue were determined, in addition to mycorrhizal colonization, nodulation, photosynthetic pigments, and oxidative stress enzyme activity. Organic fertilization increased plant growth and copper accumulation in aerial tissues. These effects were more evident with the V120, making it suitable for use in copper phytoextraction. Mycorrhization increased root and nodule dry mass, making it recommended for phytostabilization. C. ensiformis nodulation in Cu-contaminated soils depends on vermicompost fertilization and mycorrhization. Hence, the copper phytoremediation by C. ensiformis is increased by using organic fertilization and mycorrhization.

Mechanism for combined application of biochar and Bacillus cereus to reduce antibiotic resistance genes in copper contaminated soil and lettuce

The remediation of agricultural soil contaminated by antibiotic resistance genes (ARGs) is of great significance for protecting food safety and human health. Reducing the availability of copper in soil may control coresistance to ARGs. However, the feasibility of applying nano-biochar and Bacillus cereus to mitigate the spread of ARGs in Cu contaminated soil remains unclear. Therefore, this study investigated the use of biochar with different particle sizes (2 % apple branch biochar and 0.5 % nano-biochar) and 3 g m−2B. cereus in a 60-day pot experiment with growing lettuce. The effects of single and combined application on the abundances of ARGs in Cu-contaminated soil (Cu = 200 mg kg−1) were compared, and the related mechanisms were explored. Studies have shown that the addition of biochar alone is detrimental to mitigating ARGs in soil–lettuce systems. The combined application of 3 g m−2B. cereus and 0.5 % nano-biochar effectively inhibited the proliferation of ARGs in Cu-contaminated soil, and 3 g m−2B. cereus effectively inhibited the proliferation of ARGs in lettuce. Partial least squares-path modeling and network analysis showed that bacterial communities and mobile genetic elements were the key factors that affected the abundances of ARGs in rhizosphere soil, and Cu resistance genes and bioavailable copper (acid extractable state Cu (F1) + reducing state Cu (F2)) had less direct impacts. The bacterial community was the key factor that affected the abundances of ARGs in lettuce. Rhodobacter (Proteobacteria), Corynebacterium (Actinobacteria), and Methylobacterium (Proteobacteria) may have been hosts of ARGs in lettuce plants. B. cereus and nano-biochar affected the abundances of ARGs by improving the soil properties and reducing the soil bioavailability of Cu, as well as directly or indirectly changing the bacterial community composition in soil and lettuce, thereby impeding the transport of ARGs to aboveground plant parts.

Hydrogen Peroxide Mitigates Cu Stress in Wheat

Abiotic stress imposed by heavy metals (HMs) adversely influences plant growth. In crop plants, such stresses penalize grain yield and ultimately could have enduring connotations for sustainable food security. Although copper (Cu) is an essential micronutrient for crop life, excessive availability of copper impairs plant growth and/or reproductive performance. Anecdotal evidence suggests that hydrogen peroxide (H2O2) is produced in plants under either biotic or abiotic stresses to mitigate oxygen-derived cell toxicity, although the influence of H2O2 remains to be definitively quantified. Here, our aim was to investigate the effects of hydrogen peroxide (H2O2) on the growth, grain yield, and yield components, as well as copper uptake of stressed wheat grown in sandy soil. We found that applications rates of 150 or 300 mg Cu kg−1 soil significantly reduced net photosynthesis, leaf area, chlorophyll, and grain yield. Foliar application of H2O2 to plants grown under 150 and 300 mg Cu kg−1 soil had improved growth, physiological, and yield traits. For instance, foliar application of H2O2 Cu-stressed plants grown under 300 mg Cu kg−1 soil reduced detrimental effects of Cu toxicity by −12% in terms of grains per spike and −7% for 1000-grain weight in comparison to the control treatment. Foliar application of H2O2 on wheat grown under copper stress reduced accumulation of other heavy metals such as cadmium. We suggest that the potential for foliar application of H2O2 in mitigating heavy metal stress in crop plants has large global potential; however, further work is required to elucidate the environmental conditions and application rates required to attain optimal benefit.

Does the aging behavior of microplastics affect the process of denitrification by the difference of copper ion adsorption?

Riparian sediment is a hot zone for denitrification that can withhold copper and microplastics (MPs) from outside. It has been proven that MPs affect denitrification and the existing forms of copper in the environment. However, the impact of copper on sediment denitrification under exposure to MPs remains unclear. This study revealed the response of sediment denitrification to copper availability under the adsorption of MPs and the complexation of MP-derived dissolved organic matter (DOM). These results showed that MP accumulation inhibited denitrification. However, aged MPs increased the activity of nitrite reductase (12.64%), nitrogen dioxide reductase (37.68%), and electron transport (28.93%) compared with pristine MPs. The aging behavior of MPs alleviated 28.18% nitrite accumulation and 16.41–118.35% nitrous oxide emissions. Thus, the aging behavior of MPs alleviated the inhibition of denitrification. Notably, we resolved the copper ion adsorption and complexation by MPs, MP-derived DOM contributed to the denitrification process, and we found that the key nitrogen removal factors were affected by KL, KM, and K2. These results fill a gap in our understanding of biochemical synthesis of MPs during denitrification. Furthermore, it can be used to build a predictive understanding of the long-term effects of MPs on the sediment nitrogen cycle.

Changes in the Availability and Distribution of Microelement Copper in Cadmium Contaminated Soil and its Accumulation in Rice (Oryza sativa L.) After Biochar Application.

This experiment explored the side effect of biochar application on the availability and distribution of the soil microelement copper (Cu) in cadmium (Cd) contaminated soil and its uptake in rice tissues. Biochar was added at 0, 10, 20, 30, and 40 t ha-1 on topsoil. Results observed that both the concentration of available Cu in the topsoil and subsoil layer decreased by 16.3%-28.9% and 7.5%-59.3%, respectively, after biochar application. The Cu concentration in the < 0.053mm and 1-2mm soil aggregates increased as biochar application rate increased. Increasing application rate of biochar reduced the proportion of exchangeable Cu, carbonate bound Cu, and Fe/Mn oxide Cu fraction in the soil profile. However, the fraction of organic bound and residual Cu increased by 5.0%-178.4% and 7.0%-15.6%, respectively. Biochar could immobilize microelement Cu in the soil profile and limit Cu uptake by rice.

Active Sulfate-Reducing Bacterial Community in the Camel Gut

The diversity and activity of sulfate-reducing bacteria (SRB) in the camel gut remains largely unexplored. An abundant SRB community has been previously revealed in the feces of Bactrian camels (Camelus bactrianus). This study aims to combine the 16S rRNA gene profiling, sulfate reduction rate (SRR) measurement with a radioactive tracer, and targeted cultivation to shed light on SRB activity in the camel gut. Fresh feces of 55 domestic Bactrian camels grazing freely on semi-arid mountain pastures in the Kosh-Agach district of the Russian Altai area were analyzed. Feces were sampled in early winter at an ambient temperature of -15 °C, which prevented possible contamination. SRR values measured with a radioactive tracer in feces were relatively high and ranged from 0.018 to 0.168 nmol S cm-3 day-1. The 16S rRNA gene profiles revealed the presence of Gram-negative Desulfovibrionaceae and spore-forming Desulfotomaculaceae. Targeted isolation allowed us to obtain four pure culture isolates belonging to Desulfovibrio and Desulforamulus. An active SRB community may affect the iron and copper availability in the camel intestine due to metal ions precipitation in the form of sparingly soluble sulfides. The copper-iron sulfide, chalcopyrite (CuFeS2), was detected by X-ray diffraction in 36 out of 55 analyzed camel feces. In semi-arid areas, gypsum, like other evaporite sulfates, can be used as a solid-phase electron acceptor for sulfate reduction in the camel gastrointestinal tract.

Conformationally Preorganized High-Affinity Ligands for Copper Biology with Hinged and Rigid Thiophene Backbones.

Copper-selective ligands are essential tools for probing the affinity of cuproproteins or manipulating the cellular copper availability. They also harbor significant potential as antiangiogenic agents in cancer therapy or as therapeutics to combat copper toxicity in Wilson's disease. To achieve the high Cu(I) affinities required for competing effectively with cellular cuproproteins, we recently devised a ligand design based on phosphine-sulfide-stabilized phosphine (PSP) donor motifs. Building on this design strategy, we integrated two PSP donors within preorganized ligand architectures composed of either a hinged bithiophene backbone (bithipPS) or a single rigid thiophene bridge (thipPS). Extensive characterization based on X-ray crystal structures, solution NMR data, spectrophotometric titrations, and electrochemical studies established that bithipPS adapts well to the coordination preferences of Cu(I) to form a discrete air-stable mononuclear Cu(I) complex with a dissociation constant of 4 zM. In contrast, the wider bite angle of thipPS introduces some strain upon Cu(I) coordination to yield an almost 10-fold lower affinity with a Kd of 35 zM. As revealed by ICP-MS and two-photon excitation microscopy studies with the Cu(I)-selective fluorescent probe crisp-17, both ligands are effective at removing cellular copper from live mouse fibroblasts with rapid kinetics. Altogether, the stability and redox properties of PSP-ligand-Cu(I) complexes can be effectively tuned by judicious balancing of their geometrical preorganization and conformational flexibility.

Metabolic Sensing of Extracytoplasmic Copper Availability via Translational Control by a Nascent Exported Protein.

Metabolic sensing is a crucial prerequisite for cells to adjust their physiology to rapidly changing environments. In bacteria, the response to intra- and extracellular ligands is primarily controlled by transcriptional regulators, which activate or repress gene expression to ensure metabolic acclimation. Translational control, such as ribosomal stalling, can also contribute to cellular acclimation and has been shown to mediate responses to changing intracellular molecules. In the current study, we demonstrate that the cotranslational export of the Rhodobacter capsulatus protein CutF regulates the translation of the downstream cutO-encoded multicopper oxidase CutO in response to extracellular copper (Cu). Our data show that CutF, acting as a Cu sensor, is cotranslationally exported by the signal recognition particle pathway. The binding of Cu to the periplasmically exposed Cu-binding motif of CutF delays its cotranslational export via its C-terminal ribosome stalling-like motif. This allows for the unfolding of an mRNA stem-loop sequence that shields the ribosome-binding site of cutO, which favors its subsequent translation. Bioinformatic analyses reveal that CutF-like proteins are widely distributed in bacteria and are often located upstream of genes involved in transition metal homeostasis. Our overall findings illustrate a highly conserved control mechanism using the cotranslational export of a protein acting as a sensor to integrate the changing availability of extracellular nutrients into metabolic acclimation. IMPORTANCE Metabolite sensing is a fundamental biological process, and the perception of dynamic changes in the extracellular environment is of paramount importance for the survival of organisms. Bacteria usually adjust their metabolisms to changing environments via transcriptional regulation. Here, using Rhodobacter capsulatus, we describe an alternative translational mechanism that controls the bacterial response to the presence of copper, a toxic micronutrient. This mechanism involves a cotranslationally secreted protein that, in the presence of copper, undergoes a process resembling ribosomal stalling. This allows for the unfolding of a downstream mRNA stem-loop and enables the translation of the adjacent Cu-detoxifying multicopper oxidase. Bioinformatic analyses reveal that such proteins are widespread, suggesting that metabolic sensing using ribosome-arrested nascent secreted proteins acting as sensors may be a common strategy for the integration of environmental signals into metabolic adaptations.

Dichloro(2,2′-bipyridine)copper/MAO: An Active and Stereospecific Catalyst for 1,3-Diene Polymerization

Dichloro(2,2'-bipyridine)copper was synthesized by reacting copper dichloride with bypyridine, and its behavior, in combination with methylaluminoxane (MAO), in the polymerization of butadiene, isoprene, 2,3-dimethyl-1,3 butadiene, and 3-methyl-1,3-pentadiene was examined. The purpose of this study is to find catalytic systems that are more sustainable than those currently used for the polymerization of butadiene and isoprene (e.g., Co and Ni), but that are comparable in terms of catalytic activity and selectivity. Predominantly, syndiotactic 1,2 polybutadiene, crystalline syndiotactic 3,4 polyisoprene, crystalline syndiotactic 1,2 poly(3-methyl-1,3-pentadiene), and crystalline cis-1,4 poly(2,3-dimethyl-1,3-butadiene) were obtained in a manner similar to that observed with the analogous iron complex. As far as we know, the investigated catalytic system represents the first example of a copper-based catalyst in the field of stereospecific polymerization. Given the great availability of copper, its extremely low toxicity (and therefore high sustainability), and the similarity of its behavior to that of iron, the result obtained seems to us of considerable interest and worthy of further investigation.

Soil copper and manganese availability to wheat (Triticum aestivum) as affected by long-term fertilization in an Inceptisol

Copper (Cu) and manganese (Mn) are two most important micronutrients, essential to living organisms through their involvement in different enzymes and other biologically active molecules. An experiment was conducted at the research farm of ICAR-Indian Agricultural Research Institute, New Delhi to investigate the impact of various fertilizers and manurial treatments on DTPA-extractable Cu and Mn in soil and their concentration in grain and straw of wheat (Triticum aestivum L.) after 46 years of continuous cropping. The soil and plants samples were collected at wheat harvest during April, 2018 and April, 2019 following fertilizers and manurial treatments: control, N alone, NP, NPK, 150% NPK, NPK+Zn and NPK+FYM. Results revealed that DTPA-extractable Cu did not show any significant variation among the various treatments except control but DTPA-Mn was highest under FYM treated plot. In addition, nutrient management options such as 150% NPK, NPK+FYM and NPK+Zn were equally effective in enhancing the yield of wheat. The concentration of Cu and Mn in grain varied from 5.25–7.63 and 28.5–36.4 mg/kg, respectively under different nutrient management options. Highest uptake of Cu and Mn was obtained under NPK+FYM treatment while control showed the lowest, indicating importance of organic manures for improving the nutritional quality of food grains. Transfer coefficient (indicating transfer nutrients from vegetative tissue to grain) of Cu did not change by fertilization and manuring to a significant level whereas balance and integrated use of fertilizers and manure significantly increased the transfer of Mn from straw to grains.

Diversity and adaptation properties of actinobacteria associated with Tunisian stone ruins.

Stone surface is a unique biological niche that may host a rich microbial diversity. The exploration of the biodiversity of the stone microbiome represents a major challenge and an opportunity to characterize new strains equipped with valuable biological activity. Here, we explored the diversity and adaptation strategies of total bacterial communities associated with Roman stone ruins in Tunisia by considering the effects of geo-climatic regions and stone geochemistry. Environmental 16S rRNA gene amplicon was performed on DNA extracted from stones samples collected in three different sampling sites in Tunisia, along an almost 400km aridity transect, encompassing Mediterranean, semiarid and arid climates. The library was sequenced on an Illumina MiSeq sequencing platform. The cultivable Actinobacteria were isolated from stones samples using the dilution plate technique. A total of 71 strains were isolated and identified based on 16S rRNA gene sequences. Cultivable actinobacteria were further investigated to evaluate the adaptative strategies adopted to survive in/on stones. Amplicon sequencing showed that stone ruins bacterial communities were consistently dominated by Cyanobacteria, followed by Proteobacteria and Actinobacteria along the aridity gradient. However, the relative abundance of the bacterial community components changed according to the geo-climatic origin. Stone geochemistry, particularly the availability of magnesium, chromium, and copper, also influenced the bacterial communities' diversity. Cultivable actinobacteria were further investigated to evaluate the adaptative strategies adopted to survive in/on stones. All the cultivated bacteria belonged to the Actinobacteria class, and the most abundant genera were Streptomyces, Kocuria and Arthrobacter. They were able to tolerate high temperatures (up to 45°C) and salt accumulation, and they produced enzymes involved in nutrients' solubilization, such as phosphatase, amylase, protease, chitinase, and cellulase. Actinobacteria members also had an important role in the co-occurrence interactions among bacteria, favoring the community interactome and stabilization. Our findings provide new insights into actinobacteria's diversity, adaptation, and role within the microbiome associated with stone ruins.

Deficient copper availability on organoleptic and nutritional quality of tomato fruit

Copper (Cu) is an essential micronutrient for plants because it functions as a redox-active cofactor in vital processes inside the cells. Arable lands are often deficient in micronutrient contents and require the application of enriched fertilisers, whose overuse poses a high risk for human health, the environment and the food safety. Here, we aimed to decipher the effects of Cu deficiency during fruit growth on Cu and other micronutrients contents and on the fruit nutritional value and quality of tomato, the most consumed fruit worldwide, throughout the maturation process. Changes in the contents of important micronutrients for fruit physiology and human health, such as Fe and Mn, occurred in response to Cu deficient growing conditions at different fruit ripening stages, while lower Cu levels were detected in those fruit along the whole maturation process. Cu deficiency delayed changes in lycopene content and fruit colour, but increased acidity, and advanced the rise in antioxidant capacity and vitamin C content during fruit colour change from green to light red in the Moneymaker tomato; although this time lag eventually caught up in the most mature fruit stage. Cu deficiency also increased total phenolic and flavonoid contents only in green fruit.

Direct and delayed synergistic effects of extreme temperature, metals and food limitation on tropical reef-associated fish juveniles

Tropical fish are fast-growing and high energetic-demand organisms, which can be highly vulnerable to long-lasting effects of heat stress and pollution, particularly under food shortages. We tested this by assessing highly complex direct and delayed interactive effects of an extreme temperature (ET, 32 °C) from a simulated marine heatwave (MHW), copper (Cu, 0, 100, 150 and 175 μg L−1) and food availability (limited and saturated food) on early juveniles of a tropical, reef-associated seaperch (Psammoperca waigiensis). Cu, ET, and food limitation independently reduced survival and growth, partly explained by reduced feeding. The negative effect of Cu on fish survival was more substantial under ET, particularly under limited food. Delayed interactive effects of Cu, ET, and food limitation were still lethal to fish juveniles during the post-exposure period. These results indicate that reef-associated fish juveniles are highly vulnerable to these dominant stressors, impairing their ecological function as predators in the coral reefs.

Does a decade of soil organic fertilization promote copper and zinc bioavailability to an epi-endogeic earthworm?

While long-term organic fertilizer (OF) applications tend to decrease copper (Cu) and zinc (Zn) availability in agricultural soils, earthworm bioturbation has been reported to have the opposite effect. Thus, the consequences of OF amendments in earthworm-inhabited soils on Cu and Zn bioavailability to earthworms are still under debate. Here, we assessed the effect of a decade of agronomically realistic OF applications on Cu and Zn availability in earthworm-inhabited soils and the consequences on Cu and Zn bioavailability to earthworms. An epi-endogeic species (Dichogaster saliens) was exposed in microcosms to three field-collected soils that had received either no, mineral, or organic fertilization for a decade. Dissolved organic matter (DOM) properties (i.e., concentration, aromaticity, and binding properties toward Cu), pH, and Cu and Zn availability (i.e., total concentration and free ionic activity) were determined in the solution of the soil containing earthworms. Cu and Zn bioavailability was assessed by measuring the net accumulation (ng) and concentration of Cu and Zn in earthworms (mgkg-1). Despite soil Cu and Zn contamination induced by a decade of OF applications, organic fertilization induced an increase in soil pH and DOM properties that drove the reduction of Cu and Zn availability in earthworm-inhabited soils, while bioturbation had little effect on soil pH, DOM properties, and Cu and Zn availability. Consistently, Cu and Zn bioavailability to earthworms did not increase with OF applications. From an ecotoxicological perspective, our results suggest that agronomically realistic applications of OF for a decade should not pose a risk to earthworms in terms of Cu and Zn net accumulation, but further studies have to be undertaken to understand consequent long-term toxicity after exposure.

The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions.

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins.

Copper mobilisation from Cu sulphide minerals by methanobactin: Effect of pH, oxygen and natural organic matter.

Aerobic methane oxidation (MOx) depends critically on the availability of copper (Cu) as a crucial component of the metal centre of particulate methane monooxygenase, one of the main enzymes involved in MOx. Some methanotrophs have developed Cu acquisition strategies, in which they exude Cu‐binding ligands termed chalkophores under conditions of low Cu availability. A well‐characterised chalkophore is methanobactin (mb), exuded by the microaerophilic methanotroph Methylosinus trichosporium OB3b. Aerobic methanotrophs generally reside close to environmental oxic–anoxic interfaces, where the formation of Cu sulphide phases can aggravate the limitation of bioavailable Cu due to their low solubility. The reactivity of chalkophores towards such Cu sulphide mineral phases has not yet been investigated. In this study, a combination of dissolution experiments and equilibrium modelling was used to examine the dissolution and solubility of bulk and nanoparticulate Cu sulphide minerals in the presence of mb as influenced by pH, oxygen and natural organic matter. In general, we show that mb is effective at increasing the dissolved Cu concentrations in the presence of a variety of Cu sulphide phases that may potentially limit Cu bioavailability. More Cu was mobilised per mole of mb from Cu sulphide nanoparticles compared with well‐crystalline bulk covellite (CuS). In general, the efficacy of mb at mobilising Cu from Cu sulphides is pH‐dependent. At lower pH, e.g. pH 5, mb was ineffective at solubilizing Cu. The presence of mb increased dissolved Cu concentrations between pH 7 and 8.5, where the solubility of all Cu sulphides is generally low, both in the presence and absence of oxygen. These results suggest that chalkophore‐promoted Cu mobilisation from sulphide phases is an effective extracellular mechanism for increasing dissolved Cu concentrations at oxic–anoxic interfaces, particularly in the neutral to slightly alkaline pH range. This suggests that aerobic methanotrophs may be able to fulfil their Cu requirements via the exudation of mb in natural environments where the bioavailability of Cu is constrained by very stable Cu sulphide phases.