Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms.

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

The metal-binding GTPases CobW2 and CobW3 are at the crossroads of zinc and cobalt homeostasis in Cupriavidus metallidurans.

The metal-resistant beta-proteobacterium Cupriavidus metallidurans is also able to survive conditions of metal starvation. We show that zinc-starved cells can substitute some of the required zinc with cobalt but not with nickel ions. The zinc importer ZupT was necessary for this process but was not essential for either zinc or cobalt import. The cellular cobalt content was also influenced by the two COG0523-family proteins, CobW2 and CobW3. Pulse-chase experiments with radioactive and isotope-enriched zinc demonstrated that both proteins interacted with ZupT to control the cellular flow-equilibrium of zinc, a central process of zinc homeostasis. Moreover, an antagonistic interplay of CobW2 and CobW3 in the presence of added cobalt caused a growth defect in mutant cells devoid of the cobalt efflux system DmeF. Full cobalt resistance also required a synergistic interaction of ZupT and DmeF. Thus, the two transporters along with CobW2 and CobW3 interact to control cobalt homeostasis in a process that depends on zinc availability. Because ZupT, CobW2, and CobW3 also direct zinc homeostasis, this process links the control of cobalt and zinc homeostasis, which subsequently protects C. metallidurans against cadmium stress and general metal starvation.IMPORTANCEIn bacterial cells, zinc ions need to be allocated to zinc-dependent proteins without disturbance of this process by other transition metal cations. Under zinc-starvation conditions, C. metallidurans floods the cell with cobalt ions, which protect the cell against cadmium toxicity, help withstand metal starvation, and provide cobalt to metal-promiscuous paralogs of essential zinc-dependent proteins. The number of cobalt ions needs to be carefully controlled to avoid a toxic cobalt overload. This is accomplished by an interplay of the zinc importer ZupT with the COG0523-family proteins, CobW3, and CobW2. At high external cobalt concentrations, this trio of proteins additionally interacts with the cobalt efflux system, DmeF, so that these four proteins form an inextricable link between zinc and cobalt homeostasis.

Protecting the Achilles heel: three FolE_I-type GTP-cyclohydrolases needed for full growth of metal-resistant Cupriavidus metallidurans under a variety of conditions.

In Cupriavidus metallidurans and other bacteria, biosynthesis of the essential biochemical cofactor tetrahydrofolate (THF) initiates from guanosine triphosphate (GTP). This step is catalyzed by FolE_I-type GTP cyclohydrolases, which are either zinc-dependent FolE_IA-type or metal-promiscuous FolE_IB-type enzymes. As THF is also essential for GTP biosynthesis, GTP and THF synthesis form a cooperative cycle, which may be influenced by the cellular homeostasis of zinc and other metal cations. Metal-resistant C. metallidurans harbors one FolE_IA-type and two FolE_IB-type enzymes. All three proteins were produced in Escherichia coli. FolE_IA was indeed zinc dependent and the two FolE_IB enzymes metal-promiscuous GTP cyclohydrolases in vitro, the latter, for example, functioning with iron, manganese, or cobalt. Single and double mutants of C. metallidurans with deletions in the folE_I genes were constructed to analyze the contribution of the individual FolE_I-type enzymes under various conditions. FolE_IA was required in the presence of cadmium, hydrogen peroxide, metal chelators, and under general metal starvation conditions. FolE_IB1 was important when zinc uptake was impaired in cells without the zinc importer ZupT (ZIP family) and in the presence of trimethoprim, an inhibitor of THF biosynthesis. FolE_IB2 was needed under conditions of low zinc and cobalt but high magnesium availability. Together, these data demonstrate that C. metallidurans requires all three enzymes to allow efficient growth under a variety of conditions.IMPORTANCETetrahydrofolate (THF) is an important cofactor in microbial biochemistry. This "Achilles heel" of metabolism has been exploited by anti-metabolites and antibiotics such as sulfonamide and trimethoprim. Since THF is essential for the synthesis of guanosine triphosphate (GTP) and THF biosynthesis starts from GTP, synthesis of both compounds forms a cooperative cycle. The first step of THF synthesis by GTP cyclohydrolases (FolEs) is metal dependent and catalyzed by zinc- or metal-promiscuous enzymes, so that the cooperative THF and GTP synthesis cycle may be influenced by the homeostasis of several metal cations, especially that of zinc. The metal-resistant bacterium C. metallidurans needs three FolEs to grow in environments with both high and low zinc and cadmium content. Consequently, bacterial metal homeostasis is required to guarantee THF biosynthesis.

Cobalt(II)-Substituted Cysteamine Dioxygenase Oxygenation Proceeds through a Cobalt(III)-Superoxo Complex.

We investigated the metal-substituted catalytic activity of human cysteamine dioxygenase (ADO), an enzyme pivotal in regulating thiol metabolism and contributing to oxygen homeostasis. Our findings demonstrate the catalytic competence of cobalt(II)- and nickel(II)-substituted ADO in cysteamine oxygenation. Notably, Co(II)-ADO exhibited superiority over Ni(II)-ADO despite remaining significantly less active than the natural enzyme. Structural analyses through X-ray crystallography and cobalt K-edge excitation confirmed successful metal substitution with minimal structural perturbations. This provided a robust structural basis, supporting a conserved catalytic mechanism tailored to distinct metal centers. This finding challenges the proposed high-valent ferryl-based mechanism for thiol dioxygenases, suggesting a non-high-valent catalytic pathway in the native enzyme. Further investigation of the cysteamine-bound or a peptide mimic of N-terminus RGS5 bound Co(II)-ADO binary complex revealed the metal center's high-spin (S = 3/2) state. Upon reaction with O2, a kinetically and spectroscopically detectable intermediate emerged with a ground spin state of S = 1/2. This intermediate exhibits a characteristic 59Co hyperfine splitting (A = 67 MHz) structure in the EPR spectrum alongside UV-vis features, consistent with known low-spin Co(III)-superoxo complexes. This observation, unique for protein-bound thiolate-ligated cobalt centers in a protein, unveils the capacities for O2 activation in such metal environments. These findings provide valuable insights into the non-heme iron-dependent thiol dioxygenase mechanistic landscape, furthering our understanding of thiol metabolism regulation. The exploration of metal-substituted ADO sheds light on the intricate interplay between metal and catalytic activity in this essential enzyme.

Spectroscopic and computational investigations of Cobalt(II) binding to the innate immune protein human calprotectin.

Human calprotectin (CP) is an innate immune protein that participates in the metal-withholding response to infection by sequestering essential metal nutrients from invading microbial pathogens. CP is comprised of S100A8 (α subunit, 10.8 kDa) and S100A9 (ß subunit, 13.2 kDa). Two transition-metal binding sites of CP form at the S100A8/S100A9 dimer interface. Site 1 is a His3Asp motif comprised of His83 and His87 from the S100A8 subunit and His20 and Asp30 from the S100A9 subunit. Site 2 is an unusual hexahistidine motif composed of S100A8 residues His17 and His27 and S100A9 residues His91, His95, His103, and His105. In the present study, the His3Asp and His6 sites of CP were further characterized by utilizing Co2+ as a spectroscopic probe. Magnetic circular dichroism spectroscopy was employed in conjunction with electron paramagnetic resonance spectroscopy and density functional theory computations to characterize the Co2+-bound S100A8(C42S)/S100A9(C3S) CP-Ser variant and six site variants that allowed the His3Asp and His6 sites to be further probed. Our results provide new insight into the metal-binding sites of CP-Ser and the effect of amino acid substitutions on the structure of site 2.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins.

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

Metal Ion Binding to Human Glutaminyl Cyclase: A Structural Perspective.

Glutaminyl-peptide cyclotransferases (QCs) convert the N-terminal glutamine or glutamate residues of protein and peptide substrates into pyroglutamate (pE) by releasing ammonia or a water molecule. The N-terminal pE modification protects peptides/proteins against proteolytic degradation by amino- or exopeptidases, increasing their stability. Mammalian QC is abundant in the brain and a large amount of evidence indicates that pE peptides are involved in the onset of neural human pathologies such as Alzheimer's and Huntington's disease and synucleinopathies. Hence, human QC (hQC) has become an intensively studied target for drug development against these diseases. Soon after its characterization, hQC was identified as a Zn-dependent enzyme, but a partial restoration of the enzyme activity in the presence of the Co(II) ion was also reported, suggesting a possible role of this metal ion in catalysis. The present work aims to investigate the structure of demetallated hQC and of the reconstituted enzyme with Zn(II) and Co(II) and their behavior in the presence of known inhibitors. Furthermore, our structural determinations provide a possible explanation for the presence of the mononuclear metal binding site of hQC, despite the presence of the same conserved metal binding motifs present in distantly related dinuclear aminopeptidase enzymes.

Cobalt stress enhanced dendrobine-type total alkaloids biosynthesis of Trichoderma longibrachiatum UN32 through reactive oxygen species formation.

Trichoderma longibrachiatum UN32 is a well-documented mutant strain known to produce dendrobine-type total alkaloids (DTTAs). It was serendipitously observed that the addition of Co2+ to the medium resulted in a notable enhancement in DTTAs production in the T. longibrachiatum UN32 strain, accompanied by an upregulating effect on the expression of antioxidase-related genes. Hence, the objective of the present work was to ascertain whether ROS (intracellular levels of hydrogen peroxide) induced by Co2+ treatment has a beneficial or detrimental impact on DTTAs biosynthesis. A comparison of the intracellular levels of hydrogen peroxide (H2O2) and DTTAs treated with CoCl2 and CH3COOH revealed that CoCl2 was the optimal inducer for investigating the relationship between ROS formation and DTTAs production. This was due to the observation that ROS formation was reduced by approximately 4% and DTTAs production was increased by 12.55% in comparison to the CH3COOH treatment. The physiological results revealed that the introduction of Co2+ resulted in the oxidative damage and activation of the expression of intracellular superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Furthermore, it was confirmed that ROS induced by Co2+ was beneficial to DTTAs production by adding exogenous ROS scavengers. The inclusion of all ROS scavengers, including vitamin C, tocopherol, melatonin, mannitol, and sesamol, resulted in a reduction in ROS accumulation and a concomitant decrease in DTTAs production. Specifically, the addition of melatonin at a concentration of 0.4 mg/L demonstrated significant effects, resulting in a 32.53% (P < 0.01) decrease in ROS accumulation and a 45.22% (P < 0.01) reduction in DTTAs production. Subsequently, the timelines of accumulation of intracellular H2O2 and DTTAs content indicated that ROS are also crucial for normal fermentation without CoCl2 addition. Specifically, the proper H2O2 dose for DTTAs accumulation is between 8.82 and 18.86 µmol/g. The present study offers the initial experimental evidence indicating that CoCl2 enhance DTTAs production during the culture of T. longibrachiatum UN32 via leading an increase in intracellular ROS, which is conductive to DTTAs production and can be inhibited by the ROS scavengers. Our results provide insights into the mechanistic study of DTTAs biosynthesis.

A Breast Cancer Stem Active Cobalt(III)-Cyclam Complex Containing Flufenamic Acid with Immunogenic Potential.

The cytotoxic and immunogenic-activating properties of a cobalt(III)-cyclam complex bearing the non-steroidal anti-inflammatory drug, flufenamic acid is reported within the context of anti-cancer stem cell (CSC) drug discovery. The cobalt(III)-cyclam complex 1 displays sub-micromolar potency towards breast CSCs grown in monolayers, 24-fold and 31-fold greater than salinomycin (an established anti-breast CSC agent) and cisplatin (an anticancer metallopharmaceutical), respectively. Strikingly, the cobalt(III)-cyclam complex 1 is 69-fold and 50-fold more potent than salinomycin and cisplatin towards three-dimensionally cultured breast CSC mammospheres. Mechanistic studies reveal that 1 induces DNA damage, inhibits cyclooxygenase-2 expression, and prompts caspase-dependent apoptosis. Breast CSCs treated with 1 exhibit damage-associated molecular patterns characteristic of immunogenic cell death and are phagocytosed by macrophages. As far as we are aware, 1 is the first cobalt complex of any oxidation state or geometry to display both cytotoxic and immunogenic-activating effects on breast CSCs.

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase-pyrophosphatases that act via a covalent aspartyl-phosphate intermediate.

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast-Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PPi. Crystal structures of metal-free Duf89 and Co2+-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl2 and NaF in the presence of divalent cations Co2+, Ni2+, or Zn2+ generated covalent Duf8901-(Asp248)-beryllium trifluoride (BeF3)•Co2+, Duf8901-(Asp248)-BeF3•Ni2+, or Duf8901-(Asp248)-BeF3•Zn2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)-phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF3•Co2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)-AlF4-OH2•Co2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.

Dioxin-elicited decrease in cobalamin redirects propionyl-CoA metabolism to the ß-oxidation-like pathway resulting in acrylyl-CoA conjugate buildup.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent ß-oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent ß-oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5'-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent ß-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.

Metallacarborane Cluster Anions of the Cobalt Bisdicarbollide-Type as Chaotropic Carriers for Transmembrane and Intracellular Delivery of Cationic Peptides.

Cobalt bisdicarbollides (COSANs) are inorganic boron-based anions that have been previously reported to permeate by themselves through lipid bilayer membranes, a propensity that is related to their superchaotropic character. We now introduce their use as selective and efficient molecular carriers of otherwise impermeable hydrophilic oligopeptides through both artificial and cellular membranes, without causing membrane lysis or poration at low micromolar carrier concentrations. COSANs transport not only arginine-rich but also lysine-rich peptides, whereas low-molecular-weight analytes such as amino acids as well as neutral and anionic cargos (phalloidin and BSA) are not transported. In addition to the unsubstituted isomers (known as ortho- and meta-COSAN), four derivatives bearing organic substituents or halogen atoms have been evaluated, and all six of them surpass established carriers such as pyrenebutyrate in terms of activity. U-tube experiments and black lipid membrane conductance measurements establish that the transport across model membranes is mediated by a molecular carrier mechanism. Transport experiments in living cells showed that a fluorescent peptide cargo, FITC-Arg8, is delivered into the cytosol.

Functional Characterization of the Co2+ Transporter AitP in Sinorhizobium meliloti: A New Player in Fe2+ Homeostasis.

Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.

Soil Microbial Community Composition and Tolerance to Contaminants in an Urban Brownfield Site.

Brownfields are unused sites that contain hazardous substances due to previous commercial or industrial use. The sites are inhospitable for many organisms, but some fungi and microbes can tolerate and thrive in the nutrient-depleted and contaminated soils. However, few studies have characterized the impacts of long-term contamination on soil microbiome composition and diversity at brownfields. This study focuses on an urban brownfield-a former rail yard in Los Angeles that is contaminated with heavy metals, volatile organic compounds, and petroleum-derived pollutants. We anticipate that heavy metals and organic pollutants will shape soil microbiome diversity and that several candidate fungi and bacteria will be tolerant to the contaminants. We sequence three gene markers (16S ribosomal RNA, 18S ribosomal RNA, and the fungal internal transcribed spacer (FITS)) in 55 soil samples collected at five depths to (1) profile the composition of the soil microbiome across depths; (2) determine the extent to which hazardous chemicals predict microbiome variation; and (3) identify microbial taxonomic groups that may metabolize these contaminants. Detected contaminants in the samples included heavy metals, petroleum hydrocarbons, polycyclic aromatic hydrocarbons, and volatile organic compounds. Bacterial, eukaryotic, and fungal communities all varied with depth and with concentrations of arsenic, chromium, cobalt, and lead. 18S rRNA microbiome richness and fungal richness were positively correlated with lead and cobalt levels, respectively. Furthermore, bacterial Paenibacillus and Iamia, eukaryotic Actinochloris, and fungal Alternaria were enriched in contaminated soils compared to uncontaminated soils and represent taxa of interest for future bioremediation research. Based on our results, we recommend incorporating DNA-based multi-marker microbial community profiling at multiple sites and depths in brownfield site assessment standard methods and restoration.

Nutrient co-limitation at the boundary of an oceanic gyre.

Nutrient limitation of oceanic primary production exerts a fundamental control on marine food webs and the flux of carbon into the deep ocean. The extensive boundaries of the oligotrophic sub-tropical gyres collectively define the most extreme transition in ocean productivity, but little is known about nutrient limitation in these zones. Here we present the results of full-factorial nutrient amendment experiments conducted at the eastern boundary of the South Atlantic gyre. We find extensive regions in which the addition of nitrogen or iron individually resulted in no significant phytoplankton growth over 48 hours. However, the addition of both nitrogen and iron increased concentrations of chlorophyll a by up to approximately 40-fold, led to diatom proliferation, and reduced community diversity. Once nitrogen-iron co-limitation had been alleviated, the addition of cobalt or cobalt-containing vitamin B12 could further enhance chlorophyll a yields by up to threefold. Our results suggest that nitrogen-iron co-limitation is pervasive in the ocean, with other micronutrients also approaching co-deficiency. Such multi-nutrient limitations potentially increase phytoplankton community diversity.

Bacterial riboswitches cooperatively bind Ni(2+) or Co(2+) ions and control expression of heavy metal transporters.

Bacteria regularly encounter widely varying metal concentrations in their surrounding environment. As metals become depleted or, conversely, accrue to toxicity, microbes will activate cellular responses that act to maintain metal homeostasis. A suite of metal-sensing regulatory ("metalloregulatory") proteins orchestrate these responses by allosterically coupling the selective binding of target metals to the activity of DNA-binding domains. However, we report here the discovery, validation, and structural details of a widespread class of riboswitch RNAs, whose members selectively and tightly bind the low-abundance transition metals, Ni(2+) and Co(2+). These riboswitches bind metal cooperatively, and with affinities in the low micromolar range. The structure of a Co(2+)-bound RNA reveals a network of molecular contacts that explains how it achieves cooperative binding between adjacent sites. These findings reveal that bacteria have evolved to utilize highly selective metalloregulatory riboswitches, in addition to metalloregulatory proteins, for detecting and responding to toxic levels of heavy metals.

New potential transporter CIPAS8 enhances cadmium hypersensitivity and cobalt tolerance.

KEY MESSAGE: CIPAS8 is a novel Cd-influx and Co-efflux transporters, and Ser86 and Cys128 might play a decisive role in Co-binding and translocation. Cadmium (Cd) is among the most toxic heavy metals and is a widespread environmental pollutant. Cobalt (Co) is a mineral nutrient that is essential for plant growth and development, but high concentrations may be toxic. Cadmium-induced protein AS8 (CIPAS8) is widely distributed among plant species and might be induced by heavy metals, but its function has not been studied previously. In this study, Populus euphratica PeCIPAS8 and Salix linearistipularis SlCIPAS8 were investigated. The transcription of both genes was significantly enhanced under Cd and Co stresses. PeCIPAS8 and SlCIPAS8 conferred sensitivity to Cd in transgenic yeast, allowing higher quantities of Cd to accumulate within the cells, whereas SlCIPAS8 also conferred tolerance to Co and reduced Co accumulation. The determinants of substrate selectivity of the SlCIPAS8 protein were examined by site mutagenesis, which indicated that the Ser at 86th (S86) substituted for Arg (R) [S86R] and Cys at 128th (C128) substituted for Ser [C128S] mutations limited the protein's capability for Co translocation. These results suggested that PeCIPAS8 and SlCIPAS8 may be involved in Cd uptake into the plant cell. SlCIPAS8 can reduce excess Co accumulation to maintain intracellular Co homeostasis, and the site mutations S86R and C128S were essential for Co transport. These findings provide insight into the function of CIPAS8 and highlight its potential for utilization in phytoremediation applications.

Minimal cobalt metabolism in the marine cyanobacterium Prochlorococcus.

Despite very low concentrations of cobalt in marine waters, cyanobacteria in the genus Prochlorococcus retain the genetic machinery for the synthesis and use of cobalt-bearing cofactors (cobalamins) in their genomes. We explore cobalt metabolism in a Prochlorococcus isolate from the equatorial Pacific Ocean (strain MIT9215) through a series of growth experiments under iron- and cobalt-limiting conditions. Metal uptake rates, quantitative proteomic measurements of cobalamin-dependent enzymes, and theoretical calculations all indicate that Prochlorococcus MIT9215 can sustain growth with less than 50 cobalt atoms per cell, ∼100-fold lower than minimum iron requirements for these cells (∼5,100 atoms per cell). Quantitative descriptions of Prochlorococcus cobalt limitation are used to interpret the cobalt distribution in the equatorial Pacific Ocean, where surface concentrations are among the lowest measured globally but Prochlorococcus biomass is high. A low minimum cobalt quota ensures that other nutrients, notably iron, will be exhausted before cobalt can be fully depleted, helping to explain the persistence of cobalt-dependent metabolism in marine cyanobacteria.

Simultaneous effects of aluminum exposure on the homeostasis of essential metal content in rat brain and perturbation of gut microbiota.

The theory of the brain-gut axis has confirmed that gut microbiota and metabolites are involved in the progression of neurodegenerative diseases through multiple pathways. However, few studies have highlighted the role of gut microbiota in cognitive impairment induced by aluminum (Al) exposure and its correlations with the homeostasis of essential metal content in the brain. To explore the relationship between alterations in the content of essential metals in the brain and relative abundance changes in gut microbiota induced by Al exposure, the Al, zinc (Zn), copper (Cu), iron (Fe), chromium (Cr), manganese (Mn), and cobalt (Co) content level in the hippocampus, olfactory bulb, and midbrain tissue were measured by inductively coupled plasma mass spectrometry (ICP-MS) methods after Al maltolate was intraperitoneally injected every other day for exposed groups. Then the unsupervised principal coordinates analysis (PCoA) and linear discriminant analysis effect size (LEfSe) were used to analyze the relative abundance of the gut microbiota community and the structure of the gut microbiome. Finally, the correlations between gut microbiota composition and essential metal content in the different exposure groups were explored by using the Pearson correlation coefficient method. Based on the results, we indicated that the content of Al in the hippocampus, olfactory bulb, and midbrain tissue was increased and then decreased with the increasing exposure duration, with peaks occurring between 14 and 30 days. Concomitantly, Al-exposure decreased the Zn, Fe, and Mn levels in these tissues. 16 S rRNA gene sequencing results indicated that significant differences in the intestinal microbial community structure at the phylum, family, and genus levels were found in the Day 90 exposed group compared with the Day 7 exposed group. Ten enriched species in the exposed group were identified as markers at the three levels. Furthermore, ten bacteria at the genus level were identified to have a significantly strong correlation (r = 0.70-0.90) with Fe, Zn, Mn, and Co.

Comparative analysis of biochemical, hormonal, and mineral compositions of preovulatory and cystic ovarian follicles in buffalo during the non-breeding season.

This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo (Bubalus bubalis). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12-15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater (P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser (P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition.