Copper Acquisition Research Articles

A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition.

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition.

Hybridization of Particulate Methane Monooxygenase by Methanobactin-Modified AuNPs.

Particulate methane monooxygenase (pMMO) is a characteristic membrane-bound metalloenzyme of methane-oxidizing bacteria that can catalyze the bioconversion of methane to methanol. However, in order to achieve pMMO-based continuous methane-to-methanol bioconversion, the problems of reducing power in vitro regeneration and pMMO stability need to be overcome. Methanobactin (Mb) is a small copper-chelating molecule that functions not only as electron carrier for pMMO catalysis and pMMO protector against oxygen radicals, but also as an agent for copper acquisition and uptake. In order to improve the activity and stability of pMMO, methanobactin–Cu (Mb–Cu)-modified gold nanoparticle (AuNP)–pMMO nanobiohybrids were straightforwardly synthesized via in situ reduction of HAuCl4 to AuNPs in a membrane fraction before further association with Mb–Cu. Mb–Cu modification can greatly improve the activity and stability of pMMO in the AuNP–pMMO nanobiohybrids. It is shown that the Mb–Cu-modified AuNP–pMMO nanobiohybrids can persistently catalyze the conversion of methane to methanol with hydroquinone as electron donor. The artificial heterogeneous nanobiohybrids exhibited excellent reusability and reproducibility in three cycles of catalysis, and they provide a model for achieving hydroquinone-driven conversion of methane to methanol.

Histoplasma Responses to Nutritional Immunity Imposed by Macrophage Activation.

The fungal pathogen Histoplasma capsulatum resides within the phagosome of host phagocytic cells. Within this intracellular compartment, Histoplasma yeast replication requires the acquisition of several essential nutrients, including metal ions. Recent work has shown that while iron, zinc, and copper are sufficiently abundant in resting macrophages, cytokine activation of these host cells causes restriction of these metals from intracellular yeasts as a form of nutritional immunity. Faced with limited iron availability in the phagosome following macrophage activation by IFN-γ, Histoplasma yeasts secrete iron-scavenging siderophores and employ multiple strategies for reduction of ferric iron to the more physiologically useful ferrous form. IFN-γ activation of macrophages also limits availability of copper in the phagosome, forcing Histoplasma reliance on the high affinity Ctr3 copper importer for copper acquisition. GM-CSF activation stimulates macrophage production of zinc-chelating metallothioneins and zinc transporters to sequester zinc from Histoplasma yeasts. In response, Histoplasma yeasts express the Zrt2 zinc importer. These findings highlight the dynamics of phagosomal metal ion concentrations in host-pathogen interactions and explain one mechanism by which macrophages become a less permissive environment for Histoplasma replication with the onset of adaptive immunity.

Copper Utilization, Regulation, and Acquisition by Aspergillus fumigatus.

Copper is an essential micronutrient for the opportunistic human pathogen, Aspergillus fumigatus. Maintaining copper homeostasis is critical for survival and pathogenesis. Copper-responsive transcription factors, AceA and MacA, coordinate a complex network responsible for responding to copper in the environment and determining which response is necessary to maintain homeostasis. For example, A. fumigatus uses copper exporters to mitigate the toxic effects of copper while simultaneously encoding copper importers and small molecules to ensure proper supply of the metal for copper-dependent processes such a nitrogen acquisition and respiration. Small molecules called isocyanides recently found to be produced by A. fumigatus may bind copper and partake in copper homeostasis similarly to isocyanide copper chelators in bacteria. Considering that the host uses copper as a microbial toxin and copper availability fluctuates in various environmental niches, understanding how A. fumigatus maintains copper homeostasis will give insights into mechanisms that facilitate the development of invasive aspergillosis and its survival in nature.

Cu(II) adsorption onto ammonia-oxidizing bacteria and archaea

Abstract Nitrogen cycling is dependent on ammonia-oxidizing bacteria and archaea transforming ammonia to nitrite. Copper is an essential cofactor for ammonia monooxygenase in ammonia-oxidizing archaea and bacteria. Copper is also essential for several electron carriers in ammonia-oxidizing archaea. Hence, copper acquisition is a critical step for complete enzyme maturation and metabolic function. In this work, we describe copper adsorption onto the bacterial species Nitrosococcus oceani C-107 (N. oceani) and Nitrosomonas europaea C-31 (N. europaea) and archaeal species Nitrosopumilus maritimus SCM1 (N. maritimus). Surface complexation models described copper adsorption onto bacterial species with one reaction, while N. maritimus required two reactions to adequately describe adsorption. Enthalpies of copper adsorption derived from isothermal titration calorimetry combined with surface complexation modeling revealed slightly exothermic enthalpies for N. maritimus and thermoneutral to endothermic enthalpies for the bacterial species. Entropies of copper adsorption were positive and indicative of entropy-driven inner-sphere complexation reactions. Thermodynamic parameters describing copper adsorption onto N. maritimus are consistent with adsorption onto a mixture of phosphorous-bearing anionic oxygen and thiol ligands. Thermodynamic parameters describing Cu(II) adsorption onto N. oceani and N. europaea were consistent with Cu(II) adsorption onto phosphorous-bearing anionic oxygen ligands. Modeling competitive copper complexation between N. maritimus and two organic acids, to mimic dissolved organic carbon, showed that N. maritimus could compete with nitrilotriacetic acid and triethylenetetramine under conditions similar to experimental measurements of copper adsorption. These results suggest that selectivity of the N. maritimus surface may confer some advantage in low-copper environments.

Aerobic methane oxidation under copper scarcity in a stratified lake

Aerobic methane-oxidizing bacteria (MOB) substantially reduce methane fluxes from freshwater sediments to the atmosphere. Their metalloenzyme methane monooxygenase (MMO) catalyses the first oxidation step converting methane to methanol. Its most prevalent form is the copper-dependent particulate pMMO, however, some MOB are also able to express the iron-containing, soluble sMMO under conditions of copper scarcity. So far, the link between copper availability in different forms and biological methane consumption in freshwater systems is poorly understood. Here, we present high-resolution profiles of MOB abundance and pMMO and sMMO functional genes in relation to copper, methane and oxygen profiles across the oxic-anoxic boundary of a stratified lake. We show that even at low nanomolar copper concentrations, MOB species containing the gene for pMMO expression are present at high abundance. The findings highlight the importance of copper as a micronutrient for MOB species and the potential usage of copper acquisition strategies, even under conditions of abundant iron, and shed light on the spatial distribution of these microorganisms.

Macrophage activation by IFN-γ triggers restriction of phagosomal copper from intracellular pathogens.

Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.

CHCA-1 is a copper-regulated CTR1 homolog required for normal development, copper accumulation, and copper-sensing behavior in Caenorhabditis elegans

Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health. Dietary copper deficiencies or mutations in copper homeostasis genes can lead to abnormal musculoskeletal development, cognitive disorders, and poor growth. In yeast and mammals, copper is acquired through the activities of the CTR1 family of high-affinity copper transporters. However, the mechanisms of systemic responses to dietary or tissue-specific copper deficiency remain unclear. Here, taking advantage of the animal model Caenorhabditis elegans for studying whole-body copper homeostasis, we investigated the role of a C. elegans CTR1 homolog, CHCA-1, in copper acquisition and in worm growth, development, and behavior. Using sequence homology searches, we identified 10 potential orthologs to mammalian CTR1 Among these genes, we found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation. The intestinal copper transporter CUA-1 normally traffics to endosomes to sequester excess copper, and we found here that loss of chca-1 caused CUA-1 to mislocalize to the basolateral membrane under copper overload conditions. Moreover, animals lacking chca-1 exhibited significantly reduced copper avoidance behavior in response to toxic copper conditions compared with WT worms. These results establish that CHCA-1-mediated copper acquisition in C. elegans is crucial for normal growth, development, and copper-sensing behavior.

Cu‐induced assembly of methanobactin‐modified gold nanoparticles and its peroxidase mimic activity

Methanobactin (Mb) is a small copper-chelating molecule that functions as an agent for copper acquisition, uptake and copper-containing methane monooxygenase catalysis in methane-oxidising bacteria. The UV-visible spectral and fluorescence spectral suggested that Mb/Cu coordination complex as a monomer (Mb-Cu), dimmer (Mb2-Cu) and tetramer (Mb4-Cu) could be obtained at different ratios of Mb to Cu (II). The kinetics of the oxidation of hydroquinone with hydrogen peroxide catalysed by the different Mb/Cu coordination complex were investigated. The results suggested that Mb2-Cu coordination form has highest catalytic capacity. Further, Mb-modified gold nanoparticles (AuNPs) were obtained by ligand exchange and assembled into two- and three-D nanocluster structure by metal-organic coordination as driving force. It has been found that AuNPs increased the catalytic activity of Mb2-Cu on AuNPs. The more significant catalytic activity was exhibited by the nanocluster assembly with multi-catalytic centres. This may be attributed to the multivalent collaborative characteristics of the catalytic active centres in the nanocluster network assembly. The assembly of Mb-modified AuNPs can act as excellent nanoenzyme models for imitating peroxidase.

The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1.

Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiological role of CuII binding to hCtr1.

Diisonitrile Natural Product SF2768 Functions As a Chalkophore That Mediates Copper Acquisition in Streptomyces thioluteus.

A nonribosomal peptide synthetase (NRPS) gene cluster (sfa) was identified in Streptomyces thioluteus to direct the biosynthesis of the diisonitrile antibiotic SF2768. Its biosynthetic pathway was reasonably proposed based on bioinformatics analysis, metabolic profiles of mutants, and the elucidation of the intermediate and shunt product structures. Bioinformatics-based alignment found a putative ATP-binding cassette (ABC) transporter related to iron import within the biosynthetic gene cluster, which implied that the product might be a siderophore. However, characterization of the metal-binding properties by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), metal-ligand titration, thin-layer chromatography (TLC), and chrome azurol S (CAS) assays revealed that the final product SF2768 and its diisonitrile derivatives specifically bind copper, rather than iron, to form stable complexes. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that the intracellular cupric content of S. thioluteus significantly increased upon incubation with the copper-SF2768 complex, direct evidence for the copper acquisition function of SF2768. Further in vivo functional characterization of the transport elements for the copper-SF2768 complexes not only confirmed the chalkophore identity of the compound but also gave initial clues into the copper uptake mechanism of this nonmethanotrophic microorganism.

A Noncanonical Role for Yersiniabactin in Bacterial Copper Acquisition.

A Noncanonical Role for Yersiniabactin in Bacterial Copper Acquisition.

An empirical evaluation of copper procurement and distribution: elemental analysis of Scioto Valley Hopewell copper

In prehistoric North America, artifacts of copper occupy a position of prominence in the Hopewell societies of Ohio’s Scioto Valley. These artifacts also represent the social contacts and long-distance interactions that brought copper to the Scioto Valley. Yet, our understanding of Hopewell copper acquisition, and the movement of copper artifacts within the social networks of the Scioto Valley and beyond, has been limited due to the limited availability of geochemical data concerning provenance and variability. We begin to develop the foundation for understanding these important social issues by analyzing the elemental variability of Hopewell copper through the use of laser ablation inductively coupled plasma mass spectrometry. We analyze 24 samples from four known copper source regions and 52 samples from 6 prominent sites in the Scioto Valley. Results suggest that a majority of the artifacts are consistent with sources in the Great Lakes, with a plurality classified as Isle Royale. However, 21% of Scioto Hopewell copper artifacts were most consistent with southern Appalachian sources. High elemental variability argues against simple models of copper acquisition and suggests that different social groups had access to a variety of copper sources through varying social networks. Native copper regardless of source seems to have been channeled along similar social and symbolic pathways. Our results suggest that instead of being derived from the struggle to access a specific source, value is derived from the social relationships represented by the copper and the connotation of exotic connection embodied in both style and material.

Copper Acquisition and Utilization in Fungi.

Fungal cells colonize and proliferate in distinct niches, from soil and plants to diverse tissues in human hosts. Consequently, fungi are challenged with the goal of obtaining nutrients while simultaneously elaborating robust regulatory mechanisms to cope with a range of availability of nutrients, from scarcity to excess. Copper is essential for life but also potentially toxic. In this review we describe the sophisticated homeostatic mechanisms by which fungi acquire, utilize, and control this biochemically versatile trace element. Fungal pathogens, which can occupy distinct host tissues that have their own intrinsic requirements for copper homeostasis, have evolved mechanisms to acquire copper to successfully colonize the host, disseminate to other tissues, and combat host copper bombardment mechanisms that would otherwise mitigate virulence.

Copper import in Escherichia coli by the yersiniabactin metallophore system.

Copper plays a dual role as nutrient and toxin during bacterial infections. While uropathogenic Escherichia coli (UPEC) strains can use the copper-binding metallophore yersiniabactin (Ybt) to resist copper toxicity, Ybt also converts bioavailable copper to Cu(II)-Ybt in low copper conditions. Although E. coli have long been considered to lack a copper import pathway, we observed Ybt-mediated copper import in UPEC using canonical Fe(III)-Ybt transport proteins. UPEC removed copper from Cu(II)-Ybt with subsequent re-export of metal-free Ybt to the extracellular space. Copper released through this process became available to an E. coli cuproenzyme (the amine oxidase TynA), linking this import pathway to a nutrient acquisition function. Ybt-expressing E. coli thus engage in nutritional passivation, a strategy of minimizing a metal ion's toxicity while preserving its nutritional availability. Copper acquisition through this process may contribute to the marked virulence defect of Ybt transport-deficient UPEC.

Bacterial copper acquisition

Methanotrophic bacteria use methane, a potent greenhouse gas, as their primary source of carbon and energy. The first step in methane metabolism is its oxidation to methanol. In mostly all methanotrophs, this chemically challenging reaction is catalyzed by particulate methane monooxygenase (pMMO), a copper‐dependent integral membrane enzyme. Many methanotrophs acquire copper for pMMO by secreting small ribosomally produced, post‐translationally modified natural products called methanobactins. Each species produces a methanobactin (Mbn) with a unique structure, and all Mbns characterized thus far bind copper with high affinity. The copper‐loaded form (CuMbn) is then re‐internalized into the cell via an active transport process. Genome mining has led to the identification of operons containing potential Mbn biosynthesis, transport, and regulatory genes. These operons are present in a range of non‐methanotrophic bacteria as well, suggesting a broader role for Mbn and Mbn‐like molecules in and perhaps beyond copper acquisition. The roles of specific gene products have been investigated by genetic, biophysical, and structural approaches. These data provide evidence for CuMbn‐protein interactions and begin to elucidate the molecular mechanisms of its recognition, transport, and biosynthesis.Support or Funding InformationThis work was supported by NIH grants GM118035 (A. C. R.) and F32GM110934 (L. M. K. D.), a Burroughs Wellcome Fund PDEP award (L. M. K. D.), and AHA grant 14PRE20460104 (G. E. K.).

Transcription factor Afmac1 controls copper import machinery in Aspergillus fumigatus.

Copper (Cu) is an essential metal for all living organisms, although it is toxic in excess. Filamentous fungus must acquire copper from its environment for growth. Despite its essentiality for growth, the mechanisms that maintain copper homeostasis are not fully understood in filamentous fungus. To gain insights into copper homeostasis, we investigated the roles of a copper transcription factor Afmac1 in the life-threatening fungus Aspergillus fumigatus, a homolog of the yeast MAC1. We observed that the Afmac1 deletion mutant exhibited not only significantly slower growth, but also incomplete conidiation including a short chain of conidia and defective melanin. Moreover, the expressions of the copper transporters, ctrA1, ctrA2, and ctrC, and metalloreductases, Afu8g01310 and fre7, were repressed in ∆Afmac1 cells, while those expressions were induced under copper depletion conditions in wild-type. The expressions of pksP and wetA, which are, respectively, involved in biosynthesis of conidia-specific melanin and the late stage of conidiogenesis, were decreased in the ∆Afmac1 strain under minimal media condition. Taken together, these results indicate that copper acquisition through AfMac1 functions in growth as well as conidiation.

Methanobactins: from genome to function.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptide (RiPP) natural products that bind copper with high affinity using nitrogen-containing heterocycles and thioamide groups. In some methanotrophic bacteria, Mbns are secreted under conditions of copper starvation and then re-internalized as a copper source for the enzyme particulate methane monooxygenase (pMMO). Genome mining studies have led to the identification and classification of operons encoding the Mbn precursor peptide (MbnA) as well as a number of putative transport, regulatory, and biosynthetic proteins. These Mbn operons are present in non-methanotrophic bacteria as well, suggesting a broader role in and perhaps beyond copper acquisition. Genetic and biochemical studies indicate that specific operon-encoded proteins are involved in Mbn transport and provide insight into copper-responsive gene regulation in methanotrophs. Mbn biosynthesis is not yet understood, but combined analysis of Mbn structures, MbnA sequences, and operon content represents a powerful approach to elucidating the roles of specific biosynthetic enzymes. Future work will likely lead to the discovery of unique pathways for natural product biosynthesis and new mechanisms of microbial metal homeostasis.

Copper and manganese acquisition in maize (Zea mays L) under different P and K fertilization

Summary The paper demonstrates the influence of different mineral fertilization with phosphorus and potassium on the concentration of copper (Cu) and manganese (Mn) in the ear leaf of maize at the stage of flowering (BBCH 65) as well as the contents and accumulation of the nutrients studied in maize when fully ripe (BBCH 89). A single factor experiment was carried out in 5-year-cycle (2007-2011), in the randomized complete block design. The experiment was conducted as a part of a long-term stationary trial. The investigation comprised 8 different P and K treatments: the absolute control, exclusive of one of the main nutrients (P - WPN or K - WKN), reduced amount of phosphorus and potassium (to 25% - W25 and to 50% WP50, WK50) as well as recommended amounts of basic nutrients (NPKMg - W100 and NP*KMg, P* - P* as PAPR - W100 PAPR). Evaluation of the nutriational status, performed in the ear leaf of maize at flowering stage, showed that regardless of fertilization treatment applied, the concentration of copper was lower than normative values, whereas that of manganese ranged within the optimal scope. At the same time, there was found a significant relationship between the grain yield obtained and acquisition of both copper and manganese by maize at flowering stage (stronger for manganese, r = 0.614). The total accumulation of copper and manganese in fully ripe maize was significantly differentiated as a result of mineral fertilization. The total uptake of Cu and Mn was reduced under the conditions of 10-year lack of P fertilization. Uptake reduction was considerably more advanced when K fertilization was absent for 10 years. Regardless of the experimental factor effects, more than 50% of the total copper uptake was accumulated in grain, whereas the majority of manganese was accumulated in maize leaves (50-64% of the total uptake). Correlation analysis showed a significant relationship between maize grain yield and the total accumulation of copper, whereas that of manganese was observed only in 3 of 8 treatments tested (WPN, WP50 and W100 as PAPR).

Methanobactin and the Link between Copper and Bacterial Methane Oxidation.

Methanobactins (mbs) are low-molecular-mass (<1,200 Da) copper-binding peptides, or chalkophores, produced by many methane-oxidizing bacteria (methanotrophs). These molecules exhibit similarities to certain iron-binding siderophores but are expressed and secreted in response to copper limitation. Structurally, mbs are characterized by a pair of heterocyclic rings with associated thioamide groups that form the copper coordination site. One of the rings is always an oxazolone and the second ring an oxazolone, an imidazolone, or a pyrazinedione moiety. The mb molecule originates from a peptide precursor that undergoes a series of posttranslational modifications, including (i) ring formation, (ii) cleavage of a leader peptide sequence, and (iii) in some cases, addition of a sulfate group. Functionally, mbs represent the extracellular component of a copper acquisition system. Consistent with this role in copper acquisition, mbs have a high affinity for copper ions. Following binding, mbs rapidly reduce Cu(2+) to Cu(1+). In addition to binding copper, mbs will bind most transition metals and near-transition metals and protect the host methanotroph as well as other bacteria from toxic metals. Several other physiological functions have been assigned to mbs, based primarily on their redox and metal-binding properties. In this review, we examine the current state of knowledge of this novel type of metal-binding peptide. We also explore its potential applications, how mbs may alter the bioavailability of multiple metals, and the many roles mbs may play in the physiology of methanotrophs.