Oxygen‐dependent copper toxicity: targets in the chlorophyll biosynthesis pathway identified in the copper efflux ATPase CopA deficient mutant

Characterization of a copA^? mutant in the purple photosynthetic bacterium Rubrivivax gelatinosus under low oxygen or anaerobic conditions, as well as in the human pathogen Neisseria gonorrhoeae identified HemN as a copper toxicity target enzyme in the porphyrin synthesis pathway. Heme synthesis is, however, unaffected by copper under high oxygen tension because of the aerobic coproporphyrinogen III oxidase HemF. Nevertheless, in the copA^? mutant under aerobiosis, we show that the chlorophyll biosynthesis pathway is affected by excess copper resulting in a substantial decrease of the photosystem. Analyses of pigments and enzyme activity showed that under low copper concentrations, the mutant accumulated protochlorophyllide, suggesting that the protochlorophyllide reductase activity is affected by excess copper. Increase of copper concentration led to a complete lack of chlorophyll synthesis as a result of the loss of Mg‐chelatase activity. Both enzymes are widely distributed from bacteria to plants; both are [4Fe‐4S] proteins and oxygen sensitive; our data demonstrate their in vivo susceptibility to copper in the presence of oxygen. Additionally, our study provides the understanding of molecular mechanisms that may contribute to chlorosis in plants when exposed to metals. The role of copper efflux systems and the impact of copper on heme and chlorophyll biosynthesis in phototrophs are addressed.     

Additive effects of metal excess and superoxide,a highly toxic mixture in bacteria

Heavy metal contamination is a serious environmental problem. Understanding the toxicity mechanisms may allow to lower concentration of metals in the metal-based antimicrobial treatments of crops, and reduce metal content in soil and groundwater. Here, we investigate the interplay between metal efflux systems and the superoxide dismutase (SOD) in the purple bacterium Rubrivivax gelatinosus and other bacteria through analysis of the impact of metal accumulation. Exposure of the Cd²⁺-efflux mutant ΔcadA to Cd²⁺ caused an increase in the amount and activity of the cytosolic Fe-Sod SodB, thereby suggesting a role of SodB in the protection against Cd²⁺. In support of this conclusion, inactivation of sodB gene in the ΔcadA cells alleviated detoxification of superoxide and enhanced Cd²⁺ toxicity. Similar findings were described in the Cu⁺-efflux mutant with Cu⁺. Induction of the Mn-Sod or Fe-Sod in response to metals in other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio cholera and Bacillus subtilis, was also shown. Both excess Cd²⁺ or Cu⁺ and superoxide can damage [4Fe-4S] clusters. The additive effect of metal and superoxide on the [4Fe-4S] could therefore explain the hypersensitive phenotype in mutants lacking SOD and the efflux ATPase. These findings underscore that ROS defence system becomes decisive for bacterial survival under metal excess.     

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

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

Toxicity monitoring of endocrine disrupting chemicals (EDCs) using freeze-dried recombinant bioluminescent bacteria

Five different freeze-dried recombinant bioluminescent bacteria were used for the detection of cellular stresses caused by endocrine disrupting chemicals. These strains were DPD2794 (recA::luxCDABE), which is sensitive to DNA damage, DPD2540 (fabA::luxCDABE), sensitive to cellular membrane damage, DPD2511 (katG::luxCDABE), sensitive to oxidative damage, and TV1061 (grpE::luxCDABE), sensitive to protein damage. GC2, which emits bioluminescence constitutively, was also used in this study. The toxicity of several chemicals was determined on the first four freeze-dried bacteria, while nonspecific cellular stresses were measured using GC2. Damage caused by known endocrine disrupting chemicals, such as nonyl phenol, bisphenol A, and styrene, was detected and classified according to toxicity mode, while others, such as phathalate and DDT, were not detected with the bacteria. These results suggest that endocrine disrupting chemicals are toxic in bacteria, and do not act via an estrogenic effect, and that toxicity monitoring and classification of some endocrine disrupting chemicals may be possible in the field using these freeze-dried recombinant bioluminescent bacteria.     

Response of bioluminescent bacteria to sixteen azo dyes

Recombinant bioluminescent bacteria were used to monitor and classify the toxicity of azo dyes. Two constitutive bioluminescent bacteria,Photobacterium phosphoreum andEscherichia coli, E. coli GC2 (lac::luxCDABE), were used to detect the cellular toxicity of the azo dyes. In addition, four stress-inducible bioluminescentE. coli, DPD2794 (recA::luxCDABE), a DNA damage sensitive strain; DPD2540 (fabA::luxCDABE), a membrane damage sensitive strain; DPD2511 (katG::luxCDABE), an oxidative damage sensitive strain; and TV1061 (grpE::luxCDABE), a protein damage sensitive strain, were used to provide information about the type of toxicity caused by crystal violet, the most toxic dye of the 16 azo dyes tested. These results suggest that azo dyes result in serious cellular toxicity in bacteria, and that toxicity monitoring and classification of some azo dyes, in the field, may be possible using these recombinant bioluminescent bacteria.     

Abscisic acid and transpiration rate are involved in the response to boron toxicity in Arabidopsis plants

Boron (B) is an essential microelement for vascular plant development, but its toxicity is a major problem affecting crop yields in arid and semi‐arid areas of the world. In the literature, several genes involved in abscisic acid (ABA) signalling and responses are upregulated in Arabidopsis roots after treatment with excess B. It is known that the AtNCED3 gene, which encodes a crucial enzyme for ABA biosynthesis, plays a key role in the plant response to drought stress. In this study, root AtNCED3 expression and shoot ABA content were rapidly increased in wild‐type plants upon B‐toxicity treatment. The Arabidopsis ABA‐deficient nced3‐2 mutant had higher transpiration rate, stomatal conductance and accumulated more B in their shoots than wild‐type plants, facts that were associated with the lower levels of ABA in this mutant. However, in wild‐type plants, B toxicity caused a significant reduction in stomatal conductance, resulting in a decreased transpiration rate. This response could be a mechanism to limit the transport of excess B from the roots to the leaves under B toxicity. In agreement with the higher transpiration rate of the nced3‐2 mutant, this genotype showed an increased leaf B concentration and damage upon exposure to 5 mM B. Under B toxicity, ABA application decreased B accumulation in wild‐type and nced3‐2 plants. In summary, this work shows that excess B applied to the roots leads to rapid changes in AtNCED3 expression and gas exchange parameters that would contribute to restrain the B entry into the leaves, this effect being mediated by ABA.     

Effects of Dissolved and Complexed Copper on Heterotrophic Bacterial Production in San Diego Bay

Bacterial abundance and production, free (uncomplexed) copper ion concentration, total dissolved copper concentration, dissolved organic carbon (DOC), total suspended solids (TSS), and chlorophyll a were measured over the course of 1 year in a series of 27 sample “Boxes” established within San Diego Bay. Water was collected through a trace metal-clean system so that each Box’s sample was a composite of all the surface water in that Box. Bacterial production, chlorophyll a, TSS, DOC, and dissolved copper all generally increased from Box 1 at the mouth of the Bay to Box 27 in the South or back Bay. Free copper ion concentration generally decreased from Box 1 to Box 27 presumably due to increasing complexation capacity within natural waters. Based on correlations between TSS, chlorophyll a, bacterial production or DOC and the ratio of dissolved to free Cu ion, both DOC and particulate (bacteria and algae) fractions were potentially responsible for copper complexation, each at different times of the year. CuCl₂ was added to bacterial production assays from 0 to 10 μg L⁻¹ to assess acute copper toxicity to the natural microbial assemblage. Interestingly, copper toxicity appeared to increase with decreases in free copper from the mouth of the Bay to the back Bay. This contrasts the free-ion activity model in which higher complexation capacity should afford greater copper protection. When cell-specific growth rates were calculated, faster growing bacteria (i.e. toward the back Bay) appeared to be more susceptible to free copper toxicity. The protecting effect of natural dissolved organic material (DOM) concentrated by tangential flow ultrafiltration (>1 kDa), illite and kaolinite minerals, and glutathione (a metal chelator excreted by algae under copper stress) was assessed in bacterial production assays. Only DOM concentrate offered any significant protection to bacterial production under increased copper concentrations. Although the potential copper protecting agents were allowed to interact with added copper before natural bacteria were added to production assays, there may be a temporal dose–response relationship that accounts for higher toxicity in short production assays. Regardless, it appears that effective natural complexation of copper in the back portions of San Diego Bay limits exposure of native bacterial assemblages to free copper ion, resulting in higher bacterial production.     

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

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

Response of Gram-positive bacteria to copper stress

The Gram-positive bacteria Enterococcus hirae, Lactococcus lactis, and Bacillus subtilis have received wide attention in the study of copper homeostasis. Consequently, copper extrusion by ATPases, gene regulation by copper, and intracellular copper chaperoning are understood in some detail. This has provided profound insight into basic principles of how organisms handle copper. It also emerged that many bacterial species may not require copper for life, making copper homeostatic systems pure defense mechanisms. Structural work on copper homeostatic proteins has given insight into copper coordination and bonding and has started to give molecular insight into copper handling in biological systems. Finally, recent biochemical work has shed new light on the mechanism of copper toxicity, which may not primarily be mediated by reactive oxygen radicals.     

Increasing the copper sensitivity of microorganisms by restricting iron supply,a strategy for bio-management practices

Pollution by copper (Cu²⁺) extensively used as antimicrobial in agriculture and farming represents a threat to the environment and human health. Finding ways to make microorganisms sensitive to lower metal concentrations could help decreasing the use of Cu²⁺ in agriculture. In this respect, we showed that limiting iron (Fe) uptake makes bacteria much more susceptible to Cu²⁺ or Cd²⁺ poisoning. Using efflux mutants of the purple bacterium Rubrivivax gelatinosus, we showed that Cu⁺ and Cd²⁺ resistance relies on the expression of the Fur-regulated FbpABC and Ftr iron transporters. To support this conclusion, inactivation of these Fe-importers in the Cu⁺ or Cd²⁺-ATPase efflux mutants gave rise to hypersensitivity towards these ions. Moreover, in metal overloaded cells the expression of FbpA, the periplasmic iron-binding component of the ferric ion transport FbpABC system was induced, suggesting that cells perceived an ‘iron-starvation’ situation and responded to it by inducing Fe-importers. In this context, the Fe-Sod activity increased in response to Fe homoeostasis dysregulation. Similar results were obtained for Vibrio cholerae and Escherichia coli, suggesting that perturbation of Fe-homoeostasis by metal excess appeared as an adaptive response commonly used by a variety of bacteria. The presented data support a model in which metal excess induces Fe-uptake to support [4Fe-4S] synthesis and thereby induce ROS detoxification system.     

Action of 1,1-dimethylhydrazine on bacterial cells is determined by hydrogen peroxide

Seven different recombinant bioluminescent strains of Escherichia coli containing, respectively, the promoters katG and soxS (responsive to oxidative damage), recA (DNA damage), fabA (membrane damage), grpE, and rpoE (protein damage) and lac (constitutive expression) fused to the bacterial operon from Photorhabdus luminescens, were used to describe the mechanism of toxicity of 1,1-dimethylhydrazine (1,1-DMH) on bacteria, as well as to determine whether bacteria can sensitively detect the presence of this compound. A clear response to 1,1-DMH was observed only in E. coli carrying the katG’::lux, soxS’::lux, and recA’::lux-containing constructs. Preliminary treatment with catalase of the medium containing 1,1-DMH completely diminished the stress-response of the P_katG, P_recA, and P_soxS promoters. In the strain E. coli (pXen7), which contains a constitutive promoter, the level of cellular toxicity caused by the addition of 1,1-DMH was dramatically reduced in the presence of catalase.It is suggested that the action of 1,1-DMH on bacterial cells is determined by hydrogen peroxide, which is formed in response to reduction of the air oxygen level.     

RNA expression patterns of a type 2 metallothionein-like gene from rice

A type 2 metallothionein-like gene from rice, OsMT-2 (Oryza sativa metallothionein-like gene-2), was isolated in its cDNA form and sequenced. By northern analyses OsMT-2 expression was shown to be induced under stress by sucrose starvation, heat shock and, to a lesser extent, abscisic acid, but not excess metals, including copper. Its response to sucrose starvation was transient and different from OsMT-1, a type 1 metallothionein-like gene of rice inducible by copper. These results suggest that while OsMT-2 is also involved in cellular response to stress, its function may be complementary to that of OsMT-1.     

Whole animal copper flux assessed by positron emission tomography in the Long – Evans cinnamon rat – a feasibility study

Copper is an essential trace element. However, excess copper can lead to oxidation of biomolecules and cell damage and copper levels must be carefully controlled. While copper homeostasis has been studied extensively at the cellular level, short-term body copper fluxes are poorly understood. Here, we assessed for the first time the feasibility of measuring whole body copper flux by positron emission tomography, using ⁶⁴Cu. A comparative approach comparing the Long – Evans cinnamon (LEC) rat to the wild type was chosen. LEC rats are an accepted model for Wilson disease, an inherited disorder of copper excretion in humans. In LEC rats as well as in Wilson patients, the copper transporting ATPase, ATP7B, is defective. This ATPase is primarily expressed in the liver and serves in copper secretion via the bile. Dysfunction of ATP7B leads to accumulation of copper in the liver. A control and an LEC rat were transgastrically injected with 10 μg of ⁶⁴Cu and the copper flux followed for three hours by whole animal PET and concomitant collection of bile, as well as the analysis of tissue following tomography. As seen by PET, the administered copper was largely trapped in the stomach and the proximal intestine, and without a significant difference between control and LEC rat. Due to an insufficient dynamic range of the PET technology, copper which was systemically absorbed and primarily transported to the liver could only be followed by sampling and by β-counting. Biliary copper excretion ensued after 15 min in the control rat, but was absent in the LEC rat. Biliary excretion reached saturation one hour after copper administration. The trapping of orally administered copper in the gastrointestinal tract may be an important mechanism to prevent copper toxicity under conditions of a sudden, excessive copper load, which cannot be alleviated by increased biliary secretion. This trapping does however limit the utility of PET to measure whole animal copper flux. Published online December 2004     

A copper‐induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD‐yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD‐yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p‐benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4‐hydroxymuconic semialdehyde in an oxygen‐consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD‐yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.     

Interference of Copper Sulphate in Growth of Erwinia amylovora

To understand the toxicity of copper salts on Erwinia amylovora, which are used in the control of fire blight, bacterial growth and cell metabolism was assayed with copper sulphate in the presence or absence of complex-forming compounds such as various amino acids or citrate. In minimal medium without amino acids copper sulphate strongly interfered with the growth of E. amylovora. A concentration of 15 μm CuSO₄ resulted in about 50% growth inhibition. In contrast to a strong effect of streptomycin, copper ions barely killed the cells when incubated in minimal medium for 1 h. The addition of 4 g asparagine per litre relieved a‘bacteriostatic’effect of copper ions and allowed growth of the bacteria at 2 mm CuSO₄. Other amino acids had a similar effect in the protection of E. amylovora against copper ions. This was in contrast to glycine betain, which was unable to suppress growth inhibition by CuSO₄. Presumably, the free ammonium groups of amino acids participated in the protective effect. The addition of citrate, exceeding the amount of copper-ions, was also protective. Bioluminescence of E. amylovora cells was expressed via a constitutive promoter from the lux-operon of Vibrio fischeri. The light emission is dependent on active cell metabolism. In a novel approach to determine the immediate response of E. amylovora after the addition of copper sulphate, the change of bioluminescence was determined. Addition of copper ions to MM3 medium strongly affected the bioluminescence, but no change in light production was noticed, when citrate or asparagine were present in addition to copper sulphate. A decrease of bioluminescence to 50% was observed for 50 μm CuSO₄ in the absence of amino acids.