Iron and Copper Act Synergistically To Delay Anaerobic Growth of Bacteria

Transition metals are known to cause toxic effects through their interaction with oxygen, but toxicity under anoxic conditions is poorly understood. Here we investigated the effects of iron (Fe) and copper (Cu) on the anaerobic growth and gene expression of the purple phototrophic bacterium Rhodopseudomonas palustris TIE-1. We found that Fe(II) and Cu(II) act synergistically to delay anaerobic growth at environmentally relevant metal concentrations. Cu(I) and Cu(II) had similar effects both alone and in the presence of ascorbate, a Cu(II) reductant, indicating that reduction of Cu(II) to Cu(I) by Fe(II) is not sufficient to explain the growth inhibition. Addition of Cu(II) increased the toxicity of Co(II) and Ni(II); in contrast, Ni(II) toxicity was diminished in the presence of Fe(II). The synergistic anaerobic toxicity of Fe(II) and Cu(II) was also observed for Escherichia coli MG1655, Shewanella oneidensis MR-1, and Rhodobacter capsulatus SB1003. Gene expression analyses for R. palustris identified three regulatory genes that respond to Cu(II) and not to Fe(II): homologs of cueR and cusR, two known proteobacterial copper homeostasis regulators, and csoR, a copper regulator recently identified in Mycobacterium tuberculosis. Two P-type ATPase efflux pumps, along with an F_oF₁ ATP synthase, were also upregulated by Cu(II) but not by Fe(II). An Escherichia coli mutant deficient in copA, cus, and cueO showed a smaller synergistic effect, indicating that iron might interfere with one or more of the copper homeostasis systems. Our results suggest that interactive effects of transition metals on microbial physiology may be widespread under anoxic conditions, although the molecular mechanisms remain to be more fully elucidated.     

Full, reversible copper removal from ascorbate oxidase

Anaerobic treatment with cyanide of reduced ascorbate oxidase causes total depletion of copper. No significant amount of the metal is reincorporated when the apo-enzyme is incubated with cupric ions, but it is upon incubation with a stoichiometric amount (eight mol per mol of native enzyme) of a Cu(I) complex stable in air [Cu(I)(thiourea)3]Cl. The yield in reconstituted protein is higher under anaerobic conditions (85-90%) than in air (70-75%). By treatment with less than stoichiometric amounts of [Cu(I)(thiourea)3]Cl the apo-protein binds copper preferentially at the blue copper site. As a consequence the recovery of enzymatic activity is percentually lower than copper reincorporation.     

The Effect of Toxic Doses of Copper upon Respiration, Photosynthesis and Growth of Chlorella vulgaris

When cells of Chlorella vulgaris absorb copper under anaerobic conditions, subsequent respiration, photosynthesis and growth of the cells are all severely inhibited. This does not occur when the metal is absorbed under aerobic conditions. When, after aerobic absorption of copper, the cells are exposed to a period of anaerobiosis, respiratory inhibition is as profound as when the uptake is anaerobic. In this case, however, respiration must eventually recover, for growth is not affected so severely as it is when copper is taken up under anaerobic conditions. It is concluded that the extra copper absorbed under anaerobic conditions is directly or indirectly responsible for the greatly increased toxicity to growth, and that this copper is bound to sites not normally available under aerobic conditions. Some aspects of the apparently unique toxic effect of copper suggest that these extra sites are sulphydryl groups.     

Genetic and biochemical analysis of high iron toxicity in yeast: iron toxicity is due to the accumulation of cytosolic iron and occurs under both aerobic and anaerobic conditions

Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity.     

Functional properties of the anaerobic responsive element of the maize Adh1 gene

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promotor. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position –90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.     

Oxidation-reduction reactions of copper-thiolate centres in Cu-thionein.

Cu-thionein from yeast was investigated by EPR spectroscopy to probe the oxidation state of copper, and the effects on it of oxidizing and reducing agents. At pH 0.2 the copper was released, but no EPR signal from Cu(II) was observed, unless air was present. Optical experiments did not detect any disulphide groups which might have been formed during anaerobic release of copper. The mercurial, p-hydroxymercuribenzoate caused the release of EPR-detectable copper only under aerobic conditions, and EDTA caused release of Cu(II) on heating. No reduction of the copper-thiolate units in Cu-thionein by ascorbate was detected. Potentiometric titrations with hexachloroiridate(IV) or hexacyanoferrate(III) produced several different Cu(II) EPR signals at various stages of oxidation. The former oxidizing agent required a lower oxidation-reduction potential (+350 mV) to oxidize the copper, than the latter (+410 mV) and neither titration was fully reversible. The EPR signal from Cu(II) oxidized by hexachloroiridate(IV) resembled that produced by p-hydroxy-mercuribenzoate in air, suggesting that the copper was released from its thiolate ligands. It is concluded that the EPR non-detectable copper in the native protein is Cu(I). Oxidation-reduction of the copper-thiolate clusters of Cu-thionein is proposed to be decisive for controlling storage and transport of cellular copper.     

Cu(II)-reduction by <Emphasis Type="Italic">Escherichia coli</Emphasis> cells is dependent on respiratory chain components

Copper is both an essential nutrient and a toxic element able to catalyze free radicals formation which damage lipids and proteins. Although the available copper redox species in aerobic environment is Cu(II), proteins that participate in metal homeostasis use Cu(I). With isolated Escherichia coli membranes, we have previously shown that electron flow through the respiratory chain promotes cupric ions reduction by NADH dehydrogenase-2 and quinones. Here, we determined Cu(II)-reductase activity by whole cells using strains deficient in these respiratory chain components. Measurements were done by the appearance of Cu(I) in the supernatants of cells exposed to sub-lethal Cu(II) concentrations. In the absence of quinones, the Cu(II)-reduction rate decreased ~70% in respect to the wild-type strain, while this diminution was about 85% in a strain lacking both NDH-2 and quinones. The decrease was ~10% in the absence of only NDH-2. In addition, we observed that quinone deficient strains failed to grow in media containing either excess or deficiency of copper, as we have described for NDH-2 deficient mutants. Thus, the Cu(II)-reduction by E. coli intact cells is mainly due to quinones and to a lesser extent to NDH-2, in a quinone-independent way. To our knowledge, this is the first in vivo demonstration of the involvement of E. coli respiratory components in the Cu(II)-reductase activity which contributes to the metal homeostasis.     

The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli

Copper is essential but can be toxic even at low concentrations. Coping with this duality requires multiple pathways to control intracellular copper availability. Three copper-inducible promoters, controlling expression of six copper tolerance genes, were recently identified in Escherichia coli. The cue system employs an inner membrane copper transporter, whereas the cus system includes a tripartite transporter spanning the entire cell envelope. Although cus is not essential for aerobic copper tolerance, we show here that a copper-sensitive phenotype can be observed when cus is inactivated in a cueR background. Furthermore, a clear copper-sensitive phenotype for the cus system is revealed in the absence of O(2). These results indicate that the cue pathway, which includes a copper exporter, CopA, and a periplasmic oxidase, CueO, is the primary aerobic system for copper tolerance. During anaerobic growth, however, copper toxicity increases, and the independent cus copper exporter is also necessary for full copper tolerance. We conclude that the cytosolic (CueR) and periplasmic (CusRS) sensor systems differentially regulate copper export systems in response to changes in copper and oxygen availability. These results underscore the increased toxicity of copper under anaerobic conditions and the complex adaptation of copper export in E. coli.     

An investigation into copper catalyzed D-penicillamine oxidation and subsequent hydrogen peroxide generation

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.     

Morphology of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown either aerobically or anaerobically on agar plates--observation by light and laser microscopes

Growth characteristics including cell-arrangement of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown on agar plates in level 3 laboratory were observed by both light and laser microscopes. Small daughter colonies appeared on parent colonies grown on 5% sheep blood or chocolate agar plates after 12 days incubation at room temperature. Daughter colonies, stained by Wirtz-Conklin method, were composed with vegetative cells and spores. Growth of daughter colonies might be supported by the debris of cells in the parent colony. Colonies grown under anaerobic conditions were flat with smooth edges, and the cells neither formed chains of any length, nor produced any spores after 25 days incubation at room temperature. It was thought that spores of B. anthracis were produced at the terminal stage of individual cell life instead of under unfavorable conditions for the organism. Air is needed for spore formation and cell-chain formation. More nutrients, probably amino acids, are needed for anaerobic growth rather than aerobic.     

Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge

AIMS: The study investigated the physiology of Amaricoccus kaplicensis to determine whether it could outcompete polyphosphate accumulating bacteria in activated sludge systems removing phosphorus, by preferentially assimilating substrates in the anaerobic stages of these processes. METHODS AND RESULTS: The storage processes were investigated under anaerobic, anoxic and aerobic conditions in both batch and periodically fed cultures in an aerobic sequencing batch reactor (SBR). Amaricoccus kaplicensis showed a high capacity for storing aerobically large amounts of acetate as poly beta-hydroxybutyrate (PHB) at high rates. However, no acetate assimilation under anaerobic conditions and very slow assimilation under anoxic conditions could be detected. CONCLUSION: Amaricoccus kaplicensis in pure culture does not behave as polyphosphate accumulating bacteria competitor; therefore it is difficult to understand why anaerobic/aerobic systems often contain such large numbers of Amaricoccus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Amaricoccus kaplicensis is probably not responsible for the failure of activated sludge systems removing phosphorus, and other organisms capable of anaerobic substrate assimilation should be sought.     

Thiol-copper(I) and disulfide-dicopper(I) complex O2-reactivity leading to sulfonate-copper(II) complex or the formation of a cross-linked thioether-phenol product with phenol addition

In order to better understand copper mediated oxidative chemistry via ligand-Cu(I)/O(2) reactivity employing S-donor ligands for copper, O(2)-reactivity studies of the copper(I) complexes (1 and 2, Chart 2) have been carried out with a tridentate N(2)S thiol ligand (1-(N-methyl-N-(2-(pyridin-2-yl)ethyl)amino)propane-2-thiol; L(SH)) or its oxidized disulfide form (L(SS)). Reactions of [L(SH)Cu(I)](+) (1) and [L(SS)(Cu(I))(2)(X)(2)](2+) (2) with O(2) give approximately 90% and approximately 70% yields of [L(SO3)Cu(II)(MeOH)(2)](+) (3), respectively, where L(SO3) is S-oxygenated sulfonate; 3 was characterized by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. Mimicking TyrCys galactose oxidase cofactor biogenesis, a new C-S bond is formed (within new thioether moiety L(SPhOH)) from cuprous complex (both 1 and 2) dioxygen reactivity in the presence of 2,4-tBu(2)-phenolate. In addition, the disulfide ligand (L(SS)) reacts with 2equiv. cupric ion salts and the phenolate to efficiently give the cross-linked product L(SPhOH) in high yield (>90%) under anaerobic conditions. Separately, complex [L(SPhO)Cu(II)(ClO(4))] (4), possessing the cross-linked L(SPhOH), was characterized by ESI mass spectrometry and X-ray crystallography.     

Mechanism of the synergistic cytotoxicity between pentachlorophenol and copper-1,10-phenanthroline complex: the formation of a lipophilic ternary complex

When non- or sub-toxic levels of pentachlorophenol (PCP) and bis-(1, 10-phenanthroline)cupric complex, Cu(II)(OP)(2), were combined, a remarkable synergistic toxicity was observed as indicated by growth inhibition and bacterial inactivation. Similar synergistic cytotoxic effects were observed with other polychlorinated phenols and other positively charged cupric complexes. The synergism observed for these chemicals and similar reactive pairs of chemicals was found to be due to the formation of lipophilic ternary complexes which facilitated copper transport into the bacterial cells. The formation of ternary complexes of similar lipophilic character could be of relevance as a general mechanism of toxicity.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Extraction of copper from an oxidized (lateritic) ore using bacterially catalysed reductive dissolution

An oxidized lateritic ore which contained 0.8 % (by weight) copper was bioleached in pH- and temperature-controlled stirred reactors under acidic reducing conditions using pure and mixed cultures of the acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans. Sulfur was provided as the electron donor for the bacteria, and ferric iron present in goethite (the major ferric iron mineral present in the ore) acted as electron acceptor. Significantly more copper was leached by bacterially catalysed reductive dissolution of the laterite than in aerobic cultures or in sterile anoxic reactors, with up to 78 % of the copper present in the ore being extracted. This included copper that was leached from acid-labile minerals (chiefly copper silicates) and that which was associated with ferric iron minerals in the lateritic ore. In the anaerobic bioreactors, soluble iron in the leach liquors was present as iron (II) and copper as copper (I), but both metals were rapidly oxidized (to iron (III) and copper (II)) when the reactors were aerated. The number of bacteria added to the reactors had a critical role in dictating the rate and yield of copper solubilised from the ore. This work has provided further evidence that reductive bioprocessing, a recently described approach for extracting base metals from oxidized deposits, has the potential to greatly extend the range of metal ores that can be biomined.     

Acute Copper and Cupric Ion Toxicity in an Estuarine Microbial Community

Copper was acutely toxic to the estuarine microbial community of Middle Marshes, N.C. Under ambient water quality conditions, 10 μg of added total copper [Cu(II)] liter⁻¹ reduced the CFU bacterial abundance by up to 60% and inhibited the amino acid turnover rate (AATR) by as much as 30%. Copper toxicity, however, was a quantitative function of free cupric ion (Cu²⁺) activity that was not directly related to Cu(II) or ligand-bound copper. By using a nitrilotriacetic acid-cupric ion buffer to control pCu (−log Cu²⁺ activity), it was found that an in situ pCu of 10.1 was bactericidal, reducing the CFU by 60%, but inhibited the AATR by only about 10%. A bacterial bioassay that was used to estimate the pCu in Cu(II)-treated Middle Marshes samples indicated that less than 0.5% of added Cu(II) was in the free cupric ion form. CFU was a more sensitive indicator of low-level copper stress than was AATR. When tested at different times, native microbial community responses to acute cupric ion stress were quantitatively quite similar even when there were large differences in bacterial abundances and in situ metabolic rates. Variations were observed in response to Cu(II) treatments at different times, but these were likely due to differences in water quality, which would quantitatively influence the distribution of copper complexes that were present. Asymptotic response curves suggest that some degree of copper resistance exists in this community. At a pCu of 8, more than 2 orders of magnitude above the minimum inhibitory level, the CFU was still 5 to 10% and the AATR was about 3% of the control values.     

Use of 31P nuclear magnetic resonance spectroscopy and electron microscopy to study phosphorus metabolism of microorganisms from wastewaters.

We used 31P nuclear magentic resonance to study the transfer of phosphorus between Pis and polyphosphates in microorganisms involved in wastewater treatment. We showed that the transfer process is reversible and of the first order in accordance with the polyphosphate concentration. The presence of nitrates in the anoxic phase led to results similar to those obtained during the aerobic phase. (Anoxic implies absence of oxygen but presence of nitrate, whereas anaerobic implies absence of oxygen and nitrate. In bacteriology, the term anoxic is not common, and the term anaerobic implies absence of oxygen and includes the conditions under which nitrate is present.) We observed that carbon dioxide lowers the pH, which entails a hydrolysis of polyphosphates, and helium seems to stop the evolution of the cells. Further, 2,4-dinitrophenol decouples the oxidative phosphorylation and brings about a decrease in the polyphosphate pool.     

Use of 31P nuclear magnetic resonance spectroscopy and electron microscopy to study phosphorus metabolism of microorganisms from wastewaters

We used 31P nuclear magentic resonance to study the transfer of phosphorus between Pis and polyphosphates in microorganisms involved in wastewater treatment. We showed that the transfer process is reversible and of the first order in accordance with the polyphosphate concentration. The presence of nitrates in the anoxic phase led to results similar to those obtained during the aerobic phase. (Anoxic implies absence of oxygen but presence of nitrate, whereas anaerobic implies absence of oxygen and nitrate. In bacteriology, the term anoxic is not common, and the term anaerobic implies absence of oxygen and includes the conditions under which nitrate is present.) We observed that carbon dioxide lowers the pH, which entails a hydrolysis of polyphosphates, and helium seems to stop the evolution of the cells. Further, 2,4-dinitrophenol decouples the oxidative phosphorylation and brings about a decrease in the polyphosphate pool.