The surface-associated and secreted MopE protein of Methylococcus capsulatus (Bath) responds to changes in the concentration of copper in the growth medium

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Evidence for the presence and structure of asparagine-linked oligosaccharide units in the core protein of proteodermatan sulphate.

Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein.     

Acid and alkaline phosphatase activity in the cyanobacteriumAnacystis nidulans under copper stress

The effect of lethal concentration of copper ions on the activities of acid and alkaline phosphatases was investigated in the cyanobacteriumAnacystis nidulans and the cyanophage AS-1 resistant mutant. When the level of phosphate declined in the medium, the cells were induced to form alkaline phosphatase (periplasmic protein) and acid phosphatase (cytoplasmic protein). In the presence of copper, the level of enzymes was low, suggesting that synthesis and activity were not completely abolished by copper. This may be related to the permeability of cell membrane.     

Induction of alternative oxidase by excess copper in sycamore cell suspensions

Sycamore suspension cells (Acer pseudoplatanus L.) were used to investigate the effect of copper on respiratory electron transport. Alternative oxidase (AOX) protein content and enzymatic capacity increased as a function of the concentration of copper added to the culture medium, from 0.2 to 50 μM. The latter sublethal concentration, which arrests mitochondrial biogenesis and thereby decreases cell respiration rates, stimulated cyanide-insensitive oxygen uptake in cells and mitochondria isolated therefrom. This was correlated with the accumulation of two proteins (30 and 36 kDa) which reacted with monoclonal antibodies against AOX. An accumulation of a 1.6-kb AOX mRNA was also observed. The possible mechanisms through which copper affects AOX are discussed.     

Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of Dactylium dendroides

The synthesis and subcellular localization of the two superoxide dismutases of Dactylium dendroides were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 μM) copper, manganese was poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Purification and characterization of abundant secreted protein in suspension-cultured pumpkin cells : abundant secreted protein may be a chitinase

The abundant secreted protein with molecular weight of 32,000 was purified from the culture medium of suspension-cultured pumpkin (Cucurbita sp.) cells. Two steps, ammonium sulfate fractionation and Sepharose 6B column chromatography, were sufficient for purification to homogeneity. Antibodies against the pure protein were used to show that a protein of the same size is made by callus cells. There is considerable homology between the amino-terminal amino acid sequence of this secreted protein and chitinase isolated from tobacco (Nicotiana tabacum L.) or bean (Phaseolus vulgaris L.).     

Effects of Phosphate in Medium on Protein Secretion in a Protein-Producing Bacterium, Bacillus brevis 47

Bacillus brevis 47 secreted up to 1 mg of protein per ml in a chemically defined medium, depending on phosphate concentration. The composition of exoproteins was altered quantitatively by the concentration of external phosphate. Morphologically, B. brevis 47 showed a distinct three-layered cell wall structure and shed the outer two layers during growth.     

Copper accumulation and metabolism in primary monolayer cultures of rat liver parenchymal cells

The characteristics of hepatic copper accumulation and metabolism were studied using primary monolayer cultures of adult rat liver parenchymal cells. Accumulation of copper from serum-free medium was temperature dependent and strongly inhibited by cyanide and N-ethylmaleimide. Addition of various concentrations of zinc to the medium did not alter copper accumulation by the cells. Furthermore, it was found that supplementation of the cell cultures with dexamethasone significantly stimulated zinc accumulation without affecting the accumulation of copper. Cycloheximide substantially stimulated accumulation of copper from the culture medium, whereas actinomycin D had no effect. Efflux experiments showed that copper is rapidly sequestered by intracellular components and becomes unavailable for exchange soon after it is transported into the cells. Gel chromatography of liver cytosol demonstrated that most of the cooper that is initially accumulated is bound to the low molecular weight cytoplasmic protein metallothionein.     

Resistance to copper toxicity of cultured hepatoma cells. Characterization of resistant cell lines

A series of four cell lines resistant to the toxic effect of copper were developed from Morris rat hepatoma cells by gradually increasing the concentration of copper in the growth medium. The EC50, that concentration of copper that kills and/or inhibits the growth of 50% of the cells after 72 h, increased 4-fold over that for wild type cells in the most resistant cell line. These cells were also resistant to zinc, cadmium, and mercury toxicity, but not to nickel or cobalt. The amount of copper in the soluble protein pool of the resistant cells increased proportionally with the concentration of copper in the medium in which they were maintained. Associated with copper accumulation was the production of an 18-kDa cysteine-rich protein which complexes a significant amount of the metal. It is suggested that resistance to copper toxicity is due to sequestration of the metal by this protein. When resistant cells were removed from the copper-enriched environment, cellular copper levels rapidly fell to that observed for wild type cells, but no reduction in either the EC50 or the level of the cysteine-rich protein was noted. This suggests that a permanent change responsible for copper resistance had occurred which is maintained in the absence of the metal.     

Incorporation of Copper Ions into T2/T3 Centers of Two-Domain Laccases

Laccase belongs to the family of copper-containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2/T3 centers of recombinant two-domain laccase Streptomyces griseoflavus Ac-993. The occupancy of the T3 center by copper ions was found to increase with an increasing copper content in the culture medium and after dialysis of the protein preparation against a copper sulfate-containing buffer. The T2 center was filled only when overproducer strain cells were grown at a higher copper concentration in the medium. Two-domain laccases were assumed to possess a channel that serves to deliver copper ions to the T3 center during the formation of the three-dimensional laccase conformation and dialysis of the protein preparation. A narrower channel leads to the T2 center in two-domain laccases compared with three-domain ones, rendering the center less accessible for copper atoms. The incorporation of copper ions into the T2 center of two-domain laccases is likely to occur in the course of their biosynthesis or the formation of a functional trimer.     

Extracellular Heme Peroxidases in Actinomycetes: a Case of Mistaken Identity

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.     

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.     

Copper susceptibility in Acinetobacter junii BB1A is related to the production of extracellular polymeric substances

Acinetobacter junii BB1A cells, grown in different media, were differentially inhibited in the presence of the copper. The minimum inhibitory concentration of Cu²⁺ was influenced by the nutrient status of the media. The production of extracellular polymeric substances (EPS) was stimulated by the copper present in the growth medium. The nature of the EPS was anionic showing non-Newtonian pseudoplastic behaviour. The thermal behaviour of the EPS was studied by differential scanning calorimetry. The EPS was amorphous in nature with a crystalline index of 0.16. Scanning electron micrographs revealed its porous structure. Cells grown in the presence of quorum sensing inhibitor (QSI: 4-Nitropyridine-N-oxide) did not produce EPS and were found to be more sensitive to Cu²⁺ than cells which produced EPS in the absence of QSI. EPS production in different media in the presence and absence of Cu²⁺ was determined. The production of EPS was the highest in brain heart Infusion medium and the lowest in AB minimal medium. The sorption of Cu²⁺ by EPS extracted from cells grown in non-copper-complexing AB medium was demonstrated by energy dispersive X-ray spectroscopy. A pertinent functional aspect of EPS in providing protection to A. junii in copper stress condition has been revealed.     

Studies on the Production of Fungal Peroxidases in Aspergillus niger

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.     

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu²⁺ was inoculated with 10⁷ CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.     

Selection and characterization of cadmium tolerant cells in tomato

Tomato (Lycopersicon esculentum cv. VFNT-Cherry)cell lines tolerant of to 5 mM cadmium (Cd) were selected by progressively elevating the level of CdCl₂ in the culture medium (the lethal concentration of Cd for unselected tomato cells is 400 μM). Cd tolerance was not lost during long-term culture (up to 12 months) in the absence of Cd stress. In all the cell lines examined, Cd uptake was rapid and Cd concentration within the cells exceeded that in the culture medium by several fold. While Cd included the synthesis and accumulation of phytochelatins (PCs) [poly[γ-glutamyl-cysteiny)glycine], little change has been observed in protein synthesis during short term Cd stress. PCs formed complexes with Cd. However, uptake and accumulation of Cd was not affected if PC synthesis was inhibited by treatment with buthionine sulfoximine. Selected and unselected cells were compared for their growth characteristics in the presence of various other metal ions. Cd tolerant cells showed a slightly higher tolerance of copper but not of mercury, zinc, lead or silver.     

The Secretome of Alginate-Encapsulated Limbal Epithelial Stem Cells Modulates Corneal Epithelial Cell Proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P≤0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.     

Phosphatase production and activity in copper (II) accumulating Rhizopus delemar

The fungus Rhizopus delemar produced extracellular and cellular acid phosphatase during the growth in starch-supplemented medium in the presence or absence of copper ions. The levels of both AP-ase activities were maximal at the end of exponential growth phase and were dependent on copper concentrations. Copper ions in the medium provoked slight decrease of specific AP-ase activities and significant increase of the values of secreted enzyme per gram dry cells. On the other hand, an increase of copper ions in the reaction mixture leads to considerable increase of the values of cellular enzyme activity. Total uptake of copper (II) was highest at the highest copper (II) concentration, when resting cells were used. Between 27 and 30% copper (II) was not removed by acid washing, suggested that this copper was bound intracellularly by mycelium. Determination of the Michaelis constant for the cellular AP-ase gave value of 0.325 mM. The pH optimum of the enzyme was determined to be in the range of 3.5–4.5 using p-nitrophenyl phosphate (pNPP) as a substrate. The data obtained indicated a possible participation of AP-ases in the processes of heavy metal resistance and heavy metal uptake of this fungus.