The dichotomy of mechanisms of copper accumulation in yeast induced by enhanced levels of copper as well as menadione in growth media

Abstract

A high copper accumulation is induced in the yeast Saccharomyces cerevisiae either by menadione at the level of 200 μM in the growth medium, or by elevated concentrations of copper. While the uptake, as well as the toxicity of copper, strongly depend on the zinc concentration in the medium, there is no influence of zinc on copper intake induced by menadione. The copper binding ligand d-penicillamine suppresses the accumulation of copper in the menadione systen, whereas it has virtually no effect in the copper system. Within the range of non-toxic concentrations, copper is predominantly taken up by an energy-dependent mechanism. In contrast, the accumulation in the menadione system is clearly energy-independent. Thus there exist at least two different mechanisms for the uptake of copper in the yeast Saccharomyces cerevisiae.     

Studies on the production of (S)-(+)-solketal (2,2-dimethyl-1,3-dioxolane-4-methanol) by enantioselective oxidation of racemic solketal with Comamonas testosteroni

All strains of Comamonas testosteroni investigated here, produced quinohaemoprotein ethanol dehydrogenase (QH-EDH) when grown on ethanol or butanol, but one strain of C. acidovorans and of C. terrigena did not. Hybridization experiments showed that the gene for QH-EDH is absent in the latter two strains. Induction and properties of the QH-EDHs seem to be similar: all C. testosteroni strains produced the enzyme in its apo-form [without pyrroloquinoline quinone (PQQ)] and the levels were higher at growth at low temperature; preference for the R-enentiomer and similar selectivity was shown in the oxidation of solketal (2,2-dimethyl-1,3-dioxolane-4-methanol) by cells (supplemented with PQQ); the fragment of the qhedh gene gave high hybridization with the DNA of the C. testosteroni strains. Experiments with C. testosteroni LMD 26.36 revealed that the organism is well suited for production of (S)-solketal: it shows an adequate enantioselectivity (E value of 49) for the oxidation of racemic solketal; the conversion rate of (R)-solketal is only 3.5 times lower than that of ethanol; the optimal pH for conversion (7.6) is in a region where solketal has sufficient chemical stability; separation of the remaining (S)-solketal from the acid formed is simple; induction of QH-EDH, the sole enzyme responsible for the oxidation of (R)-solketal, occurs during growth on ethanol or butanol so that the presence of solketal (inhibitory for growth) is not required; production of active cells and the conversion step can be integrated into one process, provided that PQQ and solketal addition occur at the appropriate moment; the conversion seems environmentally feasible. However, since high concentrations of solketal inhibit respiration via QH-EDH, further investigations on the mechanism of inhibition and the stability of the enzyme might be rewarding as it could lead to application of higher substrate concentrations with consequently lower down-stream processing costs.     

HEAVY METAL ACCUMULATION AND THE NATURE OF EDAPHIC ENDEMISM IN THE GENUS CALOCHORTUS (LILIACEAE)

Bulb and leaf tissues from five species in the genus Calochortus and the soils from collection sites were analyzed for cation and trace element concentrations. All five species, only three of which are considered ultramafic endemics, possessed high concentrations of nickel and copper in both the bulbs and leaves, but not at levels high enough (> 1% dry wt) to be considered hyperaccumulators. Only moderate to trace amounts of cobalt and chromium were detected in both plant tissues. Calcium-magnesium ratios, typically low in ultramafic soils and plants, were low in bulb tissue, but two to 10 times higher in leaf tissue. The ability to tolerate excessively high levels of nickel and other heavy metals may be a physiological exaptation of the genus Calochortus and not necessarily an evolutionary response by several species to life on an ultramafic substrate. The nature of “serpentine” endemism is discussed in the context of heavy metal accumulation by various plant species.     

Speciation of copper in relation to its bioavailability

Abstract

Copper speciation and bioavailability for Scenedesmus quadricauda has been studied in natural waters and in synthetic culture media. Other elements were studied simultaneously. When phosphorus and nitrogen limitation were excluded by adding these elements, copper was limiting algal growth in some natural waters. In the toxic range, growth inhibition by copper was highly correlated with copper detected by electrochemical methods and with calculated free copper.

Copper was toxic to S. quadricauda when free copper concentrations roughly exceeded 10^?10.5 M, and was limiting for values somewhere lower than 10^?12.5 M. Because we found copper limitation in some natural water samples, free copper concentration in those water samples therefore must have been lower than 10^?12.5 M.

The hypothesis that the free metal concentration rather than the total concentration determines bioavailability was confirmed for copper, cobalt and zinc.     

Effects of indole‐3‐butyric acid on growth,pigments and UV‐screening compounds in Nostoc linckia

In the present research, the effect of indole‐3‐butyric acid (IBA) on the growth, and the production of some primary and secondary metabolites was studied in Nostoc linckia. In this respect, algae cultures were supplied with 0, 0.01, 0.1, 1, 10, and 100 μM IBA for 14 days. IBA at concentrations of 10 and 100 μM induced algal growth expressed as fresh weight in N. linckia. Treatment with IBA at all concentrations stimulated heterocyst formation. In addition, low concentrations of IBA (0.01, 0.1, and 1 μM) had a stimulatory effect on chlorophyll a and carotenoids accumulation. In contrast, higher concentrations of IBA induced the accumulation of phycocyanin, allophycocyanin, and phycoerythrin in the treated algae. In this case, IBA at the concentration of 10 μM was more effective. A significant decrease in protein content was observed in the algae treated by 0.01 μM IBA. All concentrations of IBA caused a decrease in sugar content, but lower concentrations were more effective. IBA application in all of the concentrations except 100 μM increased oligosaccharide‐linked mycosporine‐like amino acids (OS‐MAAs) content. Lower concentrations had a more significant effect on increasing OS‐MAAs content. However the concentrations of 10 and 100 μM IBA decreased scytonemin content. These results indicated the stimulatory impact of IBA on weight, heterocyst formation, and photosynthetic pigments in N. linckia.     

NUTRIENT REQUIREMENTS OF THE DINOFLAGELLATE PERIDINIUM GATUNENSE1

Optimum nutrient conditions for growth and photosynthesis of Peridinium gatunense (Nygaard) (Peridinium cinctum fa. westii) were investigated using axenic clones in batch cultures. Selenium (Se) had previously been found to be an indispensable growth factor for P. gatunense. Optimal, suboptimal, and supraoptimal concentrations of HCO₃^?, N, Ca, Cl, Mg, P, K, S, Si, EDTA-Na, Fe, Mo, Zn, Mn, Co, Se, B, Br, I, and various trace element mixtures were determined by measuring biomass development, growth rates, ¹⁴C uptake, and/or oxygen production at various concentration gradients of these elements. The general characteristics of the best formulation, medium-L 16, relative to other media, are its high content of NaHCO₃ (1 meq · L^?1) and Mo (0.2 μM) but low concentrations of NO_3-N (150 μM), PO_4-P (10 μM), and Fe (0.4 μM), in addition to its content of Se. The total content of trace metals, except for Se, may be reduced to one-fourth of that in medium-L 16 without altering the major growth-promoting properties of the medium. Medium-L 16 deviated considerably from Lake Kinneret (Israel) water, being much lower in macroelements except for N and P. The pH (8.1–8.4) was in the same range, but the values of conductivity (140 μS · cm^?1), alkalinity (1 meq · L^?1) and NaCl (200 μM) were > 8, 2, and 30 times higher, respectively, in the lake water. Selenium deficiency may limit the growth of P. gatunense in this lake.     

Unexpected fluctuations of trace element levels in cell culture medium in vitro: caveat emptor

Copper is an essential trace element micronutrient in human and animal nutrition. Trace amounts present even in ultrapure water, serum and other cell culture medium components are sufficient to support the health requirements of most cell types in culture. Analysis of a variety of different types of basal media from a number of different suppliers revealed large fluctuations in the levels of copper, and also of other micronutrients including zinc, iron, selenium and cobalt. Investigations on proliferating Caco-2 cells revealed reductions in growth with increasing copper concentrations within the range seen in the commercial media and changes in expression of apoptosis- and autophagy-related proteins were noted. Even at concentrations of 1 μM CuSO₄ where there was no significant change in cell growth, there was a significant decrease in procaspase-3 expression. These results stress the importance of batch testing of basal media when undertaking trace metal research since the baseline levels may vary. Batch variation of serum is well established but our results suggest that batch variation of the media may also be important.     

Trace Element Distribution in Selected Edible Tissues of Zebu (Bos indicus) Cattle Slaughtered at Jimma,SW Ethiopia

The amount of trace elements present in edible bovine tissues is of importance for both animal health and human nutrition. This study presents data on trace element concentrations in semitendinosus and cardiac muscles, livers and kidneys of 60 zebu (Bos indicus) bulls, sampled at Jimma, Ethiopia. From 28 of these bulls, blood samples were also obtained. Deficient levels of copper were found in plasma, livers, kidneys and semitendinosus muscles. Suboptimal selenium concentrations were found in plasma and semitendinosus muscles. Semitendinosus muscles contained high iron concentrations. Trace elements were mainly stored in the liver, except for iron and selenium. Cardiac muscles generally contained higher concentrations of trace elements than semitendinous muscles except for zinc. A strong association was found between liver and kidney concentrations of copper, iron, cobalt and molybdenum. Liver storage was well correlated with storage in semitendinosus muscle for selenium and with cardiac muscle for cobalt and selenium. Plasma concentrations of copper, selenium, cobalt were well related with their respective liver concentrations and for cobalt and selenium, also with cardiac muscle concentrations. The data suggest multiple trace element deficiencies in zebu cattle in South-West Ethiopia, with lowered tissue concentrations as a consequence. Based on the comparison of our data with other literature, trace element concentrations in selected edible tissues of Bos indicus seem quite similar to those in Bos taurus. However, tissue threshold values for deficiency in Bos taurus cattle need to be refined and their applicability for Bos indicus cattle needs to be evaluated.     

Trace metal concentrations in the tropical sponge Spheciospongia vagabunda at a sewage outfall: synchrotron X-ray imaging reveals the micron-scale distribution of accumulated metals

Major and trace elements were measured in sponges, seawater and sediment in Darwin Harbour (Australia) to test the hypothesis that metals are elevated in sponges closer to a sewage outfall compared with unimpacted sites. Seawater and sediment at the sewage discharge site contained high, but localised, concentrations of phosphorus (P), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As) and lead (Pb) compared with background sites. Metal concentrations in the sponge Spheciospongia vagabunda were highly elevated compared with other sponges and, although site specific, high metal concentrations were unrelated to the presence of sewage effluent. X-ray fluorescence microprobe imaging was used to investigate the metal distribution pattern in S. vagabunda. High Fe, Ni and Zn concentrations were either localised in circular patches (100–200 μm size) near water canals or in the pinacoderm, or scattered in spots (approximately 10 μm) throughout the tissue. This supports a microflora-mediated metal bioaccumulation hypothesis. In contrast, Co and Mn were highly dispersed and probably associated with aluminium- and iron-oxide rich sediment inclusions. Although the lack of association between sewage effluent and metal accumulation precludes the use of S. vagabunda as a biomonitor, the apparent differential mechanisms of metal accumulation warrants further investigation.     

Fatty acids and lysophospholipids as potential second messengers in auxin action. Rapid activation of phospholipase A2 activity by auxin in suspension-cultured parsley and soybean cells

Activation of cytosolic phospholipase A₂ is a typical signal transduction reaction in animal cells and occurs in plants in response to auxin, elicitors and wounding. Exogenously added fluorescent bis-BODIPY-phosphatidylcholine was taken up and hydrolysed by a cellular phospholipase A₂. Rapid activation of a phospholipase A₂ by auxin in suspension-cultured parsley ( Petrosilenum crispum L.) and soybean ( Glycine max L.) cells was shown by detection and quantification of fluorescent reaction products of phospholipase A₂. Hormone-triggered fluorescent fatty acid accumulation could be detected as early as 5 min. Auxins at 2 μM or higher concentrations activated phospholipase A₂ and fluorescent fatty acids accumulated 1.1- to threefold after 90–120 min, depending on the auxin concentration. Fluorescent lysolipid did not accumulate up to 150 μM auxin. Known inhibitors of phospholipase A₂ inhibited hormone-dependent fluorescent fatty acid accumulation in cell cultures and, previously, elongation growth in etiolated zucchini hypocotyl segments ( Scherer & Arnold (1997 ) Planta 202, 462–469). When lipids were labeled by [¹⁴C]-choline and [¹⁴C]-ethanolamine the corresponding lysophospholipids could be quantified in cell extracts. Radioactive lysophospholipids accumulated as rapidly as 1–2 min after auxin treatment but only at concentrations well above 100 μM auxin. We hypothesize that phospholipase A₂ activation is an early intermediate step between receptor and downstream responses. We hypothesize that fatty acid(s) could be second messengers in several auxin functions, especially in cell elongation. Lysophospholipids seem to be indicators or second messengers for stress caused by high auxin concentrations or may have different auxin-linked functions and are also known to accumulate during elicitor action.     

Effets du cuivre sur l'ultrastructure de Scenedesmus quadricauda et Chlorella vulgaris. Effect of copper on the ultrastructure of Scenedesmus quadricauda and Chlorella vulgaris

A laboratory investigation was conducted to study the effect of various concentrations of copper on the freshwater algae Scenedesmus quadricauda and Chlorella vulgaris. Electron-dense inclusions were observed in the vacuoles of S. quadricauda, and this alga showed some growth at a dose as high as 2,500 μg.1⁻¹. The occurrence of the electron-dense inclusions was correlated significantly with the copper concentration and seems to be implicated in the tolerance of the species. C. vulgaris was much more sensitive to copper than S. quadricauda and showed osmotic changes and membrane damage.     

Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.     

Two aquatic macrophytes as bioindicators for medium-high copper concentrations in freshwaters

Abstract

Plant scions of Ceratophyllum demersum L. and Potamogeton natans L. were exposed in controlled conditions to different concentrations of copper during approximately 2 weeks; Fv/Fm was monitored at regular intervals and relative growth rate (RGR) was calculated at the end of the trial. P. natans was affected by Cu concentrations starting from 2 μM; C. demersum started to show significant reductions in growth and photosynthetic efficiency from 4 μM Cu. As it results from the observed data, the two aquatic macrophytes can be used as valid bioindicators for medium-high copper concentrations in freshwaters.     

Anti-Candida Activity of Cal protect in in Combination with Neutrophils or Lactoferrin

The effect of an anti-microbial protein, calprotectin, in combination with neutrophils on the growth of Candida albicans was investigated. The growth inhibition of C. albicans by murine neutrophils was augmented by the addition of a low concentration of calprotectin prepared from rat peritoneal exudate cells. The concentrations of calprotectin causing 50% inhibition of growth of C. albicans in the absence or presence of neutrophils at an effector-to-target (E/T) ratio of 30 and 60 were estimated to be 0.45, 0.34 and 0.28 U/ml, respectively. The anti-Candida activity of calprotectin was completely inhibited by 2 μM of zinc ion, while it only partially lowered the activity of the combination of calprotectin and neutrophils. Lactoferrin, which is an anti-microbial protein released from neutrophils, strongly inhibited the growth of C. albicans in combination with calprotectin. These results suggest that calprotectin and lactoferrin released from neutrophils may cooperate to inhibit the growth of C. albicans at a local lesion of the infection where there is an accumulation of neutrophils.     

Seminal plasma trace metal levels in industrial workers

This study compares the seminal plasma trace metal levels of hospital workers with groups of industrial workers in a petroleum refinery, smelter, and chemical plant. The metals measured were the essential metals (copper, zinc, nickel, cobalt, and manganese) and the toxic metals (lead, cadmium, and aluminum). The group mean±SE metal level for each group (50 subjects per group) was calculated, and the statistical significance of the group mean differences of the industrial groups with the hospital group (control) was determined by the Student’s t-test. The differences observed in the smelter group were increased copper and zinc (p≤0.001) and decreased nickel, cobalt, and manganese (p≤0.001,≤0.01). The refinery group differences were increased copper, zinc, and nickel (p≤0.001) but decreased cobalt and manganese (p≤0.001). The chemical group differences were increased zinc (p≤0.001) and decreased cobalt (p≤0.001). The seminal plasma levels of the toxic metals lead and aluminum were increased in each of the industrial groups (p≤0.001). Concurrent differences were (1) decreased accumulation of nickel, cobalt, and manganese in the smelter group, (2) decreased cobalt and managanese in the refinery group, and (3) only decreased cobalt in the chemical group.     

TOXICITY OF COPPER TO THALASSIOSIRA PSEUDONANA IN UNENRICHED INSHORE SEAWATER1

Toxicity of copper to T. pseudonana (formerly Cyclotella nana, clone 13-1) wax examined in inshore seawater using a 96-hr bioassay method. Raw unenriched seawater wax filtered through a 0.22-μ membrane filter and then pasteurized, for 30 min at 60 C. Following this treatment, samples contained 0.68–1.14 μg Cu/liter. Copper was added as the chloride in 5-μg increments over the range of 5 to 30 μg/liter (about 0.1–0.5 μM). Population densities, mean cell, volume, and ¹⁴C bicarbonate uptake were measured. Population growth, and ¹⁴C uptake by T. pseudonana displayed inhibition over the entire range of added copper. Growth rate constant (k) of T. pseudonana decreased with increasing copper concentration and during the course of growth at each concentration. Correspondingly, mean cell volumes increased with copper concentration and time. Copper toxicity varied in different water samples. The presence of decomposed natural plankton and detritus decreased toxicity. In the absence of enrichment, bacteria had little effect on copper toxicity. H exults were influenced by glassware treatment, collection and storage of seawater, and absence of enrichments.     

Effect of cadmium on growth, proton extrusion and membrane potential in maize coleoptile segments

Cd accumulation, its effects on elongation growth of maize coleoptile segments, pH changes of their incubation medium and the membrane potential of parenchymal cells were studied. The Cd content increased significantly with exposure to increasing cadmium concentrations. Coleoptile segments accumulated the metal more efficiently in the range 10–100 μM Cd, than in the range 100–1000 μM Cd. Cd at concentrations higher than 1.0 μM produced a significant inhibition of both growth and proton extrusion. 100 μM Cd caused depolarization of the plasma membrane (PM) potential in parenchymal cells. The simultaneous treatment of maize coleoptile segments by indole-3-acetic acid (IAA) and Cd, counteracted the toxic effect of Cd on growth. Moreover, our data also showed that 100 μM Cd suppressed the characteristic IAA-induced hyperpolarization of the membrane potential, causing membrane depolarization. These results indicate that the toxic effect of Cd on growth of maize coleoptile segments might be, at least in part, caused via reduced PM H⁺-ATPase activity.     

Copper resistance in Desulfovibrio strain R2

A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of zinc accumulation potential of Hydrilla verticillata

Biofortification of foods with essential micronutrients and phytoremediation of the contaminated sites are the two sides of the same coin for metals like zinc. In the present study, Zn accumulation potential, growth and antioxidant status of Hydrilla verticillata (L.f.) Royle plants were studied upon supplementation of Zn (0–5 000 μM) for 2 and 7 d. At 5000 μM Zn, plants accumulated about 7.60 and 18.07 mg(Zn) g⁻¹(d.m.) after 2 and 7 d, respectively. Plants exposed to Zn concentrations up to 500 μM showed significantly increased contents of low molecular mass antioxidants and activities of antioxidant enzymes in comparison with controls. Only upon exposure of plants to 5 000 μM Zn, toxicity was observed after 7 d. Therefore, owing to their high Zn accumulation capacity, Hydrilla plants may be used both as a Zn source (via culturing in ca. 100 μM Zn supplemented nutrient medium) or as a phytoremediator.     

Characterization of Copper-Resistant Rhizosphere Bacteria from <Emphasis Type="Italic">Avena sativa</Emphasis> and <Emphasis Type="Italic">Plantago lanceolata</Emphasis> for Copper Bioreduction and Biosorption

Copper is a toxic heavy metal widely used to microbial control especially in agriculture. Consequently, high concentrations of copper residues remain in soils selecting copper-resistant organisms. In vineyards, copper is routinely used for fungi control. This work was undertaken to study copper resistance by rhizosphere microorganisms from two plants (Avena sativa L. and Plantago lanceolata L.) common in vineyard soils. Eleven rhizosphere microorganisms were isolated, and four displayed high resistance to copper. The isolates were identified by 16S rRNA gene sequence analysis as Pseudomonas putida (A1), Stenotrophomonas maltophilia (A2) and Acinetobacter sp. (A6), isolated from Avena sativa rhizosphere, and Acinetobacter sp. (T5), isolated from Plantago lanceolata rhizosphere. The isolates displayed high copper resistance in the temperature range from 25°C to 35°C and pH in the range from 5.0 to 9.0. Pseudomonas putida A1 resisted as much as 1,000 mg L⁻¹ of copper. The isolates showed similar behavior on copper removal from liquid medium, with a bioremoval rate of 30% at 500 mg L⁻¹ after 24 h of growth. Speciation of copper revealed high copper biotransformation, reducing Cu(II) to Cu(I), capacity. Results indicate that our isolates are potential agents for copper bioremoval and bacterial stimulation of copper biosorption by Avena sativa and Plantago lanceolata.