Heavy metal tolerance of marine phytoplankton. II. Copper tolerance of three species in dialysis and batch cultures

The tolerance to copper ions of three diatoms, namely, Skeletonema costatum, (Grev.) Cleve, Thalassiosira pseudonana (Hust.) Hasle and Phaeodactylum tricornutum Bohlin grown in dialysis and batch cultures in the local fjord water has been established. Reduction of growth rates was observed by the addition of 10, 25 and 400 μg/1 of copper ions, respectively for the three species investigated. At the higher levels of copper addition (400 and 700 MS/1) cells of P. tricornutum in dialysis culture increased their copper content to more than 200 times over those of the controls, the ratio of copper to chlorophyll in the cells increasing 150 times.All three species showed marked increases in copper content when a copper salt was added to batch cultures of the algae. The two clones of Skeletonema costatum tested showed nearly identical sensitivity to copper ions, but they differed markedly in their zinc tolerance.     

Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases in vivo, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells.     

Effect of rate of increase of water temperature on sensitivity of fish digestive glycosidases to action of copper and zinc

There was established an increase of sensitivity of glycosidases of intestinal mucosa of juvenile goldfish Carassius auratus, carp Cyprinus carpio, roach Rutilus rutilus, and common perch Perca fluviatilis to action in vitro of heavy metal ions (copper and zinc at concentrations of 0.1–25 mg/l) at heating of water at the summer period. The inhibitory effect of copper and zinc ions as a rule was higher at the heating rate of 50°C/h than at the rate of 4°C/h. The perch glycosidases were less sensitive to the elevation of temperature and to effect of the copper ions as compared with the enzymes of the carp species.     

Heavy metal tolerance of marine phytoplankton. I. The tolerance of three algal species to zinc in coastal sea water

Tolerance levels to zinc ions of three diatoms (Skeletonema costatum (Grev.) Cleve, Thalassiosira pseudonana (Hust.) Hasle and Phaeodactylum tricornutum (Bohlin) grown in dialysis culture in the local fjord water were studied. Declining relative growth rates were observed by addition of 50, 250 and 25,000μg/l of zinc ions, respectively, for the three algae. Reduced final cell concentrations were found at lower zinc levels. At least for one species a significant increase in zinc uptake by the cells took place at zinc levels which did not seem to influence the growth and development of the alga. Two clones of Skeletonema costatum studied showed significant intraspecific differences regarding the tolerance to zinc pollution. Dialysis bioassay was found suitable for monitoring heavy metal pollution of aquatic recipients.     

Resistance of <Emphasis Type="Italic">Acidiplasma</Emphasis> archaea to heavy metal ions

Effect of the ions of heavy metals (copper, zinc, and nickel) on growth of and ferrous iron oxidation by extremely acidophilic archaeal strains of the genus Acidiplasma (Acidiplasma sp. MBA-1; A. aeolicum V1^T; and A. cupricumulans BH2^T) was studied. Effect of the metals depending on their concentration was studied within the range from 5 to 1000 mM. All studied strains were able to oxidize iron in the presence of the highest tested heavy metal concentrations (1000 mM). All metal ions had, however, a noticeable inhibitory effect both on both growth and on iron oxidation. The lowest concentrations of copper, zinc, and nickel partially suppressing microbial growth were determined (5, 10, and 5 mM, respectively). These findings are of interest, since almost no literature data on resistance of Acidiplasma archaea to heavy metals are available, although these prokaryotes are among the most important groups of microorganisms used in biohydrometallurgy.     

Cell Wall Composition of Neurospora crassa Under Conditions of Copper Toxicity

The mycelia of Neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. The cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. Although all the glucosamine of the cell wall of control cultures could be released within 6 h of hydrolysis with acid, that of the blue mycelium required prolonged hydrolysis for 24 h. On removal of copper, the cell wall of the blue mycelium could quantitatively bind again to copper as well as to zinc. Although zinc binding was fivefold greater, copper alone was preferentially bound from a mixture of the two metal ions. Supplementation of iron along with copper in the culture medium resulted in the disappearance of the blue color of the mycelium and restoration of normal growth and composition of the cell wall, probably by limiting the uptake of copper from the medium. The possibility of the cell wall being a specific site of lesion in copper toxicity in the mold is discussed.     

Insights into the Role of the Unusual Disulfide Bond in Copper-Zinc Superoxide Dismutase

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30–50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.     

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.     

Selective binding behavior of zinc(II) and copper(II) ions to their native sites of apo-bovine superoxide dismutase

The four binding constants of zinc(II) ions to apo-bovine superoxide dismutase were measured by the method of equilibrium dialysis. The binding constants (10(11.1)-10(10.9) M-1) of zinc ions to the native zinc sites were much larger than those to the native copper sites (10(7.8)-10(6.5) M-1) at pH 6.25. The competitive reaction between copper(II) and zinc(II) ions for the native copper sites of copper free bovine superoxide dismutase was also investigated. The native copper sites of bovine superoxide dismutase selectively react with copper ions, because the binding constants of copper ions for the native copper sites were much larger (10(6) times) than those of zinc ions.     

Association Between Zinc, Copper, and Iron Concentrations in Breast Milk and Growth of Healthy Infants in Tabriz, Iran

The aims of this study were to determine the effect of breast milk zinc, copper, and iron concentrations on infants’ growth and their possible correlations with maternal dietary intake. Milk samples and information on food intake were collected from 182 lactating women. Concentrations of zinc, copper, and iron in milk were analyzed using atomic absorption spectrophotometry. The infant’s weight for age Z-score (WAZ) and height for age Z-score (HAZ) were calculated. The mean milk zinc, copper, and iron concentrations were 1.85?±?0.5, 0.53?±?0.3, and 0.85?±?0.2 mg/l, respectively. Only zinc mean level was lower than the recommended range. Association between zinc, copper, and iron concentrations of milk and WAZ or HAZ of infants were not significant. However, the WAZ of infants whose mothers' milk zinc was more than 2 mg/l was significantly (P?<?0.039) higher than for others. The mean dietary zinc (5.31?±?2.3 mg/day) and copper (1.16?±?0.7 mg/day) intake of mothers was significantly less than the required daily intake (RDA) recommendations (P?<?0.05). The mean dietary iron intake (11.8?±?8.2 mg/day) was significantly higher than RDA recommendation (P?<?0.001). No significant association was found between maternal mean dietary zinc, copper, and iron intakes with their concentrations in milk. Dietary consultation or/and zinc supplementation is suggested for lactating women and infants.     

INFLUENCE OF THE CLAY TYPES ILLITE AND MONTMORILLONITE ON THE UPTAKE OF65 Zn BY SCENEDESMUS OBLIQUUS.

SUMMARY

The stimulatory effect of the clays illite and montmorillonite on zinc uptake by Scenedesmus obliquus is discussed. The competitive effects of hydrogen, phosphate, magnesium and copper ions on zinc uptake are illustrated. This competition is reduced in the presence of illite and montmorillonite and zinc uptake is stimulated. Ferrous ions have no effect on zinc uptake, while calcium ions antagonize zinc uptake at high concentrations. The latter effect was remarkable in the presence of montmorillonite.     

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.     

Potentiometric stripping analysis of zinc and copper in human teeth and dental materials.

Potentiometric stripping analysis (PSA) with oxygen as the oxidant has been used to determine soluble zinc and copper levels in exfoliated human teeth (all of which required extraction for orthodontic reasons) and commercial dental materials. The soluble zinc and copper contents of teeth were slightly below the zinc and copper contents in whole teeth reported by other researchers, except in the case of tooth with removed amalgam filling. Soluble zinc and copper concentrations of the dental materials and metal ceramic crowns were 0.50-6.30, and of 2.00-4.30 microg/g, respectively. The results of this work suggest that PSA may be a good method for zinc and copper leaching studies during the investigation of dental prosthetic materials' biocompatibility. Corrosive action of acidic media as evidenced by SEM micrographs caused the leaching of metal ions from teeth.     

Thermal aggregation of beta-lactoglobulin in presence of metal ions

In this work, we report a study of the effects of zinc and copper ions on the heat-induced aggregation of beta-lactoglobulin (BLG). Kinetics investigations on aggregates growth by light scattering measurements and on secondary structure changes by FTIR absorption measurements show the different role played by the two metals during the whole process. In particular, the presence of zinc in solution promotes the formation of aggregates of BLG at a lower temperature than copper. Then, at fixed temperature, formation of a large amount of aggregates, of large dimension, is observed for Zn-BLG in shorter time; on the contrary, the presence of copper in solution does not affect the aggregation process while the secondary structure changes and the formation of different stronger intermolecular H-bonds, which probably lead to build a network of bonds that takes towards gelation. Our studies show how time evolution of aggregation process of BLG is dramatically affected by the presence of metal ions in solution and structural protein modifications are induced by different divalent metal ions.     

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein,ZIPB

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn²⁺ and Cd²⁺, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.     

Influence of zinc on the calcium carbonate biomineralization of Halomonas halophila

Background

The salt tolerance of halophilic bacteria make them promising candidates for technical applications, like isolation of salt tolerant enzymes or remediation of contaminated saline soils and waters. Furthermore, some halophilic bacteria synthesize inorganic solids resulting in organic–inorganic hybrids. This process is known as biomineralization, which is induced and/or controlled by the organism. The adaption of the soft and eco-friendly reaction conditions of this formation process to technical syntheses of inorganic nano materials is desirable. In addition, environmental contaminations can be entrapped in biomineralization products which facilitate the subsequent removal from waste waters. The moderately halophilic bacteria Halomonas halophila mineralize calcium carbonate in the calcite polymorph. The biomineralization process was investigated in the presence of zinc ions as a toxic model contaminant. In particular, the time course of the mineralization process and the influence of zinc on the mineralized inorganic materials have been focused in this study.

Results

H. halophila can adapt to zinc contaminated medium, maintaining the ability for biomineralization of calcium carbonate. Adapted cultures show only a low influence of zinc on the growth rate. In the time course of cultivation, zinc ions accumulated on the bacterial surface while the medium depleted in the zinc contamination. Intracellular zinc concentrations were below the detection limit, suggesting that zinc was mainly bound extracellular. Zinc ions influence the biomineralization process. In the presence of zinc, the polymorphs monohydrocalcite and vaterite were mineralized, instead of calcite which is synthesized in zinc-free medium.

Conclusions

We have demonstrated that the bacterial mineralization process can be influenced by zinc ions resulting in the modification of the synthesized calcium carbonate polymorph. In addition, the shape of the mineralized inorganic material is chancing through the presence of zinc ions. Furthermore, the moderately halophilic bacterium H. halophila can be applied for the decontamination of zinc from aqueous solutions.     

Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens

Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.     

Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90% of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.     

Efficient expression of the yeast metallothionein gene in Escherichia coli.

The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.     

The influence of medium composition on alkaloid biosynthesis by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The fungus P. citrinum produces secondary metabolites, clavinet ergot alkaloids (EA), and quinoline alkaloids (quinocitrinines, QA) in medium with various carbon and nitrogen sources and in the presence of iron, copper, and zinc additives. Mannitol and sucrose are most favorable for EA biosynthesis and mannitol is most favorable for QA. Maximum alkaloid production is observed on urea. Iron and copper additives in the medium containing zinc ions stimulated fungal growth but inhibited alkaloid biosynthesis. The production of these secondary metabolites does not depend on the physiological state of culture, probably due to the constitutive nature of the enzymes involved in biosynthesis of these substances.