9 Beyond biogeography: large-scale patterns of distribution and abundance of intertidal marine algae along the us west coast

This research was directed to gather a better understanding about the unicellular alga Chlamydomonas acidophila, one of the 6 algal species found in the Berkeley Pit Lake. Berkeley Pit Lake is a flodded, abandoned pit mine with a pH of 2.7 and high metal concentrations. It has been found that the effective concentrations of metals that limit the growth of C. acidophila by 50% were 9.024 mg/L for Cu2+ and 75.4 mg/L for Zn 2+. We have been able to grow C. acidophila from Berkeley Pit samples at high densities in medium containing 15.36 mg/L Cu2+ and 83.65 mg/L Zn2+. Moreover, this species is able to grow in nutrified Berkeley Pit water, which contains approximately 110 mg/L Cu2+ and 323 mg/L of Zn2+. The hypothesis is that the species found in Berkeley Pit Lake represents a genetic strain adapted to high metal concentrations environments. A comparison between the American Type Culture Collection strain of C. acidophila and the strain collected from Berkeley Pit was made. Growth rate of the two strains in Bold Basil Medium, Modified Acid Medium and Berkeley Pit nutrified water were calculated and compared. Moreover, preliminary investigations of the genome of C.acidophila from the Berkeley Pit Lake were initiated.     

Isolation, growth, ultrastructure, and metal tolerance of the green alga, Chlamydomonas acidophila (Chlorophyta)

An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance. Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5-4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50%, EC50 were measured, after 72 h of static exposure. EC50s were 14.4 microM Cd2+, 81.3 microM Co2+, 141 microM Cu2+, and 1.16 mM Zn2+ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas.     

金属离子对粪产碱杆菌C16的脱氮和亚硝酸盐积累的影响

【目的】研究不同金属离子对异养氨氧化细菌C16的生长和脱氮性能影响,探讨适于C16生长和脱氮的金属离子及其浓度。【方法】实验选用Mg2+、Mn2+、Fe2+、Cu2+、Zn2+5种金属离子,对C16的生长﹑脱氮性能﹑亚硝酸盐氮积累以及相关酶活性进行研究。【结果】Mg2+明显促进C16的生长和NH4+-N氧化速率;较高浓度Mn2+使得C16无法生长;原培养基中缺少Fe2+会抑制C16的生长和NH4+-N氧化速率;在原培养基中加入0.1 mmol/L的Cu2+对C16的生长和脱氮具有一定的促进作用,Cu2+使得培养基中基本无NO2--N和NH2OH的积累;不同浓度的Zn2+对C16的生长和氨氮去除有抑制作用。酶活实验结果显示,0.1 mmol/L Mg2+促进了羟胺氧化还原酶(HAO)的活性;0.1 mmol/L Cu2+促进了硝酸盐还原酶(Nar)和亚硝酸盐还原酶(Nir)的活性。【结论】Mg2+是C16生长和脱氮过程中的一种重要金属离子;加入Cu2+可避免过量亚硝酸盐积累。     

匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+ 胁迫的生长响应与阈限浓度

采用砂培法,研究了匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+胁迫的生长响应及阈限浓度,结果表明:种子萌发率随着4种重金属浓度的增加而下降。对株高的影响是当重金属浓度小于100mg/L时会促进株高生长,高于100mg/L则产生抑制作用。Cu2+显著抑制根系生长,并随浓度的增加抑制效应愈加显著;在Cu2+浓度为600mg/L时匍茎翦股颖的根长比对照下降了93.75%。Cu2+、Zn2+、Pb2+浓度小于200mg/L时会促进地上生物量的增加,但高于200mg/L时,地上生物量会随着3种重金属的增加而减少。Cu2+、Zn2+浓度小于100mg/L或Cd2+、Pb2+浓度小于200mg/L会增加叶绿素的含量,高浓度会降低叶绿素的含量;Cd2+在浓度为600mg/L时显著降低叶绿素含量,与对照相比,下降了43.55%。匍茎翦股颖生长的综合效应分析表明,匍茎翦股颖对Cu2+胁迫最敏感,具有较低的阈限浓度,而Zn2+胁迫对匍茎翦股颖的生长影响最小,阈限浓度相对较高。     

Isolation,Growth, Ultrastructure,and Metal Tolerance of the Green Alga,Chlamydomonas acidophila (Chlorophyta)

An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance.

Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5-4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50%, EC₅₀ were measured, after 72h of static exposure. EC₅₀s were 14.4 μM Cd²⁺, 81.3 μM Co²⁺, 141μM Cu²⁺, and 1.16 mM Zn²⁺ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas.     

ALGAL BIOREMEDIATION OF THE BERKELEY PIT LAKE

Ongoing research is unraveling the intricacies of the microbial ecology of the Berkeley Pit Lake System, with ever increasing information becoming available regarding the diversity of Algae, Protistans, Fungi and Bacteria that inhabit this mine waste site. Defining the baseline community structure has been the first step not only toward understanding the interactions of the different groups of organisms, but also toward assessing any improvement in biodiversity within the biotic community. Now that this first step has begun, some of these extremophiles, specifically algae, that have been isolated from the Berkeley Pit Lake System are being used as a potential solution for bioremediation. The specific objectives of this research are fivefold: 1) To evaluate the bioremediative potential of our four most rapidly growing species: (Chromulina freiburgensis Dofl., Chlorella ellipsoidea Gerneck, Chlorella vulgaris Beyerinck and Chlamydomonas acidophilla Negoro) in Berkeley Pit Lake System Water with the additions of NaNO₃ and NaPO₄ by using an experimental matrix. This matrix will be used to estimate the minimum nutrient concentrations that would be necessary to achieve the maximum growth of algae and maximum bioremediation of the Berkeley Pit Lake System. 2) To determine which combination of nutrients will stimulate growth of the best bioremediator of our four isolated species in natural Berkeley Pit Lake System waters. In other words, what nutrient combination will give the best bioremediator a competitive edge over the other species. If time permits, different species may be grown in combination to determine if there are synergistic effects (protocooperation) between/among species. 3) To determine a temperature profile for these four species in order to determine their optimal growth temperature in Berkeley Pit Lake System water. 4) To continue to isolate organisms from the Berkeley Pit Lake System and determine their bioremediative potential. 5) Monitor algal and bacterial counts from a profile of Pit Lake System waters. The results to date will be presented for this conference.     

Biosorption of chromium, copper, manganese and zinc by Pseudomonas aeruginosa AT18 isolated from a site contaminated with petroleum

The study describes the sorption of Cr, Cu, Mn and Zn by Pseudomonas aeruginosa AT18 isolated from a site contaminated with petroleum and heavy metals. The concentrations studied were 50, 49, 60 and 70 (mg L(-1)) for Cr, Cu, Mn and Zn, respectively. The solution pH and ionic strength were very important factors in the metal biosorption performance and the biosorption capacity of P. aeruginosa AT18 for Cr3+,Cu2+, Mn2+ and Zn2+. In aqueous solution, the biosorption increased with increasing pH in the range 5.46-7.72. The results obtained in the experimental assays show that P. aeruginosa AT18 has the capacity for biosorption of the metallic ions Cr3+, Cu2+ and Zn2+ in solutions, although its capacity for the sorption of manganese is low (22.39 mg Mn2+/g of biomass) in comparison to the Cr3+, Cu2+ and Zn2+ ions, as shown by the individual analyses. However, 20% of the manganese was removed from an initial concentration of 49.0 mg L(-1), with a Qm value similar to that obtained in solutions containing mixtures of Cr3+, Cu2+, Mn2+and Zn2+. The chromium level sorbed by P. aeruginosa AT18 biomass was higher than that for Cu, Mn and Zn, with 100% removal in the pH range 7.00-7.72 and a Qm of 121.90-200.00 mg of Cr3+/g of biomass. The removal of Cr, Cu and Zn is also a result of precipitation processes.     

Expression of human metallothionein III and its metalloabsorption capability in Escherichia coli

Human metallothionein III (MT III) gene was synthesized with Escherichia coli preference codon usage and expressed in E. coli in glutathione-S-transferase (GST) fusion form. The recombinant MT III was released by proteinase Factor Xa digestion and purified with the yield of 2 mg/L culture, and its specific Cd2+ binding capability was confirmed. E. coli strain BL21(DE3), expressing MT III, showed metal tolerance between 0.1 and 0.5 mM Cd2+ and bacterial growth was inhibited at 1 mM Cd2+. MT III expressing E. coli strain showed binding discrimination between different metal ions in combination use, with the preference order of Cd2+ > Cu2+ > Zn2+. It absorbed different metal ions with relatively constant ratio and showed a cumulative absorption capability for mixed heavy metals.     

Removal of Zn2+ and Cu2+ by a sequencing batch reactor (SBR) system

Both resting (living) and autoclaved (dead) bio-sludges showed almost the same Cu2+ and Zn2+ adsorption capacities with synthetic industrial estate wastewater (SIEWW). The resting bio-sludge showed not only Cu2+ and Zn2+ adsorption abilities but also organic matter adsorption ability. But, the organic matter (COD and BOD5) adsorption ability of bio-sludge with SIEWW containing 60 mg/L Cu2+ was about half of that with SIEWW containing 60 mg/L Zn2+. The adsorbed Cu2+ and Zn2+ were easily eluted (70-75%) from bio-sludge with 0.1 N HNO3 and 0.1 M EDTA solutions. Bio-sludge from a wastewater treatment plant could be used as an adsorbent for metal ions (Cu2+ and Zn2+). Cu2+ and Zn2+ could repress the SBR system efficiency but its efficiency could be increased with the increase of mixed liquor suspended solids (MLSS), and Cu2+ had more effect than Zn2+ to repress the system efficiency. The SBR system showed very low removal efficiencies of the pollutants with industrial estate wastewater (IEWW), but its pollutant removal efficiencies with IEWW could be increased with the addition of glucose. The Zn2+, Cu2+, BOD5, COD and TKN removal efficiencies of the system with IEWW containing 1.27 g/L glucose, 10 mg/L Cu2+ and 10 mg/L Zn2+ under MLSS of 4500 mg/L were 92.61 +/- 0.28%, 83.77 +/- 0.93%, 98 +/- 0%, 92 +/- 0% and 78.1 +/- 0.1%, respectively.     

重金属离子对凡纳滨对虾肝胰脏、鳃丝和血液SOD活力的影响

研究了3种重金属离子(Cu²⁺、Zn²⁺、Cd²⁺)在96 h内对凡纳滨对虾(Litopenaeus vannamei)对肝胰脏、鳃丝和血液超氧化物歧化酶(SOD)活力的影响.结果表明,凡纳滨对虾SOD活力在3种重金属离子作用下随取样时间变化显著(P＜0.0),Cu²⁺在实验浓度范围内(0.1～1 mg·L^-1),肝胰脏、鳃丝和血液的SOD活力随时间延长呈一峰值变化,Zn²⁺在10 mg·L^-1时对肝胰脏表现为显著抑制作用,Cd²⁺在0. mg·L^-1时对肝胰脏和鳃丝起显著抑制作用,0.2 mg·L^-1对鳃丝SOD活力无显著变化(P＞0.0),其他浓度Zn²⁺(＜10 mg·L^-1)、Cd²⁺(＜0.2 mg·L^-1)对各组织器官SOD活力的影响随时间延长均呈现先升高后下降的趋势.3种重金属离子对凡纳滨对虾肝胰脏、鳃丝、血液SOD活力的影响呈现明显的剂量-时间效应关系.其SOD活力大小顺序为肝胰脏＞鳃丝＞血液,3种重金属离子对凡纳滨对虾伤害大小顺序为Cd²⁺＞Cu²⁺＞Zn²⁺.     

煤矿区耐镉青霉菌的分离鉴定

[目的]分离鉴定煤矸石中耐Cd2+菌株.[方法]用菌落形态和18S rRNA序列分析鉴定菌株,研究菌株的重金属耐性和在酸性煤矸石浸出液的生长能力,分析其抗氧化酶活性对重金属复合污染的响应.[结果]BJKD4菌株为青霉属(Penicillium sp.)菌,能耐29 mmol/L的Cd2+,不同重金属对BJKD4的毒性大小依次为:Cu2+＞Ni2+＞Cd2+＞pb2+或Zn2+＞Mn2+.正交试验表明BJKD4菌株能在不同浓度重金属Cd、Zn、Ni和Mn等复合污染条件下生长,SOD活性在重金属复合污染时升高,CAT活性变化依重金属的种类和浓度不同而不同;此外,BJKD4能在含有煤矸石酸性浸出液的培养基中生长,并提高其pH.[结论]BJKD4菌株能耐多种重金属,具有阻止煤矸石山淋溶液酸化的应用潜力.抗氧化酶在减缓重金属诱导的氧化胁迫中起重要作用.     

一株红假单胞菌的分离及对Cu2+的去除

【背景】重金属污染对环境和人类的健康构成了重大威胁,因此,重金属污染的治理迫在眉睫。生物治理因成本低、处理效果好和无二次污染等优点,在处理重金属污染时被优先选择。【目的】从辽河入海口深13 m的水体中,利用紫色非硫细菌富集培养基筛选产色素能力强并对高浓度Cu~(2+)有高效去除能力的菌株。【方法】采用形态学、生理生化特性和分子生物学方法鉴定菌种;采用二乙基二硫代氨基甲酸钠分光光度法测定Cu~(2+)的含量。【结果】鉴定菌株为红假单胞菌属,将其命名为Rhodopseudomonas sp. gh32。菌株gh32的最适生长温度和最适pH分别为30°C和7.0,其在pH 5.0-10.0范围内可正常生长,在3 mmol/L的CuSO4溶液中能够正常生长,能利用葡萄糖、甘露糖和果糖等单糖和硫化氢。菌株gh32在24h内对Cu~(2+)的去除率均在99%以上,处理能力为1 331 g/g干菌重或167.6 g/g湿菌重。【结论】菌株gh32对Cu~(2+)具有较强的耐受性和很好的去除效果,是治理含Cu~(2+)废水的潜力菌株。本研究为生物治理重金属废水提供了支持。     

Superoxide-scavenging activity of carnosine in the presence of copper and zinc ions

In the presence of Cu2+ and Zn2+ carnosine (beta-alanyl-L-histidine) possesses a superoxide-scavenging activity. The efficiency of scavenging as measured by the inhibition of tetrazolium nitroblue reduction in superoxide anion generation systems (phenazine methasulfate/NADH and xanthine/xanthine oxidase) is concentration-dependent and shows a maximum in the presence of millimolar concentrations of carnosine and equimolar concentrations of Cu2+ and Zn2+. In the presence of Cu2+ and Zn2+ histidine also exhibits a superoxide-scavenging activity. The feasible role of the superoxide-scavenging activity of histidine-containing dipeptide complexes with bivalent metal ions in the realization of physiological function of these dipeptides in skeletal muscles is discussed.     

Characterization of Desmodesmus pleiomorphus isolated from a heavy metal-contaminated site: biosorption of zinc

Microalgae have been proven efficient biological vectors for heavy metal uptake. In order to further study their biosorption potential, a strain of Desmodesmus pleiomorphus (L) was isolated from a strongly contaminated industrial site in Portugal. Under different initial Zn²⁺ concentrations, metal removal by that strain reached a maximum of 360 mg Zn/g biomass after 7 days, at 30 mg Zn/l, after an initial rapid phase of uptake. Comparative studies were carried out using a strain of the same microalgal species that is commercially available (ACOI 561): when exposed to 30 mg Zn/l, it could remove only 81.8 mg Zn/g biomass. Biosorption experiments using inactivated biomass of the isolated strain reached a maximum Zn²⁺ uptake of 103.7 mg/g. Metal removal at various initial pH values was studied as well; higher removal was obtained at pH 5.0. The microalga strain L, isolated from the contaminated site, exhibited a much higher removal capacity than the commercial strain, and the living biomass yielded higher levels of metal removal than its inactivated form.     

BACTERIAL EFFECTS AND ALGAL BIOREMEDIATION BY CHLORELLA ELLIPSOIDEA GERNECK OF THE BERKELEY PIT LAKE SYSTEM

Mitman, G. G. Department of Biological Sciences, Montana Tech of The University of Montana, Butte, MT 59701 The Berkeley Pit Lake System is one of the largest contaminated sites in North America and is located near the headwaters of the largest superfund site in the U.S. It is filling at a rate of about 28.7 million liters per day with metal laden, acidic (pH 2.7) water. Chlorella ellipsoidea is one of the first autochthonous species of algae from the Berkeley Pit Lake System to be tested for its bioremediative potential. An experimental matrix was designed for this experiment using a completely randomized design (CRD). The matrix was set up with tissue culture flasks having the following treatments: Na2HPO4 at 0, 25, 50, 75, 100 mg/l, inoculated vs. no- inoculated with algae (187,500 algae/ml final), and filtered (0.2Fm) vs. non filtered pit water as variables, NaNO₃ amount was fixed at 53 mg/ml. Three replicates were made of each and the experiment lasted 60 days. The results of this experiment demonstrated significant remediation of most metals. These results will be presented. In addition, filtering seemed to negatively impact the bioremediative potential of the cultures. As a result, axenic cultures of Chlorella ellipsoidea will be tested in Berkeley pit water to determine if it is bacteria or filtration that caused these results.     

Influence of multiple metal ions on beta-amyloid aggregation and dissociation on a solid surface

Recently discovered evidences suggest that precipitation of Alzheimer's beta-amyloid (Abeta) peptide and the toxicity in Alzheimer's disease (AD) are caused by abnormal interactions with neocortical metal ions, especially Zn2+, Cu2+, and Fe3+. While many studies had focused on the role of a "single" metal ion and its interaction with Abeta peptides, such studies involving "multiple" metal ions have hardly been explored. Here, to explore the nature of codeposition of different metals, two or more metal ions along with Abeta were incubated over a solid template prepared by immobilizing Abeta42 oligomers. The influence of Zn2+,Cu2+, and Fe3+ on Abeta aggregation was investigated by two approaches: co-incubation and sequential addition. Our results using ex situ AFM, ThT-induced fluorescence, and FTIR spectroscopy indicated that the co-incubation of Cu2+, Zn2+, and Fe3+ significantly altered the morphology of aggregates. A concentration dependence study with mixed metal ions suggested that Zn2+ was required at much lower concentrations than Cu2+ to yield nonfibrillar amorphous Abeta deposits. In addition, sequential addition of Zn2+ or Cu2+ on fibrillar aggregates formed by Fe3+ demonstrated that Zn2+ and Cu2+ could possibly change the conformation of the aggregates induced by Fe3+. Our findings elucidate the coexistence of multiple metal ions through their interactions with Abeta peptides or its aggregates.     

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn2+. This Zn2+ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn2+. However, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no additional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu2+. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn2+ for optimal activity.     

一株耐铅、锌、铬菌株的分离鉴定及其吸附能力

【背景】采矿带来的土壤重金属污染问题对生态环境和人类健康产生了极大的危害,因此矿区重金属污染问题亟待解决。生物修复法成本低、资源广、没有二次污染,是修复土壤重金属污染的有效途径。【目的】以山东省曲阜市某煤矸石山周围土壤为材料,采用平板划线法分离筛选一株同时对铅、锌、铬有高耐性且吸附能力强的菌株。【方法】通过形态学特征、生理生化鉴定、分子生物学方法鉴定菌种;采用原子吸收分光光度计测量各重金属的浓度。【结果】鉴定菌株为蜡状芽孢杆菌(Bacillus cereus),命名为Bacillus cereus MZ-11。菌株MZ-11在pH为5.5-8.5、温度为15-45°C、NaCl质量分数为2%-8%时可正常生长。菌株MZ-11最高耐铅、锌、铬浓度高达1 000、1 200、1 600 mg/L。在培养基中铅、锌、铬3种重金属浓度分别为初始浓度(含Pb2+、Zn2+、Cr6+浓度分别为50、60、80 mg/L)、初始浓度的5倍、10倍、20倍的条件下,MZ-11对Cr6+和Zn2+的吸附比例则随浓度的提高逐渐增大,吸附百分比均在98%-99%以上。对Pb2+的吸附比例有一个最高值,并且P...     

Mixed-Species Biofilms Cultured from an Oil Sand Tailings Pond can Biomineralize Metals

Here, we used an in vitro biofilm approach to study metal resistance and/or tolerance of mixed-species biofilms grown from an oil sand tailings pond in northern Alberta, Canada. Metals can be inhibitory to microbial hydrocarbon degradation. If microorganisms are exposed to metal concentrations above their resistance levels, metabolic activities and hydrocarbon degradation can be slowed significantly, if not inhibited completely. For this reason, bioremediation strategies may be most effective if metal-resistant microorganisms are used. Viability was measured after exposure to a range of concentrations of ions of Cu, Ag, Pb, Ni, Zn, V, Cr, and Sr. Mixed-species biofilms were found to be extremely metal resistant; up to 20 mg/L of Pb, 16 mg/L of Zn, 1,000 mg/L of Sr, and 3.2 mg/L of Ni. Metal mineralization was observed by visualization with scanning electron microscopy with metal crystals of Cu, Ag, Pb, and Sr exuding from the biofilms. Following metal exposure, the mixed-species biofilms were analyzed by molecular methods and were found to maintain high levels of species complexity. A single species isolated from the community (Rhodococcus erythropolis) was used as a comparison against the mixed-community biofilm and was seen to be much less tolerant to metal stress than the community and did not biomineralize the metals.     

Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae. Kinetics and mechanism

The mechanism of copper uptake in Saccharomyces cerevisiae has been investigated using a combination of 64Cu2+ and atomic absorption spectrophotometry. A wild type copper-resistant CUP 1R-containing strain and a strain carrying a deletion of the CUP1 locus (yeast copper metallothionein) exhibited quantitatively similar saturable energy-dependent 64Cu2+ uptake when cultures were pregrown in copper-free media (medium [Cu] approximately 15 nM). The kinetic constants for uptake by the wild type strain were Vmax = 0.21 nmol of copper/min/mg of protein and Km = 4.4 microM. This accumulation of 64Cu2+ represented net uptake as confirmed by atomic absorption spectrophotometry. This uptake was not seen in glucose-starved cells, but was supported in glycerol- and ethanol-grown ones. Uptake was inhibited by both N3- and dinitrophenol and was barely detectable in cultures at 4 degrees C. When present at 50 microM, Zn2+ and Ni2+ inhibited by 50% indicating that this uptake process was relatively selective for Cu2+. 64Cu2+ accumulation was qualitatively and quantitatively different in cultures either grown in or preincubated with cold Cu2+. Either treatment resulted in the appearance of a fast phase (t 1/2 approximately 1 min) of 64Cu2+ accumulation which represented isotopic exchange since it did not lead to an increase in the mass of cell-associated copper; also, it was not energy-dependent. Exchange of 64Cu2+ into this pool was not inhibited by Zn2+. Pretreatment with Cu2+ caused a change in the rate of net accumulation as well; a 3-h incubation of cells in 5 microM medium Cu2+ caused a 1.6-fold increase in the velocity of energy-dependent uptake. Prior addition of cycloheximide abolished this Cu2(+)-dependent increase and, in fact, inhibited the 64Cu2+ uptake velocity by greater than 85%. The exchangeable pool was also absent in cycloheximide, Cu2(+)-treated cells suggesting that exchangeable Cu2+ derived from the copper taken up initially by the energy-dependent process. The thionein deletion mutant was similar to wild type in response to medium Cu2+ and cycloheximide indicating that copper metallothionein is not directly involved in Cu2+ uptake (as distinct from retention) in yeast.