Laboratory evolution of copper tolerant yeast strains
© Adamo et al; licensee BioMed Central Ltd. 2012
Received: 27 August 2011
Accepted: 3 January 2012
Published: 3 January 2012
Yeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts.
We characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L-1 CuSO4 in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts.
Following evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L-1 CuSO4 and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper-binding proteome. However, copper elicits different physiological and molecular reactions in yeasts with different backgrounds.
Keywordsyeast copper adaptation evolutionary engineering oxidative stress response micronutrients
Metal ions like copper, manganese, zinc and iron are essential micronutrients for living organisms and play a central role in the cell metabolism being the cofactors of a large number of enzymes and electron transport proteins . The metabolism of copper and the mechanisms that control its intracellular concentration are the targets of intense studies since impairments in Cu level, transport and localization have been associated with several human diseases [2, 3]. In fact, while copper deficiency impacts the function of key cell enzymes, Cu overload can generate highly reactive oxygen species (ROS) which produce peroxidation of membrane lipids, displacement of other metal cofactors from their natural ligands in signalling proteins , oxidation of proteins and cleavage of DNA and RNA molecules  resulting in general cellular damage. Moreover, ROS are thought to play a major role in cancer development and in aging . To cope with such strict constraints, all organisms have developed complex regulatory mechanisms to maintain copper homeostasis.
Yeast cells are a good tool both for the investigation and the manipulation of copper metabolism. Studies on the accumulation of metals in edible microorganisms are of relevance for the production of functional foods enriched in micronutrients (for example the ones about the inclusion of iron, cobalt, copper and manganese in yeast cells ) and the industrial production of Saccharomyces cerevisiae biomass highly enriched with organic forms of selenium [7, 8]. Yeast cells resistant to and accumulating intracellular copper have been recently patented for cleaning copper from extracellular solutions  and use in pro-biotic, cosmetic, dietary and nutraceuticals products .
It has been reported that microorganisms can acquire stress tolerance and novel metabolic abilities when exposed to the appropriate selection pressure. This approach is often reported as "evolutionary engineering", a term introduced by Butler and collaborators in 1996 , since it uses evolutionary principles based on the selection of random mutants arising in the microbial population either spontaneously or upon mutagenesis and has been applied for improving complex physiological properties whose genetic and physiological basis is not fully understood . For example, microbial cells were recently evolved to improve their resistance towards multiple stresses , cobalt , iron- and sulfur-compounds , alcohols ; and to gain the ability to ferment xylose  and lactose .
S. cerevisiae is a powerful model organism to investigate copper metabolism and homeostasis in Eukaryotes. As a consequence, a large body of knowledge is available about Cu uptake, intracellular transport and functional role in yeast cells , as well as about non-enzymatic and enzymatic mechanisms of protection from ROS and oxidative stress .
In this study, yeast strains endowed with different natural robustness towards copper were compared with strains evolved by stepwise adaptation to tolerate high metal concentrations. We report that different and overlapping physiological and molecular responses are elicited in cells with different backgrounds to allow them to tolerate challenging conditions.
The tolerance of cells to metals is relevant both for understanding the mechanisms of defence towards stress and for the production of microorganisms enriched in a given micronutrient for biotechnological applications. We applied an evolution-based approach to improve robustness towards copper in all strains, independently from their natural background. The experimental protocol relied on the stepwise cultivation of cells in media supplemented with progressively higher concentrations of copper sulphate. At each step the culture was grown for 72 hours before withdrawing aliquots to be inoculated at a higher CuSO4 concentration. The starting condition was YPD + 1 g · L-1 CuSO4, which is permissive for all strains. The following steps were in YPD + 1.5 g · L-1 CuSO4, YPD + 2 g · L-1 CuSO4 and YPD + 2.5 g · L-1 CuSO4. This last one was the highest concentration applied, since above it copper salts led to acidification of the medium resulting in the precipitation of its protein components. Single colonies isolated after the last step of adaptation displayed relatively high rates of growth when directly re-inoculated in YPD + 2.5 g · L-1 CuSO4. These cells are defined in the following as "evolved".
The increase of intracellular copper in evolved S. cerevisiae was slower and at the end of the experiments we measured 2.1 to 4.2 mg Cu · g-1 biomass (Figure 3 b, c, d). The Cu content of non-evolved and evolved Saccharomyces cells was compared also in a milder condition, (1 g ·L-1 CuSO4). Whereas both kinds of BL7 cells displayed the same kinetics of copper accumulation (Additional file 3), non-evolved EL1 and GL6 showed a faster kinetic of bioaccumulation (Additional file 3) associated to a growth kinetic slower than in their evolved counterpart (Additional file 2). As in Candida samples, also in this case the behaviour of evolved and de-adapted cells was similar.
Results obtained up to this point suggested that all Saccharomyces strains are rather homogeneous in their response to copper, but different from the Candida one. Therefore, subsequent experiments aimed at highlighting possible adaptive changes were focused on a more in-depth comparison between Candida humilis and the only Saccharomyces cells strain BL7.
Antioxidant enzyme activities in S. cerevisiae and C. humilis cells grown in YPD and in YPD + CuSO4 2.5 g · L-1 (Cu)
20.93 ± 0
39.22 ± 0
64.14 ± 0.92
132.97 ± 17.09
117.2 ± 21.26
32.34 ± 1.08
41.1 ± 8.42
3.2 ± 0.45
3.23 ± 0.45
11.82 ± 1.26
81.5 ± 5.17
73.76 ± 6.74
74.14 ± 11.46
27.7 ± 4.4
0.001 ± 0
0.0032 ± 0
0.004 ± 0
0.011 ± 0.001
0.019 ± 0.003
0.014 ± 0
0.008 ± 0
0.041 ± 0.001
0.012 ± 0.001
0.013 ± 0.009
0.19 ± 0.024
0.534 ± 0.039
0.235 ± 0.039
0.172 ± 0.07
Identification of copper-binding proteins
NADP glutamate dehydrogenase 1
NADP glutamate dehydrogenase 2
Elongation factor 1-β
Uncharacterized oxidoreductase YMR226C
Triose phosphate isomerase
Cu, Zn-superoxide dismutase
Peptidyl-prolyl cis-trans isomerase
40S ribosomal protein S26-A
Hypothetical protein YIL051C (or MMF1)
60S ribosomal protein L19
Hypothetical protein YDR032C
Eucaryotic translation initiation factor 5A-2
Glyceraldehyde-3-phosphate dehydrogenase 2
Glyceraldehyde-3-phosphate dehydrogenase 3
Ubiquitin-40S ribosomal protein S31
In this work, we obtained copper-enriched and copper-tolerant yeasts through a number of generations smaller than reported in similar recent works [13, 14, 25]. This might be explained on the basis of a different experimental set-up in which relatively few rounds of selection were applied, but with longer cultivation times (72 h) and wider intervals of metal concentration (increases of 0.5 g. L-1 at each round). The effectiveness of the method of evolution is substantiated by the preservation of metal tolerance in absence of selective pressure (de-adapted cells), meaning that stable molecular changes occurred. This view is corroborated by the amplification of the CUP1 gene, which encodes for a metallothionein, detected in evolved S. cerevisiae cells (Adamo et al., in preparation). However, it is possible that other adaptive (transient) modifications contribute to the increased resistance.
In the frame of this complex picture, the comparison of yeasts differing in their robustness towards copper allowed us to investigate physiological differences involved in natural and acquired copper tolerance and to obtain some preliminary information about the molecular determinants of this trait. Taken together, our results hint at the concurrence of different mechanisms that we briefly summarize and discuss in the following.
i) Copper uptake. C. humilis cells can grow in copper medium due to their natural tolerance that can be further improved via evolutionary engineering. This feature allowed to compare the bioaccumulation of intracellular Cu in non-evolved and evolved cells at the highest copper concentration used (2.5 g ·L-1 CuSO4), showing that copper is lower in these latter. On this basis we hypothesize that one of the mechanisms of robustness might rely on hindering metal uptake. Consistently, the toxicity of Cu incorporated in the non-evolved strain (mainly in the first hours of growth) might account for the growth impairment and the increase in the rate of propidium positive cells we observed. In this light, the variability in copper sensitivity between Candida and Saccharomyces cells might depend (at least partly) on a different ability to limit copper uptake and its overload. Such a mechanism has been reported to protect S. cerevisiae cells from copper  cadmium  and cobalt  and points to a central role of the plasma membrane [28–30] and of the cell wall  in the onset of tolerance to heavy metals.
ii) Antioxidative enzymes and ROS production. Our results indicate that the biochemical bases of copper resistance can be deeply different among yeast species. In S. cerevisiae, evolution of copper tolerance is associated with the increase of antioxidative activities, as already documented by others , and with a reduced production of ROS. On the contrary, non-evolved cells suffer severe oxidative stress, as showed by the complete inhibition of their growth and the huge percentage of propidium positive cells. Basal activities of most detoxifying enzymes are higher in C. humilis than in S. cerevisiae cells, fact that could partly explain the natural copper tolerance of Candida cells and the reduced ROS production. The response of evolved Candida cells to copper is intriguing since our results show a reduction in the production of ROS, a generalised decrease of the antioxidative activities, and non-responsiveness to copper (compare values reported for evolved cells in YPD and copper medium). Indeed, in evolved cells superoxide dismutase is always lower than in the original strain, independently from the presence of copper. We would assume that the different activity profiles and their re-shaping upon evolution might reflect different and peculiar defence mechanisms responsible for both natural and acquired copper tolerance in Saccharomyces and Candida strains.
iii) Cu-binding proteins. The hypothesis above is further corroborated by the observation that the amounts of soluble Cu-binding proteins extracted from non-evolved and evolved cells are different too. Over-expression of Cu-binding proteins is consistent with their role in the primary response to copper exposure and results in copper tolerance . We are aware that the enrichment procedure used might lead to overestimation of some proteins that carry modifications such as thiolation [34, 35] or sequence motives that increase their affinity for the chromatographic resin or to underestimation of low-affinity proteins. Nevertheless, the differences detected between the two yeasts are marked and relevant proteins identified are in agreement with literature data reported by others. Among Cu-binding proteins, enzymes involved in sulphur metabolism (i.e. cystathionine-γ-lyase and aspartate-semialdehyde dehydrogenase) were over-expressed by evolved S. cerevisiae cells during growth in copper medium, suggesting that copper might redirect the metabolic flux towards the production of GSH to balance the redox equilibrium. The over-expression of glycolytic enzymes such as triose phosphate isomerase and GAPDH3 might be a consequence of the reconfiguration of the glycolytic flux, a mechanism reported to regulate the response to oxidative stress in human , plant  and yeast  cells. Furthermore, the increase of GAPDH3 is consistent with the role of this protein as a sensor of oxidative stress in DNA repair  and apoptosis [40, 41]. GAPDH3 increase is also the most remarkable effect triggered by copper in evolved C. humilis. Besides, these cells are enriched in the peroxiredoxin Tsa1 [42, 43], known to act as an antioxidant against ROS  and to protect actively translating ribosomes from stress conditions . On this basis we propose that in evolved Candida cells the inhibition of protein synthesis associated with oxidative stress  occurs in an attenuated form in comparison to non-evolved cells, that show a strong decrease of Cu-binding proteins, without any remarkable down regulation of the ribosomal proteins and other components of the protein translation apparatus.
The comparison between yeast cells naturally resistant or experimentally evolved to tolerate high copper concentration reported in this work supports the view that copper tolerance is due to multiple responses relying on different physiological and macromolecular changes. Yeasts endowed with copper tolerance and able to accumulate metal ions can find application in the biotechnology field for example for bioremediation or as dietary supplement, being these "GRAS organisms" valuable sources of microelements in organic form. Moreover, the comprehension of physiological and molecular responses of microorganisms to metal stress and of the mechanisms triggered during evolution of tolerance could help in the identification of biomarkers for ecotoxicological studies.
Yeast strains and growth conditions
Yeasts used in this study were isolated from sourdough. Strains were identified by Random Amplification of Polymorphic DNA-PCR (RAPD-PCR) and designated as Candida humilis AL5, and Saccharomyces cerevisiae BL7, EL1 and GL6 (Veneto Agricoltura - Istituto per la Qualità e le Tecnologie Agroalimentari - VI). Growth was on YPD medium [2% (w/v) glucose, 1% (w/v) yeast extract, 2% (w/v) tryptone] or on minimal medium [2% (w/v) glucose, 0.67% (v/v) yeast nitrogen base]. Solid media contained 2% (w/v) agar.
Copper tolerance was tested on cells grown shaking over night in 3 mL of liquid YPD at 30°C and subjected to serial dilutions in physiological solution [0.9% (w/v) NaCl]. 5 μL aliquots were spotted on either YPD or minimal medium plates containing CuSO4 and incubated at 30°C for 2 days.
Adaptative evolution was performed stepwise starting from cells taken from a fresh culture on agarized YPD and grown overnight at 30°C in 3 mL of liquid YPD + 1 g · L-1 CuSO4. In the subsequent steps, 5 × 105 cells from stationary cultures were inoculated and cultivated for 72 hours in fresh medium containing each time increasing concentrations of copper (1.5 - 2 - 2.5 g · L-1). Single colonies were isolated from the culture on YPD + 2.5 g · L-1 CuSO4 by plating on solid YPD and re-inoculated in liquid YPD + 2.5 g · L-1 CuSO4. To assess the endurance of metal tolerance, evolved cells were subjected to 10 cycles of inoculation-growth in fresh YPD medium without copper (de-adaptation) prior to be cultivated again in YPD + 2.5 g · L-1 CuSO4.
Growth of non-evolved and evolved S. cerevisiae cells (BL7, EL1 and GL6) was assayed in liquid YPD medium supplemented with intermediate copper concentrations (1, 1.5, 2 g, · L-1 CuSO4).
Growth was determined as the increase of cells number using a cells counter (Particle Count & Size Analyzer, Beckman Coulter).
Cells were harvested from 2 mL culture by centrifugation at 10,000 g for 10 min, washed twice with de-ionized water and at least four times with 10 mM citric acid in 0.5% (w/v) NaCl to remove copper ions adsorbed on the cell surface. The biomass was dried by Max-Dry Iyo (Heto) for 30 min, re-suspended in 300 μL of 20% (w/v) trichloroacetic acid (TCA), transferred to a 2 mL screw cap tube containing 100 μL of glass microbeads and subjected to mechanical lysis by three cycles of 20 sec at maximum speed with a Fast Prep® - FP120 (Bio101-Savant). The crude extract was clarified by centrifugation at 10,000 g for 10 min and the supernatant transferred to a new tube. Copper was quantified according to Brenner and Harris  adapted as follow. The clarified crude extract was diluted in 500 μL of de-ionized water and added to 100 μL of 1% (w/v) ascorbic acid and 400 μL of "BCA reagent" [0.006% (w/v) 2,2'-biquinoline-4,4'-dicarboxylic acid disodium salt hydrate (Fluka), 3.6% (w/v) NaOH, 15.6% (w/v) Hepes]. After 2 min incubation at room temperature, absorbance was recorded at 354 nm with a spectrophotometer Ultraspec 1000 (Pharmacia Biotec). The amount of copper is referred as mg Cu · g-1 dry biomass.
Flow cytometry analysis
Samples containing 107 cells were withdrawn during the exponential growth phase. Cells were harvested by centrifugation, washed twice with deionized water and four times with 10 mM citric acid in 0.5% (w/v) NaCl. Cells were washed with 1 mL of PBS (3.3 mM NaH2PO4, 6.7 mM Na2HPO4, 127 mM NaCl, 0.2 mM EDTA) and stained with 1 mL of the fluorescent probe propidium iodide (5 ng . mL-1). Stained cells were sonicated for 15 sec and then analyzed using a Cell Lab Quanta™SC flow cytometer (Beckman Coulter) equipped with a diode laser (excitation wavelength 488 nm, laser power 22 mW). The fluorescence emission was measured through a 670 nm long pass filter (FL3 parameter) in logarithmic mode for propidium iodide (PI) signal. Not stained and ethanol-treated samples were used as controls. The sample flow rate during analysis did not exceed 500 cells sec-1. A total of 2 × 104 cells were measured for each sample. Data analysis was performed with WinMDI 2.8 software, build#13 01-19-2000 (Purdue University, Cytometry Laboratories http://facs.scripps.edu/software.html).
Preparation of cell-free extracts
Cells from exponential cultures were harvested by centrifugation at 4,900 g for 10 min and washed twice with cold deionised water and four times with cold 10 mM citric acid in 0.5% (w/v) NaCl. The cell pellet was finally re-suspended in 0.5 M Tris-Cl pH 8.5, 0.25 M EDTA pH 8.4 added with protease inhibitor cocktail (Sigma) and mechanically disrupted using glass microbeads. Cell debris was removed by centrifugation at 700 g for 10 min and the clarified crude extract was used for enzymatic analyses. The protein concentration in cell-free extracts was estimated according to Bradford  using bovine serum albumin as the reference.
Enzyme activities were measured on cell-free extracts by spectrophotometric assays. Activities were expressed as Units · mg-1 proteins. Catalase activity was determined according to Bergmeyer  monitoring hydrogen peroxide decrease at 240 nm. Superoxide dismutase activity was measured as the inhibition of the rate of reduction of cytochrome c by the superoxide radical, observed at 550 nm . Peroxidase activity was measured following the oxidation of pyrogallol at 420 nm . The activity of glutathione peroxidase was determined monitoring NADPH oxidation at 340 nm .
Purification of copper-binding proteins
Supernatants obtained by ultracentrifugation (18,000 g, 45 min) of cell extracts were heated at 65°C for 10 min to enrich thermostable proteins, since thermostability is common to several copper-binding proteins . Samples were then centrifugated at 10,000 g for 10 min and supernatants purified by affinity chromatography on a Sepharose Chelating resin (Sigma) loaded with 0.2 M CuSO4 (copper resin). Samples (ca. 800 μg protein) were incubated for 20 min with 0.5 mL of copper resin previously equilibrated with 1 mL of binding buffer (0.02 M Na2HPO4, 0.5 M NaCl, pH 7.2). Unbound proteins were removed by gravity flow and the column washed three times with 0.5 mL of binding buffer. Bound proteins were eluted first with 0.02 M Na2HPO4, 0.5 M NaCl, pH 3.5 and then with 0.02 M Na2HPO4, 0.5 M NaCl, 0.05 M EDTA, pH 7.2. Proteins from 200 μL of each elution fraction were precipitated with 60 μl of 20% (w/v) TCA, resuspended in 50 μL of SDS-Sample buffer (0.25 M Tris-Cl pH 6.8, 50% (v/v) glycerol, 10% (w/v) sodium dodecyl sulphate, 5% (v/v) β-mercaptoethanol, 0.25% (w/v) bromophenol blue), heated at 99°C for 5 min and applied to 18% (w/v) polyacrylamide gels. Electrophoresis in denaturing conditions (SDS-PAGE) was carried out according to Laemmli . Gels were stained by GelCode Blue (Pierce).
Bands were excised from the polyacrylamide gels, cut into small pieces and de-stained by repeated washing cycles alternating 50 mM ammonium hydrogen carbonate and pure acetonitrile. After complete destaining, gel particles were dehydrated by acetonitrile, covered with trypsin solution (12.5 ng/mL in 50 mM ammonium hydrogen carbonate, pH 8.0) and incubated 1 h on ice. Excess liquid was removed and the gel pieces covered with a solution of 50 mM ammonium hydrogen carbonate (pH 8.0) and incubated overnight at 37°C. Tryptic peptides were extracted by alternating incubation in pure acetonitrile and 1% formic acid. Samples were lyophilised, resuspended in 1% formic acid, and desalted by ZipTip (Millipore) before ESI-MS analysis.
ESI-MS experiments were performed with a hybrid Quadrupole-Time-of-Flight (q-TOF) mass spectrometer (QSTAR ELITE, Applied Biosystems) equipped with a nano-electrospray ionisation sample source. Metal-coated borosilicate capillaries (Proxeon, Denmark) with medium-length emitter tip of 1-μm internal diameter were used for off-line analysis. The instrument was calibrated with standard solution Renin (MH2+ 879.97 Da and its fragment MH+ 110.07 Da, Applied Biosystems). Peptide identification was performed using the MASCOT software with the following parameters: 2 missed cleavages, peptide tolerance 0.6 Da, MS/MS tolerance 0.6 Da, peptide charges 2+ and 3+. Only monoisotopic masses were considered as precursor ions.
Spectra of tryptic peptides were acquired in the 400-1,500 m/z range, with 1.0 sec accumulation time, ion-spray voltage 1,3000 V, declustering potential 60 V, with active Information Dependent Acquisition (IDA), using rolling collision energy to fragment peptides for MS/MS analysis.
Determination of Reactive Oxygen Species
Samples containing 5 × 106 cells were withdrawn from exponential cultures. Cells were harvested by centrifugation, washed twice with de-ionized water, four times with 10 mM citric acid in 0.5% (w/v) NaCl, once with 1 mL of PBS and finally incubated with 0.5 mL of 5 μM (v/v) dihydroethidium (Sigma, stock solution 30 mM in DMSO) for 30 min in the dark. Stained cells were washed three times with PBS and sonicated for 15 sec. The fluorescence signal was detected using a Cary Eclipse spectrofluorimeter (Varian, CA, USA) at excitation wavelength of 518 nm and emission wavelength of 605 nm. Fluorescence values were normalized against those of not stained cells.
Authors wish to thank Angiolella Lombardi of Veneto Agricoltura - Istituto per la Qualità e le Tecnologie Agroalimentari (VI) - for providing the yeast strains and Maria Šamalíková for technical support in MS analysis. G.M.A. is the recipient of a PhD fellowship of the University of Milano-Bicocca.
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