Tolerance towards copper of one Candida
and three Saccharomyces
strains was first assessed by a drop test on minimal or rich (YPD) solid medium supplemented with increasing concentrations of copper salt (CuSO4
) (Figure ). In good agreement with results from other laboratories showing that the composition of the culture medium and the growth conditions affect copper sensitivity of yeast cells [21
], we observed that on minimal medium, 0.5 g · L-1
was sufficient to inhibit the growth of all yeast strains, whereas on YPD plates all of them tolerated up to 1 g · L-1
. However, above this concentration only C. humilis
cells still proliferated, suggesting low copper tolerance in all strains assayed but C. humilis
which showed a higher tolerance.
Figure 1 Assay of copper tolerance in yeast strains. Five μl of 1:10 serial dilutions were plated on minimal medium without or with 0.5 g · L-1 CuSO4 (a) or YPD medium containing 0-2.5 g · L-1 CuSO4 (b). Plates were incubated two days at (more ...)
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".
Figure compares the growth kinetics of non-evolved and evolved cells in YPD + 2.5 g ·L-1
(for simplicity we will refer to this condition as "copper medium"). Among the natural strains (in the following "non-evolved"), only C. humilis
AL5 proliferated under this condition, even though growth started after a prolonged lag phase and a very low final cell density was achieved (Figure ). On the contrary, all evolved strains proliferated in copper medium and reached final biomass densities close to those observed in YPD medium, although with lower growth rate (Additional file 1
). Cells subjected to 10 cycles of growth/re-inoculation in YPD without Cu (referred to as "de-adapted"), retained their ability to proliferate if re-inoculated in copper medium, showing only negligible differences when compared with the corresponding evolved strain (Figure and Additional file 1
). This observation suggests that copper tolerance is maintained also in absence of selective pressure. To gain more insight into the behaviour of the copper-sensitive S. cerevisiae
strains, we compared the kinetics of growth of evolved and non-evolved cells also at 1, 1.5 and 2 g ·L-1
(Additional file 2
), highlighting a progressive decrease of the proliferation ability of natural cells at increasing copper concentrations. As expected, the same conditions were permissive for evolved Saccharomyces
Figure 2 Growth of yeast cells in YPD + 2.5 g · L-1 CuSO4. Evolved (black cirles), non-evolved (black triangles) and de-adapted (black squares) cells of C. humilis AL5 (a), S. cerevisiae BL7 (b), S. cerevisiae EL1 (c), S. cerevisiae GL6 (d). The values (more ...)
Analysis of the Cu content of Candida cell samples from the same cultures shown in Figure revealed relatively high amounts of intracellular copper, i.e. 6.5 and 7.6 mg · g-1 biomass in evolved and non-evolved cells, respectively. The kinetics of bioaccumulation was faster in non-evolved cells where Cu measured after 24 hours of growth was three times higher than in the evolved ones (Figure ). In copper medium non-evolved Candida grew poorly and contained high copper concentration from the very beginning of the experiment, while Cu was lower in evolved cells - which proliferated at the same rate as in YPD broth. In both cases, intracellular copper kept increasing up to 48 hours and then reached a plateau. The amount of copper measured in evolved and de-adapted cells grown in copper medium was always comparable, supporting once more the hypothesis that evolutionary engineering produced stable effects.
Figure 3 Intracellular copper measured during growth in YPD + 2.5 g · L-1 CuSO4. C. humilis AL5 (a); S. cerevisiae BL7 (b); S. cerevisiae EL1 (c) and S. cerevisiae GL6 (d). White bars: evolved cells; black bars: de-adapted cells; grey bars: non-evolved (more ...)
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 ). The Cu content of non-evolved and evolved Saccharomyces
cells was compared also in a milder condition, (1 g ·L-1
). 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.
Initially, the effect of copper on cell viability was evaluated by citofluorimetry (Figure ), that allows to get this information at the single cell level [23
]. Samples of C. humilis
and S. cerevisiae
cultures were harvested during exponential growth in copper and stained with propidium iodide, an intercalating agent excluded by viable cells, that can instead permeate the surface of seriously injured/dead cells [24
]. In both evolved strains the percentage of propidium-positive cells was lower (8.5% for C. humilis
and 11% for S. cerevisiae
) than in their non-evolved counterpart (28% for C. humilis
and 60% for S. cerevisiae
). The percentage of propidium-positive cells grown in absence of copper (used as a control) was around 2% (data not shown
). Altogether, these results confirm that evolution confers robustness - although not complete insensitivity - to copper. Copper was more detrimental for S. cerevisiae
than C. humilis
natural cells, while this difference fainted after adaptive evolution, in good agreement with the kinetics of growth reported in Figure .
Figure 4 Cytofluorimetric analysis. Evolved and non-evolved C. humilis (a) and S. cerevisiae (b) cells. Cells were collected during exponential growth in YPD + 2.5 g . L-1 CuSO4 and stained with propidium iodide. The x- and y-axes of the histogram display the (more ...)
Since copper is a strong oxidizing agent, we measured the activities of superoxide dismutase, peroxidase, glutathione peroxidase and catalase - all involved in the so-called copper-dependent oxidative stress response - in evolved and non-evolved C. humilis and in evolved S. cerevisiae cells harvested from copper medium in the exponential phase of growth. As a control, basal enzymatic activities were determined in YPD-grown cells (Table ). In S. cerevisiae, exposure to copper resulted in the increase of superoxide dismutase and catalase activities, while peroxidase and glutathione peroxidase activities were only marginally affected. The picture emerging from the analysis of the copper tolerant C. humilis strain is different and more exhaustive, since the data set can also include the response of non-evolved cells in copper medium. In all cultivation broths, all enzymatic activities tested in natural Candida cells were 2 to 8 fold higher than in S. cerevisiae. Interestingly enough, we detected high constitutive superoxide dismutase and catalase activities in non-evolved cells, independently from the composition of the culture broth, while peroxidase and glutathione peroxidase activities were induced only by growth in copper medium. Evolution resulted in lower levels of all tested activities in both YPD and copper medium, with the most remarkable effect on superoxide dismutase. Catalase activity that was high in YPD-grown cells (both kinds of cells), strongly decreased in evolved cells grown in copper medium.
Antioxidant enzyme activities in S. cerevisiae and C. humilis cells grown in YPD and in YPD + CuSO4 2.5 g · L-1 (Cu)
We then evaluated the production of reactive oxygen species (ROS) staining cells with dihydroethidium (Figure ). In presence of superoxide anions in the cytosolic space, this probe is oxidized to the fluorescent product ethidium. Therefore, fluorescence intensity reports on oxidative stress. While in YPD medium the amount of ROS was low in both Candida
cells, copper exposure clearly triggered oxidative stress, though with milder effects in the evolved cells. Moreover, in agreement with growth and cytofluorimetric data (Figure and ), the effect on S. cerevisiae
was stronger than on Candida
with ROS production three-fold higher. Evaluation of oxidative stress in S. cerevisiae
at intermediate metal concentration (1, 1.5 and 2 g ·L-1
) showed that while ROS production increases with copper concentration in the non-evolved sample, it remains low in the evolved cells (Additional file 4
Figure 5 Fluorimetric analysis of superoxide anion (OH-•) production. Evolved and non-evolved C. humilis (a) and S. cerevisiae (b) cells exponentially growing in YPD (white bars) and in YPD + 2.5 g . L-1 CuSO4 (grey bars). OH-• formation is expressed (more ...)
Finally we performed a preliminary analysis of the copper-binding proteins extracted from non-evolved and evolved cells grown either in YPD or in copper medium. Samples were enriched by affinity chromatography as described in Materials and Methods and then analysed by SDS-PAGE. Equal volumes of elution fractions obtained from the same amount (800 μg) of proteins applied on the column were loaded for the electrophoretic run, therefore assuring that differences detected in the gel reflect changes in composition and content of copper-binding proteins in the starting samples. Figure shows the electrophoretic analysis and Table lists proteins identified by tandem mass spectrometry. The exposure of evolved S. cerevisiae cells to copper elicited the induction of several proteins (Figure , lane 2 and 3) involved in different biochemical and metabolic functions, i.e. the pentose phosphate pathway (band 1), amino acid and sulphur metabolism (bands 2, 3 and 10), glucose metabolism (bands 4 and 7), redox reactions (bands 6 and 8), the translation machinery (bands 5 and 9) and isomerization reactions (bands 1 and 9). The same trend was detected at lower CuSO4 concentration (data not shown). The picture relevant to Candida is completely different (Figure ). Non-evolved Candida cells react to copper repressing a number of Cu-binding proteins that would be otherwise expressed during growth in YPD (Figure , lane 1 and lane 3). Among the down-regulated proteins we could identify ribosomal proteins and components of the protein translation apparatus (band 11, 13 and 16). The profile of Cu-binding proteins extracted from evolved Candida cells (Figure , lane 2 and 4) showed a massive enrichment of a protein of ~35 kDa (band 14), identified as glyceraldehyde-3-posphate dehydrogenase 3 (GAPDH). We further observed the increase of a protein of ~ 22 kDa (band 15) identified as peroxiredoxin and of a protein inducible by oxidative stress (band 12).
Figure 6 SDS-PAGE of copper-binding proteins. (a) S. cerevisiae. Lane 1: proteins from non-evolved cells grown in YPD; lane 2: proteins from evolved cells grown in YPD; lane 3: proteins from evolved cells grown in YPD + CuSO4 2.5 g · L-1. (b) C. humilis (more ...)
Identification of copper-binding proteins