Various types of genetic modification and selective forces have been implicated
in the process of adaptation to novel or adverse environments. However, the
underlying molecular mechanisms are not well understood in most natural
populations. Here we report that a set of yeast strains collected from Evolution
Canyon (EC), Israel, exhibit an extremely high tolerance to the heavy metal
cadmium. We found that cadmium resistance is primarily caused by an enhanced
function of a metal efflux pump, PCA1. Molecular analyses
demonstrate that this enhancement can be largely attributed to mutations in the
promoter sequence, while mutations in the coding region have a minor effect.
Reconstruction experiments show that three single nucleotide substitutions in
the PCA1 promoter quantitatively increase its activity and thus
enhance the cells' cadmium resistance. Comparison among different yeast
species shows that the critical nucleotides found in EC strains are conserved
and functionally important for cadmium resistance in other species, suggesting
that they represent an ancestral type. However, these nucleotides had diverged
in most Saccharomyces cerevisiae populations, which gave cells
growth advantages under conditions where cadmium is low or absent. Our results
provide a rare example of a selective sweep in yeast populations driven by a
tradeoff in metal resistance.
Unicellular microorganisms are often challenged by fluctuating environmental
conditions. Especially for those organisms having limited mobility, adaptation to
such environmental stresses is critical for survival of their populations. However,
mutations beneficial for survival in one environment may impose a cost under other
need to fine-tune the evolved gene function or regulation in order to maintain an
optimal physiology under a range of conditions. It is important to understand how
cells adapt to novel or adverse environments. Such information may not only allow us
to dissect the factors affecting evolution of organisms, but may also provide us
some insights into pathway or functional network flexibility (or evolvability) of
the cell. To address this issue, identifying the mutations responsible for the
adaptive phenotypes is the most direct approach, and yet it remains challenging even
in simple organisms such as E. coli. Moreover, even if a mutation
is identified, detailed population and phylogeny data are required in order to
deduce the evolutionary trajectory of adaptive traits.
Experimental evolution represents a simplified approach since it allows scientists to
follow the evolutionary history of populations exposed to known selective pressures.
Several adaptive mutations in microorganisms have been discovered and characterized
at the molecular level from laboratory experimental evolution, adding greatly to our
understanding of adaptive evolution , –. On the other hand, studies
related to natural adaptation are more complicated. Although the mechanistic basis
or phylogeny of adaptive traits have been revealed in several previous studies –, systematic
approaches dealing with both aspects are still rare –.
Metal ions such as copper, iron, zinc, potassium and sodium are essential nutrients
involved in a broad range of biological processes , . These essential metals
function as catalysts for biochemical reactions, stabilizers of protein structures
or cell walls, or regulators of intracellular osmotic balance. Despite their
importance, unbalanced metal concentrations can cause deleterious effects, sometimes
leading to programmed cell death , and thus represent a double-edged sword. It is important
for cells to tightly regulate homeostasis of these metal ions.
In natural environments, cells often encounter other nonessential metal ions. Some of
them such as cadmium, lead and arsenic are highly toxic to cells. Toxicity often
occurs through the displacement of essential metals from their native binding sites
or through ligand interactions, resulting in altered structural conformations or
interference with biochemical reactions . These metal ions can induce
the generation of reactive oxygen species and cause damages to various cellular
components –. Organisms have evolved several different mechanisms to
cope with metal induced stresses, including specific metal transporters, metal
sequestration proteins or compartments, and different detoxification enzymes , , , . These various
systems often cooperate with each other to quickly respond to variations in
environmental metal concentrations, indicating the importance of metal ion balance
In this study, we observed that a subset of diploid yeast strains collected from
different locations of the EC could tolerate a heavy metal, cadmium, to a level
unseen in most known yeast strains. We found that the cadmium-resistant phenotype is
primarily caused by regulatory changes in the PCA1 gene, which
encodes a P-type ATPase required for cadmium efflux , . By performing functional assays
and phylogenetic analyses, we show that PCA1 has experienced
several rounds of selective adaptation during yeast evolution. More strikingly, we
observe that a weak PCA1 allele spread to most S.
cerevisiae populations, probably due to a tradeoff between metal
resistance and fitness under low cadmium conditions.
One subset of EC yeast strains is highly resistant to cadmium
Evolution Canyon is an east-west-oriented canyon at Lower Nahal Oren, Israel. It
originated 3–5 million years ago and is believed to have experienced
minimal human disturbance , . In contrast to other wild yeast, the strains
collected from EC are often polyploid and most of them are heterothallic ,. Previous
studies have revealed high allelic diversity among EC yeast strains , . To
assess whether these strains also carry specific adaptive phenotypes, we
performed a panel of phenotypic assays including cell growth under several
stress conditions. Only diploid strains were included in this study since
triploid and tetraploid strains are less amenable to further genetic analyses.
The results showed that a subset of EC strains (EC9, 10, 35, 36, 39, 57, and 58)
was resistant to a very high concentration of cadmium (0.8 mM CdCl2),
while all other strains analyzed were unable to grow on plates containing 0.2 mM
CdCl2 (Figure S1A).
Because chromosomal rearrangement has been suggested to be involved in adaptive
evolution , ,, we first examined the karyotype of 14 diploid EC
strains. Pulsed-field gel electrophoresis (PFGE) analysis revealed that these EC
strains comprised three major karyotypes, EC-C1, EC-C2 and EC-C3 (with some
minor deviations)(Figure S1B). Interestingly, all
cadmium-resistant strains belong to EC-C1, suggesting that the metal-resistant
phenotypes have already evolved before the EC-C1 populations split. Therefore,
we chose to use mainly one strain (EC9) from EC-C1 for subsequent genetic
The cadmium-resistant phenotype is caused by a mutant allele of
We performed a genetic analysis to assess how many genetic loci are involved in
the cadmium-resistant phenotype. A haploid Cd-resistant clone (EC9-8) was
crossed with two Cd-sensitive strains (EC13 from EC-C2 and lab strain S288C).
Both hybrid diploids were Cd resistant, indicating that the Cd-resistant
phenotype was dominant. The hybrid diploids were then induced to sporulate, and
their haploid segregants were examined for their cadmium tolerance. These
segregrants showed a 2∶2 (resistant to sensitive) segregation pattern,
indicating that the Cd-resistant phenotype was primarily controlled by a single
To screen for the gene responsible for cadmium resistance, a genomic DNA library
constructed from EC9 genomic DNA was transformed into Cd-sensitive cells. All
Cd-resistant colonies carried plasmids containing PCA1.
Sequencing the PCA1 allele (PCA1-C1) of the
EC-C1 strains revealed many mutations in both promoter and protein-coding
regions as compared with the PCA1 sequences from other strains
S1). Next, we directly tested whether PCA1-C1 alone
is able to improve the cadmium tolerance of cells. Plasmids carrying
PCA1-C1 were transformed into three Cd-sensitive strains
(S288C, SK1, and YJM789) in which the PCA1 gene had been
deleted. As shown in Figure
1A, the different strains carrying PCA1-C1 all
exhibited a level of cadmium resistance close to the level of EC-C1. By
contrast, when the PCA1 alleles from Cd-sensitive strains were
transformed into an EC9 pca1Δ mutant, the transformants
remained cadmium sensitive (Figure
1B). Finally, we sequenced the PCA1 alleles of the
segregants obtained from the previous genetic analysis and confirmed that all
Cd-resistant segregants carry the PCA1-C1 allele.
A previous study by Adle and co-workers has shown that the PCA1-dependent cadmium
resistance is mainly a consequence of active cadmium export (efflux) . To determine
whether cadmium efflux is higher in cells containing PCA1-C1, cells carrying
PCA1-C1 or PCA1-SK1 were pretreated with
cadmium, washed to remove extracellular cadmium, resuspended in fresh media, and
collected at different time points to measure the cellular cadmium content using
inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The
PCA1-SK1 allele from the SK1 strain was chosen for
comparison because this allele is phylogenetically more related to
PCA1-C1 based on an analysis of the corresponding ORF
sequences (Figure S2D) and because it is a cadmium-sensitive allele lacking the
G970R mutation (which abolishes the activity of Pca1) present in other
laboratory strains. Indeed, cells carrying PCA1-C1 could reduce
the intracellular cadmium concentration more quickly than cells carrying
PCA1-SK1. These data indicate that cells containing
PCA1-C1 have a very efficient cadmium efflux (Figure 1C).
Mutations in both the promoter and coding regions of PCA1-C1
contribute to cadmium resistance
In order to understand how PCA1-C1 has evolved a high cadmium
resistance, chimeric proteins with regions from PCA1-C1 and
PCA1-SK1 were constructed and assayed for their ability to
complement cadmium sensitivity of the pca1Δ mutant. We
found that swapping the promoters drastically affected cadmium resistance (Figure 2A, compare C1 with H6
and SK1 with H3), whereas swapping the region between amino acids 207 and 1216
had a mild effect (C1 vs. H1 and H2 vs. H3). Four nonsynonymous mutations are
present in this coding region: N223, T358, T363, and G365. Previous studies have
identified a few domains important for the stability or function of Pca1 , . However,
none of these four mutations are located within these functional domains.
To assess whether the different levels of cadmium resistance resulted from
differences in promoter strength, we fused the PCA1-C1 or
PCA1-SK1 promoters to a luciferase reporter and assayed the
luciferase activity of these constructs. The expression driven by the
PCA1-C1 promoter was about four-fold higher than that
driven by the PCA1-SK1 promoter in the absence and presence of
cadmium treatments, suggesting that mutations in the PCA1-C1
promoter increased the degree of cadmium resistance by increasing the
PCA1 gene expression without destroying its regulation
(Figure 2B). We also
performed quantitative PCR to determine the level of PCA1 mRNA
in EC9 and SK1 strains. The data were consistent with the results from the
luciferase reporter gene assay.
The increase in PCA1-C1 expression is mainly caused by three
point mutations in the promoter region
By comparing the promoter sequences (including 600 bp upstream of the initiation
codon) of PCA1-C1 and PCA1-SK1, we observed 18
single nucleotide polymorphisms (17 single nucleotide substitutions and one 1-bp
deletion) and one 10-bp insertion (Table S1A). Because altered
PCA1-C1 expression plays a key role in enhancing cadmium
resistance, we sought to understand how this gene evolved and the degree to
which changes in its promoter contribute to expression differences. To address
the latter issue, we fused chimeric promoters with regions from
PCA1-C1 and PCA1-SK1 promoters to a
luciferase reporter and assayed their expression levels. We found that only the
region immediately upstream of the initiation codon (−213 to −1)
contributed significantly to the enhanced gene expression (Figure 2B, p5-2). Only 6 of the 18 single
nucleotide polymorphisms are present in this region. To determine which
mutations led to the enhanced promoter activity, we introduced the
PCA1-C1 version of each of these sites into the
PCA1-SK1 promoter and assayed the reporter gene expression.
Only mutations in three nucleotides (PCA1-SK1 to
PCA1-C1: −97T > C, −148T > G, and
−159G > T) had obvious effects (Figure 2C; Table
To confirm that the changed expression is important for the cadmium resistance,
we introduced the same mutations into the PCA1-SK1 allele and
then measured the cadmium resistance of cells carrying these mutant alleles. The
expression level of the PCA1-SK1 mutants was indeed correlated
with the cadmium resistance (Figure
2D). When all three mutations were combined together into a single
mutant clone (PCA1-SK1+2/5/6), cells carrying this clone
were as resistant to cadmium as the cells carrying PCA1-H3, in
which the PCA1-C1 promoter is fused to the
PCA1-SK1 ORF. This result demonstrated that the enhanced
cadmium resistance of EC-C1 strains is mainly caused by three single nucleotide
substitutions in the promoter of PCA1-C1. Currently, it is
still unclear how PCA1 transcription is regulated. Although we
could not identify any transcription factor binding motif in the sequences where
the critical mutations (−97C, −148G and −159T) are located, it
is quite possible that these regions contain some of the regulatory elements of
Cadmium resistance is an ancestral phenotype
To determine whether the Cd-resistant phenotype is specific to EC-C1 strains, we
examined the cadmium sensitivity of two closely related species, S.
paradoxus and S. mikatae. Two S.
mikatae strains and 28 S. paradoxus strains
isolated from different niches were tested . Although both species could
not tolerate, unlike EC-C1, a high level of cadmium (2.0 mM CdCl2),
they exhibited much higher Cd resistance (0.4–0.8 mM CdCl2)
than the other S. cerevisiae strains (Figure S3).
To confirm that the medium level of cadmium resistance in S.
paradoxus was also mediated through PCA1, we
cloned the S. paradoxus PCA1 gene (Sp-PCA1)
into a plasmid, transformed the resulting vector into S. cerevisiae
pca1Δ mutant cells, and then assayed the transformants for
cadmium sensitivity. The result showed that Sp-PCA1 was able to
generate a medium level of cadmium resistance in S. cerevisiae
pca1Δ mutants (Figure S3B). Consistent with these results,
deletions of PCA1 in S. paradoxus strains
resulted in a Cd-sensitive phenotype (Figure S3C).
Interestingly, comparison between S. cerevisiae-, S.
paradoxus-, and S. mikatae-PCA1 promoter sequences
revealed that those critical residues (−97C, −148G and −159T)
identified in the previous experiment are conserved between EC-C1 strains,
S. paradoxus (28 strains) and S. mikatae
(2 strains)(Figure 3). When
we mutated these nucleotides of Sp-PCA1 (−100C,
−149G and −162T) to non-EC-C1 Sc-PCA1 sequences,
the mutant allele became cadmium sensitive (Figure
S3D), indicating that these nucleotides were also critical for the
function of Sp-PCA1. Since both S. paradoxus
and S. mikatae can tolerate a medium level of cadmium, it is
likely that this phenotype represents an original phenotype of the common
ancestor of S. cerevisiae, S. paradoxus, and
S. mikatae, which has been lost in most S.
cerevisiae populations (Figure S4).
Loss of cadmium resistance provides a fitness advantage under cadmium-free
conditions in S. cerevisiae
If the common ancestor of S. cerevisiae and S.
paradoxus was cadmium resistant, why did most S.
cerevisiae populations become cadmium sensitive? By comparing the
promoter sequences of PCA1 from EC-C1, EC-C2, EC-C3, and 38
other S. cerevisiae strains (collected from various habitats on
different continents; see ), a striking pattern was revealed: most S.
cerevisiae strains carry a weak PCA1 promoter
similar to the one in the SK1 strain (Figure 4A). Hence, reduced promoter strength
accounts for the cadmium-sensitive phenotype observed in these strains (Figure S4).
The only exceptions are EC-C1, UWOPS87_2421, and UWOPS83_787_3. Interestingly,
the PCA1 promoters of UWOPS87_2421 and UWOPS83_787_3 also
contain the mutations critical for cadmium resistance (−97C and
−159T in UWOPS87_2421 and −97C in UWOPS83_787_3), and both strains
show cadmium-resistant phenotypes (Table S1).
A previous study showed that cells overexpressing PCA1 in a
medium without cadmium suffered reduced fitness . To determine whether
expression of PCA1-C1 also imposes a high fitness cost on
cells, we conducted a competition assay to measure the fitness of cells
containing PCA1-C1, PCA1-SK1 or
PCA1-S288C. Plasmids carrying either
PCA1-C1, PCA1-SK1 or
PCA1-S288C were transformed into S288C
pca1Δ mutants. The resulting transformants were then
mixed with a reference strain carrying a green fluorescent protein-tagged Pgk1
protein and grown in a medium without cadmium. The results showed that cells
containing PCA1-C1 had a lower fitness than cells containing
PCA1-SK1 or PCA1-S288C
(p<0.001, two-tailed t-test), suggesting a
tradeoff between high Cd resistance and the fitness of cells under Cd-free
conditions (Figure 4B). It
is possible that Sc-PCA1 was selected to reduce the fitness
cost, thus resulting in lower Cd resistance if most S.
cerevisiae cells were constantly living in environments containing
low levels of cadmium. Alternatively, Sc-PCA1 might have been
selected, at a cost of reduced Cd resistance, to enhance other activities.
Previous studies have suggested that Pca1 is also involved in copper resistance
However, we found that cells carrying PCA1-C1 or
PCA1-SK1 showed a similar level of copper resistance,
indicating that the mutations in PCA1-C1 are specific to
cadmium resistance (Figure S5).
It is unclear why cells carrying PCA1-C1 have a lower fitness
under Cd-free conditions. Since Pca1 is not a highly abundant protein, the
fitness reduction is unlikely due to the energy cost for producing extra amounts
of the Pca1 protein. PCA1 belongs to a P-type ATPase family
whose members have been shown to transport metal ions such as cadmium, copper,
zinc, cobalt, and lead , . Hence, the fitness cost of PCA1-C1
under non-cadmium conditions may result from a depletion of essential metal ions
caused by enhanced PCA1 expression. In S.
cerevisiae, it has been shown that Pca1 exports cadmium, not copper
However, a study by Adle et al. has also demonstrated that Pca1 affects copper
balance by chelating copper ions in a manner analogous to metallothionine . Thus, it is
possible that the high expression of PCA1-C1 depletes copper or
other unidentified vital metal ions by metal sequestration or by metal
In nature, S. paradoxus and S. cerevisiae were
found to occasionally coexist in the same ecological niches . We
have shown that most S. cerevisiae strains have lost the
ancestral cadmium-resistant phenotype probably due to its fitness cost. Why do
S. paradoxus populations still maintain this phenotype?
Using the aforementioned competitive fitness assay, we found that S.
paradoxus cells carrying either wild-type or low-expression alleles
of Sp-PCA1 exhibit similar fitness under Cd-free conditions
S6). These data suggest that S. paradoxus has
evolved other mechanisms to offset the fitness cost of high
Evolution of PCA1 in S. cerevisiae
In EC-C1 cells, the increase in the expression of PCA1-C1 was
caused by three nucleotide substitutions in the promoter that were also shared
by the S. paradoxus and S. mikatae PCA1 genes.
Horizontal gene transfer between different species of yeast has been observed in
previous studies , . One possible explanation for the high Cd resistance
of EC-C1 strains is that the ancestor of EC-C1 strains acquired a S.
paradoxus PCA1 allele through a horizontal gene transfer event, and
that the transferred Sp-PCA1 function was reinforced later on
by natural selection in EC-C1 strains. If that was the case, we would expect to
see that the sequence of PCA1-C1 is more similar to that of
Sp-PCA1 than to sequences of PCA1 alleles
from other S. cerevisiae strains. Phylogenetic analyses using
the PCA1 coding or promoter sequences, however, showed that the
distance between Sp-PCA1 and PCA1-C1 is
farther than that between Sp-PCA1 and other S.
cerevisiae PCA1 alleles, suggesting that PCA1-C1
was not derived from Sp-PCA1 (Figure 3 and Figure S7).
Moreover, we can rule out the possibility that gene conversion of a small region
between Sc-PCA1 and Sp-PCA1 has occurred in
the ancestor of EC-C1 strains. At alignment of PCA1 promoter
sequences, we found that even in the region containing the critical nucleotides
several nucleotides (−99, −112, −115, −119, −124,
and −137) were shared by all S. cerevisiae strains, but
did not exist in S. paradoxus strains (Figure 3); it should be noted, however, that
these sequences were also conserved in all 28 S. paradoxus
We have shown that, unlike S. paradoxus and S.
mikatae, most S. cerevisiae strains except for
EC-C1 and two other strains (UWOPS87_2421 and UWOPS83_787_3) are highly
sensitive to cadmium. Intriguingly, we found that sequences of the
PCA1 promoter in Cd-sensitive strains showed high identity
S1A). When population data were analyzed, a dramatic decrease in the
frequency of DNA polymorphisms was observed in this region, inconsistent with
the phylogenetic relationship observed in both upstream and downstream regions
(Figure 4A and Figure S2;
Tajima's D = −1.94, p<0.05).
This result suggests that the cadmium-sensitive phenotype did not evolve
independently in different strains. Instead, it was caused by a selective sweep
of a weak PCA1 promoter in S. cerevisiae
populations. A selective sweep of nonfunctional aquaporin alleles in S.
cerevisiae populations has been reported recently . However,
unlike the aquaporin case, the selective sweep of S. cerevisiae
PCA1 is mainly caused by a single allele and covers a wider range
of populations. In addition, the PCA1 allele involved in the
sweep is still functional. In S. cerevisiae populations, only
the PCA1 alleles from S288C and W303 have lost the function
completely due to a mutation (R970G) in the catalytic domain , . Expression
of PCA1-SK1 is upregulated when cells sense environmental
cadmium and deletion of PCA1 in other Cd-sensitive S.
cerevisiae strains makes mutant cells at least 20-fold more
sensitive to cadmium.
The reduced cadmium resistance in most S. cerevisiae strains is
probably a result of regulatory fine-tuning that allows cells to maintain a
certain level of cadmium efflux activity, without compromising their fitness,
under normal conditions. Such an ‘optimized’ PCA1
might explain why this weak allele spread so efficiently to different S.
cerevisae populations. On the other hand, the EC-C1 strains
maintained and even reinforced the ancestral Pca1 activity, probably due to
constant selection in their living environments. We measured the soil cadmium
concentrations at the collection sites of Evolution Canyon using inductively
coupled plasma-atomic emission spectroscopy (see Materials and Methods) and found that they ranged from 2.5 to 4.2
ppm, which is about 17–28-fold higher the median soil cadmium
concentration in Europe . It is intriguing that some cadmium-sensitive strains
(EC-C2 and EC-C3) were isolated from the same areas as the EC-C1 strains. We
found that the PCA1 sequences of EC-C2 and EC-C3 strains are
more closely related to those in the European isolates than to those in EC-C1
strains (Figure 4A and Figure S2).
One possible explanation is that these cadmium-sensitive strains arrived in
Evolution Canyon more recently and have not yet adapted to the environment. An
in-depth study combining genomics and population distribution of the EC strains
will help us address this issue.
Phenotypic studies in budding yeast have suggested that resistance to various
metal ions in different yeast strains is quite diverse , , . A recent genomic analysis
of both promoter and coding regions of three S. cerevisiae
strains also indicates that metal ion transporter genes are significantly
enriched in the gene group showing signatures of positive selection . Our data with
PCA1 provide a clear example how a metal transporter gene
evolves after experiencing various types of selection that occurred at both
inter- and intra-species levels.
In the present study, we found that mutations in the regulatory and coding
regions both contribute to the adaptive phenotype. However, the mutations in the
regulatory region have a more profound effect as compared to those in the coding
region. Recent studies in a variety of organisms have suggested that regulatory
changes are critical for adaptive evolution , –. It is possible that the
promoter is more flexible to accommodate functional changes since it has less
structural constraints than the coding region . Our results showed that,
by fine-tuning the PCA1 gene expression, Cd tolerance and cell
growth could be dramatically affected in natural yeast isolates, further
emphasizing the importance of regulatory changes in evolution.
Materials and Methods
Strains and genetic procedures
EC diploid strains consist of S. cerevisiae collected from an
east-west-oriented canyon (Evolution Canyon) at Lower Nahal Oren, Israel , . Our
strain numbers are the same as the numbers shown in Figure 2 of reference 26. In brief, EC3, 5,
23, 33, 34, 35 and 36 were isolated from the south-facing slope (SFS), EC7, 9,
10, 39, 40, 45 and 48 were isolated from the valley bottom (VB), and EC13, 14,
57, 58, 59, 60 and 63 were isolated from the north-facing slope (NFS). S
paradoxus, S. mikatae and other S.
cerevisiae strains were obtained from the collections of Dr. Duncan
Greig (University College London, UK) and Dr. Edward Louis (University of
Nottingham) . Substitutive and integrative transformations were
carried out by the lithium acetate procedure . Media, microbial, and genetic
techniques were as described .
Karyotyping of EC strains
A total amount of ∼2×108 yeast cells was used for plug
preparation. Cells were washed with 1 ml EDTA/Tris (50 mM EDTA, 10 mM Tris-HCl,
pH 7.5) and transferred into EDTA/Tris containing 0.13 mg/ml zymolyase
(Seikagaku America Inc., St. Petersburg, FL). The cell mixtures were incubated
for 30 s at 42°C and then embedded in low melting point agarose
(Sigma-Aldrich, St. Louis, MO). The agarose plugs were then incubated at
37°C overnight for zymolyase digestion. After digestion, the agarose plugs
were placed in LET solution (0.5 M EDTA, 10 mM Tris-HCl (pH 7.5), 2 mg/ml
protease K, and 1% N-lauroylsarcosine) at 50°C overnight. This step
was repeated three times. The plugs were transferred to EDTA/Tris solution and
dialyzed four times for 1 h at 37°C. Yeast chromosomes were separated in
0.7% agarose gels by pulsed-field gel electrophoresis (PFGE) using a
Rotaphor Type V apparatus (Biometra, Göttingen, Germany). Electrophoresis
was performed for 48 h at 13°C in 0.5× TBE buffer at a fixed voltage
of 120 V and an angle of 115°, with pulse time intervals of 30 s.
Genomic DNA library screening
To construct an EC9 genomic DNA library, yeast genomic DNA was extracted using
the Qiagen Genomic-Tip 100/G kit (Qiagen, Valencia, CA), digested with
restriction enzymes, and ligated into a yeast vector pRS416 as described . To screen
for Cd-resistant genes, a Cd-sensitive lab strain (S288C) was transformed with
the EC9 genomic DNA library and plated on cadmium-containing plates (0.4 mM
CdCl2). Plasmids isolated from the Cd-resistant colonies were
sequenced to identify the insert-containing genes.
To measure the intracellular cadmium concentration, log-phase cells carrying
different alleles of PCA1 were pretreated with 0.4 mM
CdCl2 for 1 h, washed with PBS containing 10 mM EDTA, resuspended
in rich medium without cadmium, and then collected at indicated time points.
Collected cells were immediately washed with PBS containing 10 mM EDTA. Total
intracellular cadmium levels were measured by inductively coupled plasma-atomic
emission spectroscopy (ICP-AES).
After cadmium treatment (0.1 mM CdCl2) for 2 h at 28°C, total RNA
was isolated from cells using the Qiagen RNeasy Midi Kit (Qiagen, Valencia, CA).
First-strand cDNA was synthesized for 2 h at 37°C using the High Capacity
cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA). A 20-fold
dilution of the reaction products was then subjected to real-time quantitative
PCR using gene-specific primers, the SYBR Green PCR master mix, and an ABI-7000
sequence detection system (Applied Biosystems). Data were analyzed using the
built-in analysis program.
To construct the luciferase reporter plasmids, different promoter regions (up to
668 bp upstream of the start codon) of PCA1-C1 and
PCA1-SK1 were amplified by PCR. The luciferase coding
region (from Renilla reniforms) was also amplified by PCR. The
PCR fragments were co-transformed with pRS416 digested with XhoI and SacI into
the lab strain S288C. The genomic DNA of Ura+ colonies was isolated and
transformed into component E. coli cells. The plasmids from
ampicillin-resistant clones were isolated and sequenced. The constructed
luciferase reporter plasmids were transformed into an EC9
Yeast cells carrying different luciferase reporter plasmids were treated with 0.1
mM of CdCl2 for 2 h. After the treatment, 0.5×107
cells were harvested for detection of the luciferase activity on a luminometer
(PE Victor3 luminometer plus autojector, Perkin Elmer, Waltham, MA). To the test
samples, 100 µl of 1 µM substrate (coelenterazine) was added.
Following a 5 second equilibration time, luminescence was measured with a 10
second integration time.
Competitive relative fitness assay
We measured the fitness of the testing strains by competing them against a
reference strain expressing PGK1::GFP in CSM-URA media at 28°C. The testing
cells and reference cells were inoculated in the CSM-URA medium individually and
acclimated for 24 h. The cells were subsequently diluted and refreshed in new
media for another 4 h. The reference and testing cells were then mixed at a
1∶1 ratio, diluted into fresh medium at a final cell concentration of
5×103 cells/ml, and allowed to compete for 21 h, which
represents about 12 generations of growth. The ratio of the two competitors was
quantified at the initial and final time points using a fluorescence activated
cell sorter (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). Five
independent replicates for each fitness measurement were performed.
Phylogenetic tree construction
The evolutionary history of the PCA1 ORF (3651 bps),
SUL1 ORF (2580 bps), PCA1 promoter (213
bps) and PCA1-SUL1 intergenic region (820 bps) was inferred
using the Neighbor-Joining method . Sequences were obtained
from previously released data . The percentage of replicate trees in which the
associated taxa were clustered together in the bootstrap test (500 replicates)
is shown next to the branches, analogous to a previous study . The
tree was drawn to scale, with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree. The evolutionary
distances were computed using the Maximum Composite Likelihood method  and are
expressed as number of base substitutions per site. All positions containing
gaps and missing data were eliminated from the dataset (Complete deletion
option). Phylogenetic analyses were conducted in MEGA4 . Tajima's D of the
PCA1 promoter (213 bp) was calculated by DnaSP V5 .
Measurement of soil cadmium concentrations
Soil samples were collected at 7 locations of Evolution Canyon corresponding to
the collection sites of the EC yeast strains (3 at the SFS, one at the VB, and 3
at the NFS). Soil cadmium levels were measured by inductively coupled
plasma-atomic emission spectroscopy (ICP-AES) using at least 200 g of individual