www.sciencemag.org/cgi/content/full/334/6061/1408/DC1
Supporting Online Material for
The Competitive Advantage of a Dual-Transporter System
Sagi Levy, Moshe Kafri, Miri Carmi, Naama Barkai*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 9 December 2011, Science 334, 1408 (2011)
DOI: 10.1126/science.1207154
This PDF file includes:
Materials and Methods
SOM Text
Figs. S1 to S9
Tables S1 to S4
References
Supporting Online Material
Materials and Methods
Media and strains
All strains of S. cerevisiae used in this study were constructed from a BY4741
background (MATa his3-1 leu2-0 met15-0 ura3-0) by standard genetic methods
(see Supplementary Text) and are listed in Supplementary Table S1. Strains were
grown in SC medium (29) or in SC medium depleted of a specific nutrient, as
described in the main text. Glucose limiting media contained 0.2% glucose. Other SC
limiting media were prepared from YNB without the relevant nutrient (ForMedium;
phosphate- CYN0804, zinc- CYN2301, iron- CYN1101, ammonium sulfateCYN0510, copper- CYN0901). Phosphate depleted media was prepared by adding
separately phosphate in the form of KH2PO4 to the specified levels. The level of
potassium was preserved by adding KCl instead of KH2PO4 in proper amount. Zinc
limiting medium was prepared as described previously (30) . Specifically, YNB
without zinc was supplemented by EDTA (1mM), Na3 citrate (10mM), MnCl2
(25μM), FeCl3 (10μM) and proper amount of zinc in the form of zinc sulfate.
Nitrogen limiting medium was prepared from YNB without ammonium sulfate
(CYN0510) by adding separately 50µM of ammonium sulfate. The pH of phosphate
and glucose media was set to 5. Media for chemostat experiments included also
500µl/liter of antifoam (Sigma A5758).
Competition assays
Cells were grown to logarithmic phase in SC medium (OD~0.5) and washed in the
relevant media. GFP and mCherry strains were then co-incubated in the specified
media in 30˚C. The list of competition assays, strains, conditions and repeats are listed
in Supplementary Table S2. The initial OD of the inocula was set to 0.05, and the WT
initial frequency was ~50%. Generation times were calculated from the measured OD
(
). Frequencies of GFP
versus mCherry cells were measured by flow cytometry. The design of the cells‟
dilution time-scales was set from the cells growth rate in the specified condition, and
the desired final OD. Specifically, for competition experiments in conditions that cells
reach stationary phase, cells were diluted once a day (except of low-zinc conditions
for which the slow growing cells were diluted every 1-2 days), and for log phase
conditions, cells were diluted twice a day (low-zinc: once a day). Experiments were
done for WT strains expressing GFP and constitutive strains expressing mCherry, and
vice versa, as listed in Supplementary Table S2. During competition in fluctuating
phosphate, standard phosphate assay was used to validate the shift between phosphate
starvation before dilution (<5μM) and intermediate phosphate after dilution. A linear
fit for the logarithm of the WT frequency dynamics was used to calculate the slope for
each competition assay. The relative WT fitness advantage is calculated from the
slope divided by log(2). As a control, competition assays between similar strains
expressing different fluorescence markers (e.g. WT-GFP vs. WT-mCherry) were done
in different experimental conditions, and no fitness advantage was detected.
Chemostat growth conditions and steady state measurements
Cells were grown in BioFlo 110 chemostats (New Brunswick Scientific) with the
following parameters: working volume = 750ml, temperature = 30ºC, agitation speed
= 500 RPM, air flux = 1 LPM, dilution rate 0.3 h-1. The pH was set on 5 by titration
with NaOH (0.25M). Cell samples were regularly validated under the microscope.
The steady state profiles for different phosphate feeding levels (Figures 2, S1-2) were
characterized by measurements of Pho84 reporter expression (FACS), cell density
(OD600) and residual phosphate levels. 3 technical repeats were done for each
phosphate level. Biological repeats scanning all phosphate levels were done as
follows: wild-type (YMC200)- 4 repeats, PHO84c (YMC201)- 2 repeats, Δpho84PHO84c (YMC202)- 1 and Δpho84Δspl2 (YMC203)- 1 repeat. For figure S1,
experiments were repeated as follows: WT (YMC200)- 4 biological repeats, 25
different steady state conditions, PHO84c (YMC201)- 2 biological repeats, 12 steady
states. The measured extracellular phosphate was above 200μM in all steady states
used for Figure S1.
The steady state fraction of cells that activate the PHO-regulon (Figure 2) was
calculated from the steady state FACS distribution of a YFP marker driven by PHO84
promoter (see also Figure S2). Specifically, a threshold level separating between the
two distinct "induced" and "un-induced" cell distributions was set to compute the
active cells fraction. For figure 2F, lines were added by fitting the data to a Hill
function: Y  A  K n / K n  X n  . Similarly, for figure 2G and S2 lines were obtained
by fitting to the following Hill function: Y  X n / K n  X n  , with n=3. Each point in
figure 2G represents an average over all steady states OD with similar measured
extracellular phosphate levels. For steady-states by which extracellular phosphate is
below the assay detection level (~5μM, Figure 2G-shaded regions), OD values were
averaged over those with similar phosphate feeding levels (see Figure S2 for raw
data). Guide-lines were calculated from the raw data and not from the displayed
averages.
Starvation experiments and lag-time calculation
Cells were grown to logarithmic phase in SC medium (OD~0.5), washed twice and
then incubated in media depleted of a single nutrient as specified in the main text. See
Supplementary Table S3 for the full list of starvation experiments. For glucose
starvation, cells were grown in intermediate glucose medium (1 day in 0.2% glucose)
prior to their transfer into no-glucose medium. Experiments with the strains YMC200
(WT) and YMC201 (Pho84c) in no-phosphate medium were done with (Fig. S3A, P1)
and without (Fig. S3A, P4) an intermediate phosphate phase (1 day in 0.5mM
phosphate medium) prior to their incubation in no-phosphate medium. After 1-10
days of starvation cells were washed and inoculated in 96 well plates with rich media.
OD was measured every 5-15 minutes using an absorbance reader (Tecan Sunrise). 469 independent measurements were conducted for each strain and condition, where
each batch curve was calculated from an average over 4-48 technical repeats. Lag
time for different strain backgrounds are shown in Figure S3, and their averages is
shown in Figure 2. For the calculation of the lag time difference, the lag time of each
constitutive strain was compared to the lag time of the WT strain grown in the same
experiment. Lag time values were calculated by finding the time it takes for the initial
cell density to increase 5 fold (Fig. 3, S3- raw data). Notably, the results are
insensitive to the fold difference parameter chosen.
Measurements of reporter activation dynamics
Cells were grown to logarithmic phase in SC medium (OD~0.5), washed and then
incubated in the limiting media as specified in the main text. The dynamics of PHO84
and ZRT1 promoter activation was measured by fluorescence markers driven by
exogenous PHO84 (YMC200, and YMC201 strains) and ZRT1 (YMC300 and
YMK301) promoters. Fluorescence of cells was measured regularly by flow
cytometry. For experiments of PHO84 activation, medium samples were regularly
taken for phosphate measurements.
Flow cytometry
FACS analysis was done by BD LSRII system (BD Biosciences). Flow cytometry
was conducted with excitation at 488nm and emission at 525±25nm for GFP samples.
For mCherry markers excitation was conducted at 594nm and emission at 610±10nm.
Phosphate measurement
Cell samples were filtered and the media was assayed by a standard photometric assay
(31) with minor modifications. Specifically, stock mix was prepared from DDW,
1.25% ammonium molybdate (Sigma, 277908), and 10% ascorbic acid (Sigma,
A5960) with relative volumes of 5:4:2, respectively. 800μl of the stock was mixed
with 920μl of samples and 280μl of 70% perchloric acid (Sigma, 24,425-2), and then
heated 5 minutes in 100˚C. The samples absorbance was measured at 820nm and
normalized with respect to samples of standard phosphorus (Sigma, P3869) in nophosphate medium. At least 13 calibration samples were used for each assay. Minimal
detection level was estimated by measuring phosphate levels of DDW, mimicking the
depletion of non-phosphate nutrients in our samples. DDW measurements were found
to be 2.7 ± 1.6μM lower than no-phosphate medium (calculated from 57 reactions,
each one with two technical repeats), and the estimated detection level is therefore
~5μM.
Microarrays analysis
WT (YMC102) and PHO84c (YMC201) cells were grown to logarithmic phase in SC
medium (OD=0.5), washed and then incubated in SC medium with intermediate
phosphate level (0.5mM, initial OD=0.05). During the experiment (0, 1, 5, 8 and 23
hours) cells were harvested, pelleted and frozen for further analysis. Total RNA was
extracted using MasterPure™ yeast RNA purification Kit (Epicentre). The samples
were amplified, labeled, hybridized to yeast dual color expression microarrays
(8x15K, G2509F) and scanned, all using standard Agilent protocols, reagents, and
instruments. The scanned images were analyzed using SpotReader software (Niles
Scientific). Problematic probes (flagged by SpotReader, or highly variable, with no
signal, or at regions of spatial bias) were declared 'missing values'. Dye-dependent
(Cy3 vs. Cy5) and array-dependent (same color in different arrays) biases were
corrected by Lowess normalization. The relative expression levels (log2-ratio) shown
in Figure 2 were normalized with respect to the control transcription profiles
(logarithmic growing cells in rich media). The control profile was calculated from an
average over 4 array repeats (2 for each dye). An additional microarray experiment
was done for an additional PHO84c strain (YMC301), and similar results were
obtained. Microarrays data have been deposited in GEO (accession number
GSE32067).
Supporting text
Strains construction
All strains of S. cerevisiae used in this study were constructed from a BY4741
background (MATa his3-1 leu2-0 met15-0 ura3-0) by standard genetic methods,
and listed in Supplementary Table S1. All strains were validated by PCR with
flanking primers, and sequencing of the relevant fragments.
The strain YMC101 was constructed as follows: the fragment containing the NATTEF2pr-mCherry was amplified from pFA6a-NAT-TEF2pr-mCherry plasmid.
Primers were designed with 20bp homology to the plasmid's fragment and 40bp
homology upstream and downstream of HO ORF, and inserted to HO locus by
transformation. YMC102 strain was constructed as mentioned above with the pFA6aNAT-TEF2pr-GFP plasmid. The strain TDH3-GFP was taken from the yeast-GFP
library. The strain YMC305 was constructed by amplifying the mCherryhygromycinB fragment from pBS35 (Yeast Resource Center) and transforming it the
the C-teminus of TDH3 ORF.
The strain YMC200 was generated as follows: a BglII-EcoRI fragment containing the
HIS5 gene from pDH5 (Yeast Resource Center) was used to replace the BglII-EcoRI
fragment containing the kanMX gene of pBS7 (Yeast Resource Center). The promoter
of PHO84 (1000bp upstream to the start codon) was amplified from the genome with
primers containing restriction sites SalI and BamHI at the upstream and downstream
part of the promoter, respectively. The PCR product was digested with SalI and
BamHI and inserted into the respective restriction sites of pBS7. The fragment
containing the PHO84pr-Venus-HIS5 was amplified from pBS7 using the plasmid's
canonical forward and reverse primers (Yeast Resource Center) with 40bp homology
upstream and downstream of his3 ORF, and inserted to his3 locus by transformation.
The strain YMC201 was generated from YMC200 as follows: PHO84 ORF was
inserted to pYM-N14 (32) at the XbaI site, the fragment kanMX-TDH3-PHO84 was
amplified and inserted into the YMC200 by transformation. The strain YMC202 was
generated from YMC201 by replacing the endogenous PHO84 ORF with NAT
resistance. The strain YMC203 was generated from YMC200 by replacing PHO84
ORF with NAT and SPL2 ORF with kanMX.
The strain YMC104 was generated from YMC200 by replacing the PHO84 promoter
with kanMX-TDH3pr fragment (pYM-N14(32)). The strain YMC105 was generated
from YMC200 by replacing the PHO84 promoter with kanMX-TEF1pr fragment
(pYM-N18(32)). Fragments were amplified by the following forward primer:
AATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGATGAGAT
CTGTTTAGCTTGCCTC. Reverse primers were designed with 40bp homology to the
YFP marker and 21bp homology to the C-teminus of either TDH3 or TEF1
promoters. Specifically, the following reverse primers were used: for YMC104GAAAAGTTCTTCTCCTTTACTGTTCATTAACCCGGGGATCCGAAAACT
TAGATTAGATTGC, and for YMC105- GAAAAGTTCTTCTCCTTTACTGTTCAT
TAACCCGGGG ATCCTCGAAACTAAGTTCTTGGTG.
The strains YMC301, YMC302, and YMK309 were generated from the strains
YMC101, YMC102 and BY4741 as follows: the fragment kanMX-TDH3pr was
amplified from pYM-N14(32) and transformed before the PHO84 ORF. Fragments
were amplified using the following primers: Forward-CAGGGCACACAACAAA
CAAAACTCCACGAATACAATCCAAGCTGCAGGTCGACGGATCC, and
reverse- TTTCAGCAACATGAATAGTATCTTTATTGACGGAACTCAT
CCACTAGTTCTAGAATCCG. YMC303, YMC304 and YMK310 were generated
from the strains YMC101, YMC102 and BY4741 by amplifying the fragment kanMXTEF1pr from pYM-N18(32) and transforming it before PHO84 ORF. Fragments were
amplified using the above primers.
YMK301, YMK302, YMK305 and YMK314 were generated from the strains
YMC101, YMC102 and BY4741, by amplifying the fragment kanMX-TEF1pr from
pYM-N18(32) and transforming it before the ZRT1 or ZRT2 ORF. Fragments were
amplified using the following primers: forward- TAGACA ATAAAACAACAGCAC
AAATATCAAAAAAGGAATTGCTGCAGGT CGACGGATCC, reverseGGTCCCATTGTTTCCACCACGGCGTAGTAACGTT
GCTCATCCACTAGTTCTAGAATCCG. The strains YMK303, YMK304, YMK311
were generated from the strains BY4741 and YMC102, by amplifying the fragment
kanMX-TDH3pr from pYM-N14(32) and transforming it before the ZRT1 or ZRT2
ORF. Fragments were amplified using the above primers.
The strain YMK306 was generated from BY4741 by amplifying the fragment kanMXTDH31pr from pYM-N14(32) and transforming it before the MAP2 ORF. The
fragment was amplified using the following primers: forward- TAATATATCATA
CTTAA TATATTACAATACAATATCAACAGCTGCAGGTCGACGGATCC,
reverse- TTCCTTCGCCTGTAGGCGTACCTGTAAAATTGTAAGACATCCACT
AGTTCTAGAATCCG.
The strain YMK307 was generated from BY4741 by amplifying the fragment kanMXTEF1pr from pYM-N18(32) and transforming it before the HXT7 ORF using the
following primers: forward- AAACACAAAAACAAAAAGTTTTTTTAATTT
TAATCAAAAAGCTGCAGGTCGACGGATCC, reverse- CCACAGGAGTTTGC
TCTGCAATAGCAGCGTCTTGTGACATCCACTAGTTCTAGAATCCG.
The strains YSL101 and YSL102 were generated from the strains YMC101 and
YMC102 as follows: kanMX from pYM-N14(32) was replaced with hygromycinB to
generate a hygromycinB-TDH3pr fragment. This fragment was amplified and then
transformed before PHO90 ORF using the following primers: Forward- AGTTGTTT
TTAGGATAAACGAGTAAGTGGTAGCTGGTACAGGATCCGTACGCTGCAGG
TCGAC, and reverse- CCATTCTGGGACAGCATTGTACTTCAAGAAGTGTGAA
AATCTCATCCACTAGTTCTAGAATCCG. The strainYMK323 was generated
from YMK314 by transformation of the fragment hygromycinB-TDH3pr before
ZRC1 ORF using the following primers: forward- CGTCCCTCCCTCTGTTTTCCT
TCTTCAGATGGCCTTGAAGGTGAGcgtacgctgcaggtcgac, reverse- CATGACTC
TATTAGTTTCCTGGTATTCCTGACGGATATCGATAGGgggggatccactagttctag.
The strains YMK321 and YMK325 were generated from the strains YMK314 and
YMK302, respectively, as follows: kanMX from pYM-N18(32) was replaced with
hygromycinB to generate a hygromycinB-TEF1pr fragment. This fragment was
amplified and then transformed before ZRC1 ORF using the following primers:
forward- CGTCCCTCCCTCTGTTTTCCTTCTTCAGATGGCCTTGAAGGTGAG
cgtacgctgcaggtcgac, reverse- CATGACTCTATTAGTTTCCTGGTATTCCTGACGG
ATATCGATAGGgggggatccactagttctag.
The strain YMC300 was generated from BY4741 as follows: 600bp of ZRT1
promoter was inserted to pBS35 (Yeast Resource Center) at the restriction sites: SalI
and BamHI. The fragment containing the ZRT1pr-mCherry-hygromycinB was
amplified using the plasmid's canonical forward and reverse primers (Yeast Resource
Center) with 40bp homology upstream and downstream of his3 ORF, and inserted to
his3 locus by transformation.
The strains YSL201-7 were constructed from a previously described plasmid
(EB1264, (23)) containing a PHO4 mutant allele (contains Serine to Alanine
mutations in 4 phosphorylation sites- SA1234), followed by GFP and URA3
selection. Strains with PHO4SA1234 allele were constructed using EB1264 and the
following primers: forward-AACAAGAGTAGCAGAAAGTCATGGGCCGT
ACAACTTCT GAGGGAATACACGGTTTTGTGG, reverse- AGTCCGATATG
CCCGGAACGTGCTTCCCATTGGTGCACGGGGAGAGCTTTTTCTTTCCAA.
For strains with PHO4WT allele, we used the following forward primer:
TCACTAAAAACAAGA GTAATAGTAGTCCGTATTTGAACAAGCGCAAA
GGTAAACCCGGGC. This primer is upstream to SA1234 mutations. Sequencing of
Pho4 in the YSL207 strain revealed 3 point mutation, not in phosphorylation sites,
and Pho4 protein was correctly localized to the nucleus in all experimental conditions,
as previously described (23).
The strains YMK350-351 and YMC310-311 were generate as follow: a BglII-EcoRI
fragment containing the HIS5 gene from pDH5 (Yeast Resource Center) was used to
replace the BglII-EcoRI fragment containing the kanMX gene of pBS7 (Yeast
Resource Center). The GFP-HIS5 fragment was amplified and transformed to the end
of ZRT1 (YMK) or PHO84 (YMC) before the stop codon, using the following
primers: forward (ZRT1)– TTTCGGTGCTGGTATCATGGCTTTGATCGGTAAG
TGGGCTGGTCGACGGATCCCCGGG, reverse (ZRT1)- AAATAGAATCTATAT
GGAACATGCAGAATTTCGCTTTGGTATCGATGAATTCGAGCTCG, forward
(PHO84)- CATTGAATCTTCCAGCCCATCTCAACTTCAACATGAAGCAGGTC
GACGGATCCCCGGGTT , reverse (Pho84)- TTTGTTCTAGTTTACAAGTTT
TAGTGCATCTTTGAGGCTTTCGATGAATTCGAGCTCGTT.
Supporting Figures
Figure S1
Constitutive expression does not impair competitive growth under a variety of conditions:
(A) Competition experiment design: wild-type (WT) and constitutive expression cells
(PHO84c or ZRT1c, driven by either TDH3 or TEF1 promoters) were differently labeled and
incubated together in different media. The cells were diluted periodically and the fraction of
wild-type vs. constitutive cells was measured using flow-cytometry.
(B-C) The fraction of wild-type cells during competitive growth: Competition was performed
in various non-limiting phosphate (B, PHO84C) or non-limiting zinc (C, ZRT1C) media.
Nonetheless, the various experimental conditions conferred different growth rates due to
reduced levels of non-related nutrients, and different growth conditions. Shown are several
competition time courses, as indicated. The color code denotes the log2-ratio between the
fraction of wild-type (WT) and constitutive cells, normalized with the log2-ratio at the time of
incubation. Cells were diluted twice a day, to maintain in constant logarithmic growth (log),
or once a day, to enable them to reach stationary phase (stat). Level of phosphate and zinc are
as shown, glucose 0.2%, zinc-10μM, phosphate-0.5mM, nitrogen-50μM and no copper. The
shown per-generation fitness advantage of wild-type cells is an average over multiple
experiments with standard errors. Note that green corresponds to higher fraction of the
constitutive strain (the color-map dynamic range is similar to Figure S3B), and that in most
experimental conditions the wild-type and constitutive strains grew practically at the same
rate. See Supplementary Table S2 for the full list of experiments.
(D) Growth of constitutive cells in continuous culture: wild-type (WT) or PHO84c cells were
grown in chemostats with different non-limiting phosphate levels (Methods). Shown is the
average cell-density at steady state. Standard errors are shown in error-bars. Note that the
steady state density of PHO84c strains is similar to that of wild-type cells (p=0.72; paired Ttest) and no fitness disadvantage was observed.
Figure S2
Advanced preparation to nutrient starvation is impaired by constitutive expression of high
affinity transporters in continuous cultures:
(A) Response to phosphate depletion in continuous cultures: Cells were grown in chemostats
with different levels of phosphate in the feeding vessel and reached a steady state. The level
of phosphate in the chemostat was quantified using standard assays (Methods), and the
activation of the phosphate response was monitored in single cells by flow-cytometry analysis
of a PHO84-YFP reporter. Experiments were repeated for wild-type cells, PHO84c
constitutive strains, and cells expressing only low affinity transporters (Δpho84Δspl2), as
indicated. The color of each PHO84 reporter activation pattern corresponds to a different
measured extracellular phosphate level (blue for rich media, red low phosphate).Note the bistable pattern of activation. Note also that wild-type and Δpho84Δspl2 cells fully activate the
PHO-regulon at Pi ~ 400μM (orange), while PHO84c cells are still fully inactive.
(B) The level of phosphate inducing growth limitation in continuous culture: The normalized
cell density is shown as a function of phosphate level in the chemostat. For wild-type and
PHO84c cells, phosphate becomes limiting for growth only when it is reduced to below
10μM, comparable with the Kd of the high affinity transporters. In contrast, growth of
Δpho84Δspl2 cells become sensitive to phosphate levels at much higher levels, comparable to
the low affinity transporters Kd (~300μM). The shaded regions correspond to phosphate levels
that are below our minimal detection limit of 5μM. Each dot in the graph was generated by
averaging 3 technical repeats (3 measurements for each axis). Lines were added to guide the
eye (Methods).
(C) Negative correlation between cell density and the level of Pho84 reporter activation:
Each dot represent a steady state for which the cells are at least partially induced (>10%). The
induction level of the induced cells is shown as a function of the steady state cell density. A
clear negative correlation is observed (r= -0.8 ,p=2·1012), suggesting that the growth
limitation observed by the decrease in cell density reflects the stress to phosphate depletion
shown in the reporter activity.
(D) Steady state phosphate measurements in various phosphate feeding levels: Each dot
represents a steady state value averaged over 3 technical repeats (Methods). The limiting
factor of a chemostat must obey the shown equation ((33); D- dilution rate;
growth characteristics of the strain such that the growth rate obey:
.
Therefore, phosphate becomes limiting only when its steady state values (
does
not depend on the phosphate feeding level (
. For the low affinity strains, phosphate
obey the limiting factor equation when Psteady-state ~ 250µM (arrow on upper panel),
comparable to the Kd of low affinity transporter (13). For the wild-type and PHO84C strains,
phosphate is the limiting factor only when the steady state phosphate is below our detection
level (Psteady-state <5µM, arrows on lower panel), comparable to the Kd of the high affinity
transporters (13).
(E) Phosphate yield is constant: Shown are steady state values in the regime for which
phosphate is the chemostat limiting factor (see Figure S2D). Each dot is an average over 3
technical repeats (Methods). The steady state cell density (Nsteady-state) within the chemostat
should fulfill the shown equation ((33); Y-phosphate yield; Pfeed- feeding Pi level; Psteady-statesteady state Pi level). Note the linear relationship between the cell density and the phosphate
consumption (Pfeed - Psteady-state), as expected for the case of a constant phosphate yield. The
expected cell density for the case of a constant yield was calculated from the average yield
(dotted line).
Figure S3
Constitutive expression of high-affinity transporters impairs competitive growth in fluctuating
nutrients:
(A) Prolonged recovery from starvation: Cells were incubated in a medium depleted of a
single nutrient, as indicated, and cell density was measured following their dilution back to
rich media. Representative examples of cell density are shown for wild-type (dashed) and
constitutive strains (solid). Lag times differences are shown in the bar-graphs (mean +/SEM). Starvation experiments were repeated with over expression of different high affinity
transporters, including: PHO84c (phosphate, black, P1-P4), ZRT1c (zinc, grey, Z1-Z2),
MAP2c (nitrogen, white, M) and HXT7c (glucose, white, H). Numbered letters (P1-P4, Z1Z2) correspond to different strains or experimental conditions (see Supplementary Table S3
for list of experiments). Note the significant longer recovery of the PHO84c cells from
phosphate starvation and of ZRT1c cell from zinc starvation. The recovery time was unaltered
or even shortened for strains that constitutively express glucose (HXT7) or nitrogen (MAP2)
high affinity transporters. Note that the average lag time difference between PHO84c and WT
cells in phosphate starvation is about 1.4h (0.91±0.13 doubling time) and 2.5h (1.7±0.57
doubling times) for the TDH3 and TEF1 strains, respectively. These values are consistent
with competition assays results measuring the fold change difference during exit from
phosphate starvation (Figure 3C, 0.86±0.06 and 2.05±0.2 for the TDH3 and TEF1 strains,
respectively). See Fig. S9 for the dependence of lag time on the time in starvation.
(B) Competition in fluctuating nutrient conditions: wild-type (WT) and constitutive cells
(PHO84c or ZRT1c) were differently labeled and co-incubated into media containing
intermediate levels of nutrient, as indicated. Cells were then grown until the relevant nutrient
was exhausted, before their dilution back into rich (“low to high”) or intermediate
(“fluctuating”) nutrient levels. The fraction of wild-type vs. constitutive cells was measured
using flow-cytometry, and log2-ratio values are shown in color-code as in Figure S1B-C.
White-bars indicate the time of the cells dilution and flow-cytometry measurement. Note the
rapid increase of the fraction of wild-type vs. constitutive high affinity transporter cells
following recovery from the relevant nutrient starvation (red color, PHO84C in phosphate and
ZRT1C in zinc). This fitness advantage was not observed when low affinity transporters were
constitutively expressed (PHO90 or ZRT2) or during fluctuations of a non-relevant nutrient.
(C-D) Per-generation fitness advantage of wild-type cells: Shown is the average fitness
advantage over multiple experiments in different phosphate (C) and zinc (D) conditions (See
Figure 3B for raw data examples and experiment design). Black bars designate the high
affinity transporters relevant to the nutrient conditions (PHO84c in phosphate or ZRT1c in
zinc), grey bars designate non-relevant high affinity transporters (PHO84c in zinc or ZRT1c in
phosphate) and white bars are assigned to the relevant low affinity transporters (PHO90c in
phosphate or ZRT2c in zinc). Fitness advantage was calculated upon the cells entry into
starvation (“Entry”), during their recovery back to rich media (“Exit”, measured after 1 day
for phosphate and 5 hours for zinc), for fluctuating conditions between intermediate levels
and starvation (“Fluctuations”) and in constant conditions (“Constant”). Experiments were
repeated with different initial intermediate levels (X-axis) and for constitutive strains driven
by either TDH3 or TEF1 promoters (left and right bars, respectively; PHO90c is driven by
TDH3 promoter). Standard errors are shown in error bars. Note the large fitness advantage of
wild-type vs. constitutive high affinity transporter cells upon recovery from starvation for the
relevant nutrient (“Exit”, black bars). See Figure 3C for fold-change differences during
recovery from starvation (not normalized by the number of generation). See Supplementary
Table S2 for the list of competition experiments.
(E-F) Activation of starvation responses in various experimental conditions: Phosphate
starvation responses were measured using a YFP marker driven by PHO84 reporter (E) and
zinc starvation responses were measured using a mCherry marker driven by ZRT1 promoter
(F). Fluorescence was measured by flow-cytometry. Representative examples are shown for
the indicated conditions, and for both wild-type (dashed) and constitutive (solid; PHO84c in
phosphate or ZRT1c in zinc) genetic background. Phosphate steady states: logarithmic growth
in SC (Rich-log), cells arrested after growing 10 generations in media containing 20mM P i
(Rich-stationary) or in media that initially contained 0.5mM Pi (0.5mM, stationary, final
Pi<5μM), and cells diluted frequently in 0.5mM Pi media and kept in logarithmic phase
(0.5mM-log). Notably, consistent with our model, PHO84c strain doesn‟t induce Pho84
promoter in intermediate phosphate levels, while wild-type cells in the same condition are
partially active with a bistable activation pattern. From phosphate starvation to rich medium:
Reporter dynamics of starved cells (3 days in no phosphate) upon their transfer back to rich
media (SC). Zinc steady states: logarithmic growth in SC (Rich-log), cells arrested after
growth in SC medium (Rich-stationary) or in media that initially contained 25μM Zn (25μM,
stationary). From zinc starvation to rich medium: Reporter dynamics of starved cells (1 day in
25μM Zn) upon their transfer back to rich media (SC).
Per-transporter flux is significantly higher for low affinity than for high-affinity transporters
in both the zinc and the phosphate system.
For the phosphate system, O‟shea and colleague (13) measured the phosphate influx (Vmax) in
cells expressing a single transporter type only. Those measurements show that the total influx
of phosphate into the cell is similar in cells expressing high-affinity or low affinity
transporters. Those similar fluxes clearly indicate that the per-transporter flux is much lower
for the high-affinity transporters, since Pho84 is expressed at levels significantly higher than
the low affinity ones, even prior to its 100-fold induction by phosphate limitation (see (34)).
From this we conclude that the per-transporter flux is significantly higher for the low-affinity
transporters than the high-affinity ones.
For the zinc system, measurements of total zinc flux in the presence of a single transporter
type are not available. As is shown in the figure below, the constitutive expression of the lowaffinity transporters under conditions of high zinc, where toxicity is known to play a role,
reduces cell fitness whereas constitutive expression of the high-affinity transporters does not.
We attribute this reduced fitness to toxicity, as it can be rescued by over expressing Zrc1
which transport zinc into the vacuoles. From this we conclude that also here, the pretransporter flux is significantly higher for low affinity transporter than for high affinity ones.
Figure S4
In the figure, the indicated strains, and wild-type cells were differentially labeled and coincubated in rich media conditions (cells are kept in logarithmic phase in SC media). Relative
number of cells was monitored regularly by flow-cyotmetry. The growth rate of ZRT1C
strains was similar to wild-type (ZRT1CZRC1WT), while ZRT2C strains were significantly
slower (ZRT2CZRC1WT). Up-regulating the zinc intracellular storage system by constitutive
activation of the ZRC1 gene, reduces ZRT2C growth defects dramatically. Note that this
rescue is observed when ZRC1 is constitutively activated by either TEF1 or TDH3 promoter,
but is stronger when ZRC1 is driven by the stronger TDH3 promoter (see Figure 2).
Figure S5
Competitive fitness disadvantage of PHO84c is rescued by activating the starvation response
also in rich media:
Shown is the average per-generation fitness advantage of wild-type vs.PHO84c cells,
calculated over multiple experiments for cells with a wild-type (PHO4-WT, white) or a
mutated (PHO4-SA1234, black) PHO4 allele. Fitness advantage was calculated upon the cells
entry into phosphate starvation (“Entry”), during their recovery back to rich media (“Exit”,
measured after 1 day), for fluctuating phosphate conditions between intermediate levels and
starvation (“Fluctuations”) and in constant rich medium (“Rich”). See Figure 4B for raw data
examples and experiment design. Experiments were repeated with different initial
intermediate phosphate levels (X-axis). Standard errors are shown in error bars. Note that the
large fitness advantage of wild-type vs.PHO84c cells upon recovery from starvation is rescued
by the partially activated PHO4 allele (“Exit”, compare white to black bars). See Figure 4C
for fold-change differences during recovery from starvation (not normalized by the number of
generation). See Supplementary Table S2 for the list of competition experiments.
Figure S6
Abundance and localization of Pho84 protein in wild type and PHO84C strains:
The high-affinity phosphate transporter was fused to YFP and expressed using either its
endogenous promoter (Wild-type cells) or constitutively using the TDH3 promoter (PHO84C
cells). Both cells carried also an mCherry driven by a constitutive TEF2 which labeles the
cytoplasm; Cells were grown in rich phosphate conditions and then transferred to fresh media
with rich (7.3mM), intermediate (0.1mM) or no phosphate conditions for 3 hours. The
abundance and localization of Pho84 protein was monitored by microscopy (left, mCherry is
labeled red and YFP is labeled green) or using flow cytometry (right, showing two biological
repeats). Images were obtained using a Delta-Vision microscope using the same settings.
Contrast was adjusted to avoid saturation.
Figure S7
Abundance and localization of Zrt1 protein in wild type and ZRT1C strains:
The high-affinity Zinc transporter was fused to YFP and expressed using either its
endogenous promoter (Wild-type cells) or constitutively using the TDH3 promoter (ZRT1C
cells). Both cells carried also an mCherry driven by a constitutive TEF2 which labeled the
cytoplasm. Cells were grown in rich zinc conditions and then transferred to fresh media with
either rich (3 hours in SC) or zinc limiting (5 hours in LZM supplemented with 5µM zinc)
conditions. The abundance and localization of Zrt1-YFP fused protein was monitored using
microscopy (left) and flow cytometry (right). Images were obtained using Delta-Vision
microscope using the sane settings. Contrast was adjusted to avoid saturation.
Fusion of YFP to the Zrt1 protein likely reduced its function, as the cells grew quite poorly in
low-zinc media. Such a limitation was not observed for the Pho84-YFP fusion.
Figure S8
Functionality of constitutively expressed transporters:
(A) PHO84C strain consumes more phosphate than wild-type cells in non-limiting phosphate
chemostats: Shown is the steady state phosphate consumption (Pfeed – Pextracellular) as a function
of the feeding phosphate levels (Pfeed). In these phosphate conditions, growth is not limited by
phosphate (cell density is similar to rich conditions, and also similar for both strains, see also
Figure 2G). Note that in this regime, PHO- regulon is at least partially induced for wild-type
cells, but not induced for constitutive strain.
(B) The fitness advantage of ZRT1C in SC medium is lost by addition of zinc: We find that
constitutive expression of ZRT1 is beneficial for growth at rich media (e.g. SC). This benefit,
however, is lost by adding a high concentration of zinc, likely inducing toxicity. In the
experiment shown, fluorescently labeled ZRT1C and wild-type were co-incubated in the
specified media and their relative frequency was measured by flow cytometry. The fitness
advantage of wild-type cells (left) was calculated from the raw data (left) as indicated
(Methods).
Figure S9
Lag time upon recovery depends on the length of the starvation period:
Cells were incubated in a medium depleted of a single nutrient and their recovery time was
measured upon dilution back to rich media (Methods). Lag times are shown as a function of
the times cells were starved. Each point represents a single experiment. Filled points
correspond to constitutive strains and empty points to wild-type. Starvation to the relevant
nutrient is shown in red (circles- PHO84c in phosphate; squares- ZRT1c in zinc), and to nonrelevant nutrients in grey (circles- PHO84c in zinc, squares- ZRT1c in phosphate, trianglesPHO84c or ZRT1c in glucose).
Supporting tables
Table S1: List of strains used in this study
No
Strain
Type
Fluorescence
1
2
3
4
5
6
7
8
9
10
11
12
13
YMC101
YMC102
TDH3-GFP
YMC104
YMC105
YMC200
YMC201
YMC202
YMC203
YMC301
YMC302
YMC303
YMC304
WT
WT
WT
WT
WT
WT
PHO84c
PHO84c
pho84 spl2
PHO84c
PHO84c
PHO84c
PHO84c
mCherry
GFP
GFP
Venus
Venus
Venus
Venus
Venus
Venus
mCherry
GFP
mCherry
GFP
14
YMC305
PHO84c
mCherry
YMK309
YMK310
YMC310
YMC311
YSL101
YSL102
YSL201
YSL202
YSL203
YSL204
YSL205
YSL206
YSL207
YMK101
YMK102
YMC300
YMK201
YMK301
YMK302
YMK303
YMK304
YMK305
YMK306
YMK307
YMK311
YMK314
YMK321
YMK323
YMK325
YMK350
YMK351
c
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
PHO84
PHO84c
WT
PHO84c
PHO90c
PHO90c
PHO4WT
PHO4WT
PHO4SA1234
PHO4SA1234
PHO84c-PHO4WT
PHO84c PHO4WT
PHO84c-PHO4SA1234
WT
WT
WT
ZRT1c
ZRT1c
ZRT1c
ZRT1c
ZRT1c
ZRT1c
MAP2c
HXT7c
ZRT2c
ZRT2c
ZRT2cZRC1TEF1
ZRT2cZRC1TDH3
ZRT1cZRC1TEF1
WT
ZRT1c
None
None
mCherry+Venus
mCherry+Venus
mCherry
GFP
mCherry+GFP
GFP
mCherry+GFP
GFP
mCherry+GFP
GFP
mCherry+GFP
mCherry
mCherry
mCherry
mCherry
mCherry
GFP
GFP
None
None
None
None
mCherry
GFP
GFP
GFP
GFP
mCherry+Venus
mCherry+Venus
Genotype
BY4741 ho::NAT-TEF2pr-mCherry
BY4741 ho::NAT-TEF2pr-GFP
BY4741 TDH3-GFP-HIS5
BY4741 his3:: kanMX-TEF1pr-Venus-HIS5
BY4741 his3:: kanMX-TDH3pr-Venus-HIS5
BY4741 his3::PHO84pr-Venus-HIS5
YMC200 kanMX-TDH3pr-PHO84
YMC200 pho84::NAT, kanMX-TDH3pr-PHO84
YMC200 pho84::NAT, spl2::kanMX
YMC101 PHO84pr::kanMX-TDH3pr
YMC102 PHO84pr::kanMX-TDH3pr
YMC101 PHO84pr::kanMX-TEF1pr
YMC102 PHO84pr::kanMX-TEF1pr
BY4741 TDH3- mCherry-hygromycinB,
PHO84pr::kanMX-TDH3pr
BY4741 PHO84pr::kanMX-TDH3pr
BY4741 PHO84pr::kanMX-TEF1pr
YMC101 PHO84- Venus -HIS5
YMC301 PHO84- Venus -HIS5
YMC101 PHO90pr::hygromycinB -TDH3pr
YMC102 PHO90pr::hygromycinB -TDH3pr
YMC101 PHO4::PHO4WT-GFP-URA3
YMC102 PHO4::PHO4WT-GFP-URA3
YMC101 PHO4::PHO4SA1234-GFP-URA3
YMC102 PHO4::PHO4SA1234-GFP-URA3
YMC301 PHO4::PHO4WT-GFP-URA3
YMC302 PHO4::PHO4WT-GFP-URA3
YMC301 PHO4::PHO4SA1234-GFP-URA3
BY4741 his3::kanMX-TEF1pr-mCherry- hygromycinB
BY4741 his3::kanMX-TDH3pr-mCherry-hygromycinB
BY4741 his3::ZRT1pr-mCherry-hygromycinB
YMC300 ZRT1pr::kanMX-TEF1pr
YMC101 ZRT1pr::kanMX-TEF1pr
YMC102 ZRT1pr::kanMX-TEF1pr
YMC102 ZRT1pr::kanMX-TDH3pr
BY4741 ZRT1pr::kanMX-TDH3pr
BY4741 ZRT1pr::kanMX-TEF1pr
BY4741 MAP2pr::kanMX-TDH3pr
BY4741 HXT7pr::kanMX-TEF1pr
YMC102 ZRT2pr::kanMX- TDH3pr
YMC102 ZRT2pr::kanMX-TEF1pr
YMK314 ZRC1pr:: hygromycinB - TEF1pr
YMK314 ZRC1pr:: hygromycinB -TDH3pr
YMK302 ZRC1pr:: hygromycinB - TEF1pr
YMC101 ZRT1- Venus -HIS5
YMC101 ZRT1pr::kanMX-TDH3pr ZRT1- Venus -HIS5
Source
This study
This study
Yeast-GFP library(35)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study, EB1264 from (23)
This study, EB1264 from (23)
This study, EB1264 from (23)
This study, EB1264 from (23)
This study, EB1264 from (23)
This study, EB1264 from (23)
This study, EB1264 from (23)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
All strains were derived from BY4741 MATa his3-1 leu2-0 met15-0 ura3-0.
Table S2: List of competition assays
No
Transporter
Constitutive
promoter
Media
Culture
condition
WT marker
Repeats
(total)
1
PHO84
TDH3
20mM Pi
stationary
mCherry/GFP
60
2
PHO84
TDH3
20mM Pi
log
mCherry/GFP
18
3
4
PHO84
PHO84
TDH3
TDH3
7.3mM Pi
7.3mM Pi
stationary
log
GFP
GFP
12
4
5
PHO84
TDH3
0.5mM Pi
stationary
mCherry/GFP
57
6
PHO84
TDH3
0.5mM Pi
log
mCherry/GFP
24
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO84
PHO90
PHO90
PHO90
PHO90
PHO90
PHO90
PHO90
PHO84-PHO4WT
PHO84-PHO4WT
PHO84-PHO4WT
PHO84-PHO4WT
PHO84-PHO4WT
PHO84-PHO4WT
PHO84-PHO4WT
PHO84-PHO4SA1234
PHO84-PHO4SA1234
PHO84-PHO4SA1234
PHO84-PHO4SA1234
PHO84-PHO4SA1234
PHO84-PHO4SA1234
PHO84-PHO4SA1234
TDH3
TDH3
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TEF1
TEF1
TEF1
TDH3
TDH3
TDH3
TEF1
TEF1
TEF1
TDH3
TDH3
TEF1
TEF1
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
0.2mM Pi
0.1mM Pi
20mM Pi
20mM Pi
0.5mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
Glucose
Glucose
Nitrogen
Nitrogen
10μM Zn
25μM Zn
75μM Zn
150μM Zn
1500μM Zn
10μM Zn
25μM Zn
1500μM Zn
0.5mM Pi
0.2mM Pi
0.1mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
10μM Zn
25μM Zn
10μM Zn
25μM Zn
20mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
20mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
20mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
stationary
stationary
stationary
log
stationary
log
stationary
stationary
log
stationary
log
stationary
stationary
stationary
stationary
stationary
stationary
stationary
stationary
stationary
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
stationary
stationary
stationary
stationary
Exit to SC
Exit to SC
Exit to SC
stationary
stationary
stationary
stationary
Exit to SC
Exit to SC
Exit to SC
stationary
stationary
stationary
stationary
Exit to SC
Exit to SC
Exit to SC
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
GFP
GFP
GFP
GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry
mCherry
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
GFP
GFP
GFP
GFP
GFP
GFP
GFP
16
12
10
2
5
2
8
8
4
12
4
12
4
16
12
6
4
2
1
2
12
12
12
3
8
8
4
4
1
1
18
10
12
8
10
8
8
15
11
7
7
4
7
7
13
11
5
5
3
5
5
Strains (repeats)
YMC301 (23), YMC302 (25),
YMC305 (12)
YMC301 (7), YMC302 (7),
YMC305 (4)
YMC305 (12)
YMC305 (4)
YMC301 (22), YMC302 (23),
YMC305 (12)
YMC301 (10), YMC302 (10),
YMC305 (4)
YMC301 (8), YMC302 (8)
YMC301 (6), YMC302 (6)
YMC303 (5), YMC304 (5)
YMC303 (1), YMC304 (1)
YMC303 (2), YMC304 (3)
YMC303 (1), YMC304 (1)
YMC303 (4), YMC304 (4)
YMC303 (4), YMC304 (4)
YMC305 (4)
YMC305 (12)
YMC305 (4)
YMC305 (12)
YMC301 (2), YMC302 (2)
YMC301 (8), YMC302 (8)
YMC301 (6), YMC302 (6)
YMC301 (3), YMC302 (3)
YMC301 (2), YMC302 (2)
YMC303 (1), YMC304 (1)
YMC304 (1)
YMC303 (1), YMC304 (1)
YMC301 (6), YMC302 (6)
YMC301 (6), YMC302 (6)
YMC301 (6), YMC302 (6)
YMC303 (1), YMC304 (2)
YMC303 (4), YMC304 (4)
YMC303 (4), YMC304 (4)
YMC301 (2), YMC302 (2)
YMC301 (2), YMC302 (2)
YMC304 (1)
YMC304 (1)
YSL101(9), YSL102 (9)
YSL101(5), YSL102 (5)
YSL101(6), YSL102 (6)
YSL101(4), YSL102 (4)
YSL101(5), YSL102 (5)
YSL101(4), YSL102 (4)
YSL101(4), YSL102 (4)
YSL205(6), YSL206(9)
YSL205(4), YSL206(7)
YSL205(3), YSL206(4)
YSL205(3), YSL206(4)
YSL205(2), YSL206(2)
YSL205(3), YSL206(4)
YSL205(3), YSL206(4)
YSL207(13)
YSL207(11)
YSL207(5)
YSL207(5)
YSL207(3)
YSL207(5)
YSL207(5)
No
Transporter
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT1
ZRT2
ZRT2
ZRT2
ZRT2
ZRT2
ZRT2
ZRT2
ZRT2
ZRT2
ZRT2
ZRT2
ZRT1 + ZRC1TEF1
ZRT2 + ZRC1TEF1
ZRT2 + ZRC1TDH3
Constitutive
promoter
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TDH3
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
TDH3
TDH3
TDH3
TDH3
TDH3
TEF1
TEF1
TEF1
TEF1
TEF1
TDH3
TDH3
TDH3
TEF1
TEF1
TEF1
TDH3
TDH3
TEF1
TEF1
TEF1
TEF1
TEF1
TEF1
Media
1500μM Zn
500μM Zn
500μM Zn
300μM Zn
300μM Zn
150μM Zn
150μM Zn
25μM Zn
10μM Zn
SC
SC
Glucose
Glucose
Copper
20mM Pi
0.5mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
1500μM Zn
500μM Zn
500μM Zn
300μM Zn
300μM Zn
150μM Zn
150μM Zn
25μM Zn
10μM Zn
SC
SC
Glucose
Glucose
Copper
20mM Pi
0.5mM Pi
0.5mM Pi
0.2mM Pi
0.1mM Pi
25μM Zn
10μM Zn
0.5mM Pi
0.2mM Pi
0.1mM Pi
25μM Zn
10μM Zn
0.5mM Pi
0.2mM Pi
0.1mM Pi
1500μM Zn
25μM Zn
10μM Zn
1500μM Zn
25μM Zn
10μM Zn
25μM Zn
10μM Zn
25μM Zn
10μM Zn
SC
SC
SC
SC
Culture
condition
stationary
stationary
log
stationary
log
stationary
log
stationary
stationary
stationary
log
stationary
log
stationary
stationary
stationary
log
stationary
stationary
stationary
stationary
log
stationary
log
stationary
log
stationary
stationary
stationary
log
stationary
log
stationary
stationary
stationary
log
stationary
stationary
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
Exit to SC
stationary
stationary
stationary
stationary
stationary
stationary
Exit to SC
Exit to SC
Exit to SC
Exit to SC
log
log
log
log
WT marker
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
mCherry
mCherry
mCherry
mCherry
mCherry
GFP
GFP
mCherry/GFP
mCherry/GFP
mCherry/GFP
GFP
GFP
GFP
mCherry
mCherry
mCherry
GFP
GFP
mCherry
mCherry
mCherry
mCherry
mCherry
mCherry
Repeats
(total)
2
2
2
2
2
2
2
8
2
8
10
8
4
2
6
11
4
6
6
4
4
4
4
4
4
4
13
4
18
8
16
8
4
8
23
8
8
8
8
11
3
6
6
4
4
8
8
4
4
4
4
4
4
4
4
8
2
6
4
7
6
6
Strains (repeats)
YMK303 (2)
YMK303 (2)
YMK303 (2)
YMK303 (2)
YMK303 (2)
YMK303 (2)
YMK303 (2)
YMK303 (8)
YMK303 (2)
YMK303 (8)
YMK303 (10)
YMK303 (8)
YMK303 (4)
YMK303 (2)
YMK303 (6)
YMK303 (11)
YMK303 (4)
YMK303 (6)
YMK303 (6)
YMK301 (2), YMK302 (2)
YMK301 (2), YMK302 (2)
YMK301 (2), YMK302 (2)
YMK301 (2), YMK302 (2)
YMK301 (2), YMK302 (2)
YMK301 (2), YMK302 (2)
YMK301 (2), YMK302 (2)
YMK301 (6), YMK302 (7)
YMK301 (2), YMK302 (2)
YMK301 (8), YMK302 (10)
YMK301 (4), YMK302 (4)
YMK301 (7), YMK302 (9)
YMK301 (4), YMK302 (4)
YMK301 (2), YMK302 (2)
YMK301 (4), YMK302 (4)
YMK301 (11), YMK302 (12)
YMK301 (4), YMK302 (4)
YMK301 (4), YMK302 (4)
YMK301 (4), YMK302 (4)
YMK303 (8)
YMK303 (11)
YMK303 (3)
YMK303 (6)
YMK303 (6)
YMK301 (4)
YMK301 (4)
YMK301 (4), YMK302 (4)
YMK301 (4), YMK302 (4)
YMK301 (2), YMK302 (2)
YMK311 (4)
YMK311 (4)
YMK311 (4)
YMK314 (4)
YMK314 (4)
YMK314 (4)
YMK311 (4)
YMK311 (8)
YMK314 (2)
YMK314 (6)
YMK314 (4)
YMK325 (7)
YMK321 (6)
YMK323 (6)
Notes:





A total of 933 competition assays were conducted in 119 different conditions and
strains (not including the controls described below).
GFP strains were competed against the WT-mCherry strain (YMC101), and
mCherry strains were competed against the WT-GFP strain (YMC102), with the
following exceptions:
o PHO84C strain with a PHO4SA1234 allele (YSK207) was competed against
WT with a similar allele (YSK204).
o PHO84C strains with a PHO4WT allele (YSL205, YSL206) were competed
against WT with a similar allele (YSL201, YSL202).
o PHO84C strain with mCherry fused to the TDH3 protein (YMC305) was
competed against a WT strain with GFP fused to the TDH3 protein
(TDH3-GFP).
Measurements for recovery experiments („exit to SC‟) were done after 5 hours or
1 day for the zinc and phosphate experiments, respectively.
For the ZRT1c strains, the variability between biological repeats in different
experiments was significantly larger than the variability of between biological
repeats within an experiment, possibly due to sensitivity to fluctuations in zinc
level in the media.
Many additional control experiments were conducted between strains with similar
background but different fluorescence markers, and no fitness advantage was
detected in these cases. The control experiment included the following
competition assays: YMC101-YMC102, YMC301-YMC302, YMC303-YMC304,
YMK301-YMK302. Control experiments were repeated in all relevant
experimental conditions.
Table S3: List of starvation experiments
No
Transporter
Promoter
Constitutive
Strain
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
PHO84
PHO84
PHO84
PHO84
ZRT1
ZRT1
MAP2
HXT7
PHO84
PHO84
PHO84
ZRT1
ZRT1
MAP2
HXT7
PHO84
PHO84
ZRT1
TDH3
TDH3
TEF1
TDH3
TDH3
TEF1
TDH3
TEF1
TDH3
TDH3
TEF1
TDH3
TEF1
TDH3
TEF1
TDH3
TEF1
TEF1
YMC201
YMK309
YMK310
YMC201
YMK304
YMK305
YMK306
YMK307
YMC201
YMK309
YMK310
YMK304
YMK305
YMK306
YMK307
YMC201
YMK310
YMK305
WT
reference
strain
YMC200
BY4741
BY4741
YMC200
BY4741
BY4741
BY4741
BY4741
YMC200
BY4741
BY4741
BY4741
BY4741
BY4741
BY4741
YMC200
BY4741
BY4741
Starvation
Media
Intermediate
phase
Independent
Measurements
Independent
colonies
Name in
Fig. S2
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Phosphate
Zinc
Zinc
Zinc
Zinc
Zinc
Zinc
Zinc
Glucose
Glucose
Glucose
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
69
3
4
66
4
4
4
4
66
6
4
6
5
5
6
67
6
4
66
6
6
3
6
6
6
6
8
6
6
6
3
6
3
7
6
6
P1
P2
P3
P4
Z1
Z2
M
H
P1
P2
P3
Z1
Z2
M
H
P1
P3
Z2
Notes:



Each independent measurement corresponds to OD measurements of a cell culture
upon transferring the cells back to rich medium after 1-10 days in starvation. Each
independent measurement consisted of 4-48 technical repeats, which were
averaged for further analysis.
Independent colonies correspond to different starters.
In experiments with intermediate phase, the starter cells were transferred from rich
media to a limiting medium, and after 1 day the cells were transferred to full
starvation medium.
Table S4: Activation of phosphate responsive genes upon phosphate starvation
PHO84
SPL2
PHO89
PHO5
PHM6
PHM4
PHO11
PHM2
PHM3
PHO12
1h
1.73
1.84
2.93
0.74
1.77
0.92
1.33
1.50
0.99
1.18
Wild-type
5h
8h
1.84
2.21
1.58
2.48
2.50
2.44
1.12
1.62
1.23
2.14
0.84
1.17
0.87
0.98
1.33
1.34
0.99
1.06
1.14
0.93
23h
4.21
4.09
6.08
4.39
3.75
3.23
2.32
1.10
0.87
0.21
1h
0.16
-0.14
1.55
-0.80
-0.73
-1.47
-2.62
-0.48
-0.32
-1.51
PHO84C
5h
8h
-0.44
0.31
1.69
0.21
0.36
-0.95
-1.70
-0.34
0.21
-0.78
-0.20
0.58
0.63
0.65
0.72
-0.74
-1.11
-0.22
-0.08
-0.54
Cells were transferred to medium containing 0.5mM phosphate. Activation of the
phosphate starvation response was measured repeatedly using microarrays (Methods).
Values are log2 ratios, the reference being cells grown in rich medium.
23h
-0.63
5.20
5.78
3.59
3.65
3.43
2.32
1.14
0.70
0.19
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