RESEARCH ARTICLE
The adaptive response of anaerobically grown Saccharomyces
cerevisiae to hydrogen peroxide is mediated by theYap1and Skn7
transcription factors
Anthony G. Beckhouse1, Chris M. Grant2, Peter J. Rogers3, Ian W. Dawes1 & Vincent J. Higgins4
1
Ramaciotti Centre for Gene Function Analysis, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia;
Faculty of Life Sciences, University of Manchester, Manchester, UK; 3Foster’s Group Ltd, Abbotsford, Australia; and 4School of Biomedical and Health
Sciences, University of Western Sydney, Penrith South DC, Australia
2
Correspondence: Vincent J. Higgins, School
of Biomedical and Health Sciences, University
of Western Sydney, Locked Bag 1797, Penrith
South DC 1797, Australia. Tel.: 161 2 4620
3206; fax: 161 2 4620 3025; e-mail:
[email protected]
Received 9 June 2008; revised 22 July 2008;
accepted 12 August 2008.
First published online 15 September 2008.
DOI:10.1111/j.1567-1364.2008.00439.x
Editor: Isak Pretorius
Keywords
Saccharomyces cerevisiae ; oxidative stress;
anaerobic; aerobic.
Abstract
The molecular mechanisms involved in the ability of cells to adapt and respond to
differing oxygen tensions are of great interest to the pharmaceutical, medical and
fermentation industries. The transcriptional profiles reported in previous studies
of cells grown under anaerobic, aerobic and dynamic growth conditions have
shown significantly altered responses including induction of genes regulated by the
oxidative stress transcription factor Yap1p when oxygen was present. The present
study investigated the phenotypic changes that occur in cells when shifted from
anaerobic to aerobic growth conditions and it was found through mutant analyses
that the elevated activity of Yap1p during the shift was mediated by the
phospholipid hydroperoxide-sensing protein encoded by GPX3. Cell viability and
growth rate were unaffected even though anaerobically grown cells were found to
be hypersensitive to low doses of the oxidative stress-inducing compound
hydrogen peroxide (H2O2). Adaptation to H2O2 treatment was demonstrated to
occur when anaerobically grown wild-type cells were aerated for a short time that
was reliant on the Yap1p and Skn7p transcription factors.
Introduction
Aerobic organisms primarily respire to generate energy
through the breakdown of organic molecules such as
glucose, with oxygen as a vital cofactor. Most oxygen
molecules are reduced to water during the respiratory
process but c. 5% can undergo incomplete reduction to
form reactive oxygen species (ROS). ROS are well-known
cellular toxicants that induce DNA damage, protein oxidation and lipid peroxidation. To reduce the toxicity of such
species, organisms have evolved antioxidant defence systems
to counter the damaging effects. If the balance between
antioxidant and oxidant is perturbed, the organism is
known to have undergone oxidative stress (Jamieson, 1998;
Dawes, 2004). Oxidative stress is of interest in many fields
such as the medical and pharmaceutical industries where it
has been implicated in the pathology of diseases such as
cancer, cardiovascular disease, arthritis and the ageing
process (Halliwell, 1997; Halliwell & Gutteridge, 1999;
Finkel & Holbrook, 2000).
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c
Under anaerobic conditions, organisms are spared the
toxic effects of oxidative stress; however, it has been noted in
the yeast Saccharomyces cerevisiae that antioxidant proteins
are reduced in level rather than absent and this is thought to
be a protective mechanism against possible introduction to
an aerobic environment and ROS (Ohmori et al., 1999). In
most environmental niches, organisms experience a dynamic flux in oxygen availability and must be able to adapt
rapidly for survival. Saccharomyces cerevisiae is an ideal
model organism for studying the effect of altering oxygen
tension due to its ability to survive in aerobic and anaerobic
environments along with its accessible genome sequence
(Goffeau et al., 1996) and the availability of single-gene
deletion mutants in nonessential genes (Winzeler et al.,
1999). Many transcriptional studies have been performed
on cells in steady-state anaerobiosis or aerobiosis (ter Linde
et al., 1999; Piper et al., 2002), as well as shifts between the
two oxygen tensions (Kwast et al., 1998, 2002; Lai et al.,
2005, 2006), which identified that differential regulation of
genes involved the oxidative stress response, ergosterol and
FEMS Yeast Res 8 (2008) 1214–1222
1215
Adaptation of anaerobic yeast to hydrogen peroxide
haem biosynthesis, cell-wall maintenance and amino acid
biosynthesis. A recent study has also identified up- and
downregulated proteins within the proteome under
anaerobic and aerobic conditions (de Groot et al., 2007).
Such studies led to the observation that transcription factors
such as Yap1, Mot3, Upc2 and Sut1 may coordinately
regulate transcription of genes during steady state or
dynamic oxygen tensions. Altering oxygen tensions in live
cells is of interest in the medical field where procedures such
as open heart surgery, organ transplant, myocardial infarct
and stroke all deprive tissues of oxygen for periods and the
resultant reintroduction of oxygenated blood introduces
ROS, which can further damage tissues (Fellstrom et al.,
1998; Kaminski et al., 2002; Crack & Taylor, 2005). Similar
damaging effects occur in the brewing industry where
oxygen fluctuations can affect fermentation kinetics,
flavour profiles, shelf-life and yeast viability due to ROS
(O’Connor-Cox & Ingledew, 1990; Higgins et al., 2003;
Rosenfeld et al., 2003).
The upregulation of oxidative stress response genes
following a shift from anaerobic to aerobic conditions
indicates that cells are exposed to damaging oxidants, which
may affect cell viability or growth kinetics. The shift to
aeration and subsequent exposure to ROS may also allow
cells to adapt to exogenous oxidative stress-causing agents
such as hydrogen peroxide (H2O2). Adaptation occurs
when cells are exposed to sublethal doses of an oxidant
that induces a transient protective response that confers
resistance to subsequent doses that would normally be lethal
to the cells (Collinson & Dawes, 1992; Jamieson, 1992;
Flattery-O’Brien et al., 1993).
The aims of the present study were to determine the
sensitivity of anaerobically grown S. cerevisiae to H2O2,
investigate the viability and growth kinetics of cells shifted
from anaerobic to aerobic conditions and elucidate any
adaptive response and role of transcription factors in the
adaptive response during the shift.
Materials and methods
Strains and plasmids
Saccharomyces cerevisiae strains were obtained from the
homozygous diploid single deletion collection (Euroscarf
http://www.uni-frankfurt.de/fb15/mikro/euroscarf). Strains
were derivatives of wild-type BY4743 (MATa/MATahis31/
his3D1 leu2D0/leu2D0 met15/met15D0 lys2D0/LYS2ura3D0/
ura3D0) with the following mutations: rox1, upc2, mot3,
sut1, hap4, yap1, skn7, msn2, msn4, gcn4, skn7yap1 and
msn2,4. Plasmid pyDJ73 containing GSH1<lacZ was
provided by Derek Jamieson, pSC99 and pCEP12 containing
TRX2<lacZ YRE-CYC1<lacZ, respectively, were provided
FEMS Yeast Res 8 (2008) 1214–1222
by W. Scott Moye-Rowley and TRX1 lacZ containing
TRX1<lacZ was supplied by Chris M. Grant.
Growth conditions
Cells were grown in synthetic complete (SC) medium
supplemented with components (ergosterol and unsaturated fatty acids) essential for anaerobic growth (Andreasen
& Stier, 1953, 1954). Anaerobic cultures were grown using
methods modified from Skoneczny & Rytka (2000), Panozzo et al. (2002) and Passoth et al. (2003). Essentially, SC
medium (Alic et al., 2001) was supplemented with the
redox-indicator dye resazurin (2 mg mL 1; Sigma-Aldrich,
St. Louis, CA) and the pH was adjusted to 4.5. Media were
aliquoted into 100-mL penicillin bottles before degassing
with high-purity nitrogen gas (BOC gases, Sydney, NSW,
Australia) and sealed with butyl-rubber stoppers and aluminium cap seals (Wheaton Science, Millville, NJ). Ergosterol
(Sigma-Aldrich) and Tween 80 (Sigma-Aldrich) were
supplemented from a fresh stock (ergosterol 4 mg mL 1;
Tween 80, 40% v/v in ethanol) at a volume of 0.5 mL per
100 mL of medium. The reducing agent sodium dithionite
(Sigma-Aldrich) was added to the media before inoculation
at a concentration of 100 mg L 1 to reduce trace amounts of
oxygen. Aerobic cultures were grown in media identical to
the anaerobic cultures including the supplements (except
nitrogen gas). All incubations of liquid cultures were
performed at 30 1C with shaking at 250 r.p.m. Anaerobic
manipulations and anaerobic incubation of solid
media were performed in an anaerobic hood (Modular
Atmosphere Controlled System) (DW Scientific, Shipley,
Yorkshire, UK) flushed with a gas mixture of N2 : CO2 : H2
at a ratio of 75 : 20 : 5.
Aeration of anaerobically grown cells was performed by
adding the culture to a sterile prewarmed (30 1C) flask
where the culture volume was at least 1/5 of the flask
volume. Aeration was performed in a 30 1C incubator with
shaking at 250 r.p.m.
YEPD medium (2% w/v glucose, 2% w/v Bacto peptone,
1% yeast extract) (Sigma-Aldrich) was used for dilution of
cells and YEPD agar (YEPD plus 2% w/v type 3 agar) was
used for cultivating cells during viability determination.
Viability measurement of cultures treated with
H2O2
Anaerobic and aerobic cultures were grown to an OD600 nm
of 1 and subsequently treated with 0, 0.1, 0.25, 0.5, 0.75 and
1.5 mM H2O2 for 10 min. An aliquot of 1 mL was removed
and serially diluted appropriately in liquid YEPD. Serial
dilutions (200 mL) were then spread onto YEPD agar in
triplicate and incubated at 30 1C for 3 days to obtain
viable counts.
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1216
To test the viability and growth kinetics of cells when
shifted from anaerobic to aerobic growth conditions,
exponential-phase cells (OD600 nm = 1) were sampled in an
anaerobic hood and serially diluted. For viability tests,
appropriate dilutions (200 mL) were spread on YEPD agar
in duplicate sets of triplicates. One triplicate set was
removed to an aerobic incubator at 30 1C; the other set
remained in the anaerobic hood for incubation at 30 1C for 3
days. Growth kinetics of anaerobic and aerated samples were
monitored by measurement at an OD600 nm.
Adaptation to H2O2 treatment with a period of
aeration
Cultures were grown under anaerobic conditions to the
exponential phase (OD600 nm = 1) and an aliquot (10 mL)
was removed for aeration in a prewarmed 50-mL flask.
The anaerobic culture was then treated with 1.5 mM H2O2
(Ajax Chemicals, Sydney, NSW, Australia) for a period of
60 min. Samples were removed, serially diluted in YEPD and
spread on YEPD agar in triplicate every 15 min. Following
incubation for 30 min, the aerated cultures were treated with
1.5 mM H2O2 for 60 min, with samples taken every 15 min.
Viability was determined as a percentage of the mean
number of cells before H2O2 treatment.
b-Galactosidase assays
Specific activity of the b-galactosidase in extracts of cells
containing the Yap1p reporter construct in pCEP12 was
assayed essentially as described previously (Rose & Botstein,
1983). Cell protein in the extract was measured with bovine
serum albumin (Sigma-Aldrich) was used as a standard and
protein assay dye reagent (Bio-Rad, Hercules, CA) was used
to bind proteins as per the manufacturer’s protocol (Bradford, 1976). Specific activity was expressed as nanomoles
of o-nitrophenyl-b-D-galactopyranoside (ONPG) (SigmaAldrich) hydrolysed per minute per microgram of total
protein. Each data point was obtained from triplicate
cultures and results were expressed as the mean and
SD. Statistical analyses were performed using t-tests to
determine significance at a 95% confidence level.
Cell harvesting, RNA extraction and Northern
blotting
Cultures were grown under anaerobic conditions to the
exponential growth phase (OD600 nm = 1) and then transferred to a prewarmed 250-mL flask and incubated with
shaking at 30 1C for indicated periods of time. Anaerobic
samples were harvested immediately in a prechilled tube
containing ice by centrifugation at 4 1C. Cells were
snap-frozen in liquid nitrogen and stored at 80 1C until
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A.G. Beckhouse et al.
RNA extraction was performed. The aerated cells were
harvested in the same manner.
Total RNA was obtained by breaking cells with acidwashed glass beads in the Trizol reagent (Invitrogen,
Carlsbad, CA) using a mini-bead beater and extracted
according to the manufacturer’s instructions. The quality of
RNA was determined using agarose gel electrophoresis and
ethidium bromide staining as well as by spectrophotometric
analysis (A260 nm /A280 nm) using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE). Northern blotting was performed as described in Sambrook et al.
(1989). Probes were generated by random primer labelling
of PCR-amplified regions of the TRR1, ARG4 and ACT1
ORFs with a-32P dCTP and a-32P dATP (Perkin Elmer Life
Sciences, Melbourne, Vic., Australia). Prehybridization and
hybridization were performed at 65 1C in a RapidHyb
buffer (Amersham Biosciences, UK). Radioactivity of
membrane-bound probes was imaged and quantified
using an FLA-5100 phosphorimager and Image Gauge V4
(Fujifilm, Japan).
Results
Activation of Yap1p during the shift from
anaerobic to aerobic conditions
Lai et al. (2006) reported that a rapid transcriptional
response occurred when anoxic yeast cultures were shifted
into an aerobic environment. Of the transcripts identified to
alter within 5 min of the shift, many are known to belong to
the oxidative stress regulon, which is regulated by the Yap1p
transcription factor; therefore, we measured the activation
of the Yap1p transcriptional activator during aeration of
anaerobically grown cells. A wild-type strain harbouring the
YRE<lacZ plasmid (Wu & Moye-Rowley, 1994) containing
multiple Yap1p response elements (YRE’s) located upstream
of a minimal CYC1<lacZ fusion gene reporter was used. As
a control, the plasmid was also transformed into a strain
(Dgpx3) that is unable to sense the presence of H2O2 and
therefore unable to mount a Yap1p-mediated response to
H2O2 (Delaunay et al., 2002). The strains were grown
anaerobically and then shifted to aerobic conditions.
Samples were taken at the intervals indicated and assayed
for b-galactosidase activity. It can be seen in Fig. 1a that
there was a significant induction of the reporter construct
within 30 min of the shift, which remained at a high level for
120 min. The activity of the Yap1 reporter construct in the
Dgpx3 strain remained at relatively low levels during the
shift compared with the wild type; however, there was a
statistically significant increase (P o 0.05) in the level of the
reporter construct during the shift compared with the levels
seen in anaerobically grown cultures (t = 0 min). Control
cultures of each strain harbouring the reporter construct
FEMS Yeast Res 8 (2008) 1214–1222
1217
Adaptation of anaerobic yeast to hydrogen peroxide
(b) 80
gpx3
WT
30
*
20
*
*
10
*
0
Specific activity (nmol mg min)
Specific activity (nmol mg min)
(a) 40
TRX1::lacZ
TRX2::lacZ
GSH1::lacZ
60
*
*
40
*
*
20
0
0
30
60
90
120
O
Time (min)
0
30
60
90
120
O
Time (min)
Fig. 1. b-Galactosidase assays of Yap1 activity and Yap1-regulated genes during a shift from anaerobic to aerobic conditions. Anaerobic cultures of
yeast containing reporter constructs were grown in triplicate to exponential phase and shifted to aerobic conditions. Samples were taken at the
indicated times. Aerobic cultures were grown to the exponential phase and harvested as a control. (a) WT yeast strain BY4743 and mutant gpx3
containing the Yap1p activity reporter construct YRE-CYC1-lacZ. (b) WT yeast strain BY4743 containing TRX1<lacZ, TRX2<lacZ or GSH1<lacZ reporter
constructs. Average measurements are indicated with error bars ( SD) included. Indicates the first time-point where a significant (P o 0.05)
difference from time zero occurred or whether the aerobic level of b-galactosidase activity was significantly (P o 0.05) greater than anaerobic levels.
were grown under aerobic conditions and both strains
showed a significant increase in activation during aerobic
growth compared with anaerobic growth but not at the
levels observed during the shift. We also tested whether
known marker genes in the oxidative stress regulon were also
upregulated in response to the aeration. The lacZ reporter
constructs for TRX2 and GSH1 were transformed into wildtype yeast and assayed for b-galactosidase activity over an
aeration time course of 2 h. A control reporter construct was
also included (TRX1<lacZ) in the experiment because it is
known to be unresponsive during oxidative challenge (Lee
et al., 1999; Garrido & Grant, 2002). The results in Fig. 1b
clearly show that both TRX2 and GSH1 reporter levels were
significantly increased (P o 0.05) after 30 min of aeration
compared with anaerobic cells, indicating that the response
to aeration includes an oxidative stress response.
Viability and growth kinetics of shifted yeast
cells
The results presented thus far indicate that cells transcribe
oxidative stress response genes rapidly (but not exclusively)
and antioxidant genes are upregulated when cells are shifted
from anaerobic growth to aerobic conditions. Therefore, it
was of importance to determine the viability and growth
kinetics of the cells in which oxidative stress was signalled
following the shift in oxygen tension. To test whether
aeration of anaerobically grown samples would affect
the viability or kinetics of cell growth, CFU plate tests
were performed. Anaerobic cultures were grown to an
OD600 nm = 1.0 and placed a Modular Atmosphere
Controlled System anaerobic chamber at 30 1C. Aliquots
were removed from the flasks, serially diluted and plated
FEMS Yeast Res 8 (2008) 1214–1222
onto solid YEPD media and incubated in either an anaerobic chamber or removed and incubated aerobically at 30 1C.
There was no significant difference in viability as indicated
in Fig. 2a, but due to the length in time required for CFUs to
arise, kinetics of the cultures were also measured after the
aliquots were removed to determine whether any perturbation in growth was observed. A further aliquot was removed
from anaerobic cultures into prewarmed flasks and compared with the anaerobic culture growth kinetics. ODs were
measured for 9 h after shifting and the results are illustrated
in Fig. 2b. No differences in kinetics were observed between
shifted and anaerobic cultures until 6 h postshift where
aerobic cells changed the carbon source and began to
generate energy through respiration. This is a slightly
unexpected result as the transcriptional profiles reported by
Lai et al. (2006) indicated that cells underwent cellular
reprogramming, which may have resulted in some growth
retardation but this was clearly not the case. Because no
effect was observed on the viability or the growth kinetics of
shifted yeast cultures but the transcriptional response and
level of antioxidant reporter protein indicated that cells may
experience oxidative challenge, it was then of interest to
determine whether addition of an oxidant at low concentrations to anaerobic cells would affect cell viability.
Anaerobically grown cells are hypersensitive to
H2O2
Previous studies have shown that anaerobic yeast cells do
express basal levels of antioxidant enzymes that are hypothesized to prepare cells against sudden exposure to oxygen and
its radicals (Hortner et al., 1982; Ohmori et al., 1999). The
sensitivity of anaerobically grown cells to oxidative challenge
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1218
A.G. Beckhouse et al.
(b)
80
10
60
log OD600 nm
40
20
Shifted
Anaerobic
1
0
Aerobic
Anaerobic
Incubation condition
0
2.5
5
7.5
The postshift (h)
is not well understood. Susceptibility to H2O2 was used to
determine whether cells grown anaerobically were more
sensitive to the oxidant than cells grown under aerobic
conditions. The exponential-phase cells cultured under
anaerobic or aerobic conditions were treated with different
concentrations of H2O2 for 10 min and their viabilities were
determined. Cells that had been grown under anaerobic
conditions showed a marked reduction in viability (85%) at
a relatively low dose of H2O2 (0.1 mM) compared with
untreated cells. As the concentration of H2O2 increased to a
maximum of 1.5 mM, the viability of the anaerobically
grown cells decreased dramatically (Fig. 3). Aerobically
grown cultures did not show any decrease in viability across
the H2O2 concentrations used. Such a large difference in
viabilities at very low concentrations indicates that aeration
of anaerobic cells introduces a very small amount of
oxidative stress that elucidates a transcriptional and
physiological response but is sufficiently low that it does
not affect the growth and viability of cells. Therefore, it may
be possible that aeration of anaerobically cultured cells may
be sufficient to induce an adaptive response to an exogenous
oxidative challenge.
Anaerobic cultures of yeast adapt to H2O2
toxicity when aerated
Yeast elicits an adaptive response that confers resistance to
normally toxic levels of H2O2 when pretreated with low
doses of the oxidant (Collinson & Dawes, 1992). Cultures of
aerobic and anaerobic yeast cells were treated with 1.5 mM
H2O2 and samples were removed and tested for viability at
15-min intervals for a total of 60 min. Additional cultures of
anaerobically grown cells were aerated for a period of 30 min
before addition of 1.5 mM H2O2 and samples were then
removed and measured for viability in the same manner as
the anaerobic and aerobic cultures. As shown in Fig. 3,
1.5 mM H2O2 treatment for 10 min was lethal to anaerobically grown cells but did not affect aerobically cultured cells.
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10
Fig. 2. Viability and growth kinetics of yeast
shifted from anaerobic to aerobic conditions.
Anaerobic, exponential phase yeast cultures were
shifted to aerobic conditions. (a) The number of
viable cells measured using a CFU plate test of
triplicate samples plated onto solid YEPD and
incubated in aerobic or anaerobic conditions. (b)
The growth of triplicate cultures measured using
OD600 nm after the exponential phase had been
reached in anaerobic conditions and
samples were split into aerobic and anaerobic
environments. Error bars are included ( SD).
100
Log percent survival
Number of cells (CFU mL –1 × 106)
(a)
Anaerobic
Aerobic
10
0
0.5
1
Concentration H2O2 (mM)
1.5
Fig. 3. Sensitivity of anaerobic and aerobic exponential phase yeast
cultures to H2O2. Wild-type yeast strain (BY4743) was inoculated and
grown to exponential phase in aerobic or anaerobic conditions then
treated with indicated concentrations of H2O2 for 10 min. CFU plate tests
were used to quantify viable cells. Survival is expressed as the colonyforming ability in each incubation condition relative to that before
treatment. Data are the means of three independent experiments with
error bars ( SD) included.
Similarly, Fig. 4 shows that anaerobic cells were hypersensitive to treatment with an oxidant compared with aerobic
cells when treated for 60 min. Cells that were aerated before
oxidant addition behaved differently. The period of aeration
generated a resistance to the oxidative challenge in a manner
similar to an adaptive response. Transcription factors play
a major role in eliciting an adaptive response to stress;
therefore, we tested many transcription factor mutants
identified through transcriptional and proteomic studies.
Transcription factor involvement in anaerobic to
aerobic adaptive response
Transcriptional analyses of the anaerobic to aerobic shift by
Lai et al. (2006), identified clustered sets of genes with
FEMS Yeast Res 8 (2008) 1214–1222
1219
Adaptation of anaerobic yeast to hydrogen peroxide
similar expression profiles. These sets allowed in silico
identification of consensus promoter-binding motifs that
may play a role in regulation of the genes within the cluster.
A set of transcription factors identified in that study and a
Log percent survival
100
10
1
0.1
0.01
0
20
40
Time posttreament (min)
60
(a) 100
(b) 100
Log percent survival
Log percent survival
Fig. 4. Survival of H2O2 treated anaerobic, aerobic and anaerobic shifted
yeast cultures. Exponential phase cultures of yeast strain BY4743 were
grown anaerobically, aerobically and anaerobically with a 30-min aeration
were treated with 1.5 mM H2O2. Survival was measured using CFU plate
tests and is expressed as the colony-forming ability relative to that before
treatment. Data are the means of three independent experiments with
error bars ( SD) included but not visible due to low error.
proteomic study of de Groot et al. (2007) included Rox1,
Upc2, Mot3, Sut1, Hap4, Yap1, Skn7, Msn2, Msn4 and
Gcn4, and strains with a single deletion of each of these
transcription factors were obtained from the yeast single
nonessential deletion collection (Winzeler et al., 1999).
Strains harbouring double mutations (skn7 yap1 and msn2
msn4) were also obtained as the two transcription factors
often function together in stress responses. Mutants were
then used to test the ability of aerated cells to adapt to an
oxidant challenge after a period of aeration. When compared with the wild-type response in Fig. 4, all mutant
strains behaved in a manner similar to the wild type, except
for strains that harboured knock-outs of yap1, skn7 and the
double skn7 yap1 (Fig. 5a–c, respectively). The data clearly
show that the Dyap1 strain was defective in its ability to
mount an adaptive response to H2O2 after a period of
aeration. A similar response was seen in the Dskn7 strain
shown in Fig. 5b; however, the Dskn7 mutant did demonstrate a level of adaptive response that was absent in the
Dyap1 strain, but was still impaired relative to the wild type.
The Dskn7 Dyap1 double mutant had a similar phenotype
compared with the single Dyap1 strain when grown anaerobically; however, it was more sensitive to the oxidant when
grown under aerobic conditions (Fig. 5c). Anaerobic
cultures of the three mutant strains (Fig. 5) were only
slightly more sensitive to H2O2 treatment than anaerobic
wild-type cells (Fig. 4), indicating that Yap1p and Skn7p do
10
1
Anaerobic
0.1
10
1
0.1
Aerobic
Anaerobic + Aeration
0.01
0.01
0
FEMS Yeast Res 8 (2008) 1214–1222
60
0
(d)
100
4
× 107)
(c)
10
3
Number of cells (cfu mL
Log percent survival
Fig. 5. Survival of H2O2 treated anaerobic,
aerobic and shifted yeast strains. Exponential
phase cultures of yeast were grown in the above
conditions and treated with 1.5 mM H2O2. (a)
Dyap1, (b) Dskn7, (c) Dskn7 Dyap1. Survival is
expressed as the colony-forming ability relative to
that before treatment. (d) Exponential phase
cultures of the WT BY4743 strain and three
mutants were grown to exponential phase
and plated onto solid YEPD under anaerobic
conditions in duplicate. Cultures were incubated
either aerobically or anaerobically. Data are the
means of three independent experiments with
error bars ( SD) included.
20
40
Time posttreament (min)
1
0.1
20
40
Time posttreament (min)
60
WT
skn7
yap1
DKO
2
1
0
0.01
0
20
40
Time posttreament (min)
60
Anaerobic
Aerobic
Incubation
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1220
A.G. Beckhouse et al.
not have a major role when growing anaerobically.
Conversely, the double mutant was more sensitive to
oxidant challenge than the wild type and each single
mutant under aerobic conditions, demonstrating the
importance of Yap1p and Skn7p for aerobic growth.
The strains harbouring mutations were also tested for their
ability to survive exposure to air relative to continued
anaerobic growth, and Fig. 5d indicates that the shift to
an aerobic environment did not affect the ability of the cells
to survive. Interestingly, the Dgcn4 mutant was unable
to grow under the anaerobic conditions used. To determine
whether Gcn4p was activated under anaerobic conditions,
we measured the abundance of Gcn4p target gene
ARG4 using Northern analysis. Figure 6 clearly shows
that ARG4 is rapidly downregulated upon aeration of
anaerobically grown cells whereas an oxidative stress gene
representative TRR1 was rapidly upregulated and
house-keeping gene ACT1 expression remained constant in
response to aeration.
Relative expression
10
1
0.1
ACT1
ARG4
TRR1
0.01
0
10
20
30
45
60
O2
ACT1
ARG4
TRR1
Time (min)
Fig. 6. Northern blot analysis of ACT1, ARG4 and TRR1 during a shift
from an aerobic to anaerobic environment. Wild-type yeast strain
BY4743 was grown in anaerobic conditions to exponential phase then
aerated for a period of 60 min with samples taken at the intervals
indicated. Total RNA was extracted and run on a denaturing agarose gel
then transferred to nylon filters. Radioactively labelled probes for ACT1,
ARG4 and TRR1 were hybridized to the filters. Scanning and quantification were performed using a phosphorimager. Top panel is the relative
level of expression of each gene compared with that in an anaerobically
growing sample and is a representative example of a triplicate experiment. Lower panel is the scanned image produced using the phosphorimager of the labelled filters.
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Discussion
The ability of organisms to adapt rapidly to environmental
change is extremely important for survival. The genetic and
molecular mechanisms involved in this phenomenon are
described here for the response of S. cerevisiae to aeration
after a period of anaerobic growth. The above data show that
this yeast has the capacity to adapt very rapidly to the shift
from anaerobiosis to aerobiosis without adjusting its growth
rate or losing viability. This indicates the presence of active
homeostatic systems, including one based on the Yap1p
transcription factor associated with ensuring that ROS do
not present a problem to the cell.
The thorough microarray experiment performed by Lai
et al. (2006) identified a rapid transcriptional response for
yeast cells shifted from anaerobic to aerobic conditions with
in silico clustering of gene families that may show coregulation
through transcription factors. A similar study by Higgins et al.
(2003) performed on an industrial-scale shift of yeast cells from
anaerobic to aerobic conditions also identified genes involved
in the oxidative stress response and ergosterol biosynthesis, but
they included knock-out studies to further suggest that ergosterol content and oxidative stress can affect the viability of cells.
The results presented here show that aeration of anaerobically
growing cells produced no effect on cell viability or growth rate
when a single stress was introduced, indicating that the
transcriptional response to oxygenation may play a role in
adaptation of cells to further oxidative challenge. Reporter gene
assays were performed to measure the activity of the oxidative
stress response transcription factor Yap1 and also expression of
genes under its control during the shift. There was rapid
upregulation of Yap1 activity and Yap1 target genes during the
shift to an aerated environment and the values measured were
significantly higher than those found in steady-state aerobic
cultures, indicating that cells react to overwhelm oxidative
challenges transiently before steady-state homeostasis. It has
been shown previously that genes involved in the response to
oxidative stress (thioredoxin, thioredoxin reductase and glutathione reductase) play an important role in maintaining the
redox potential within the cell (Drakulic et al., 2005). The
upregulation of genes involved in the oxidative stress response
following a shift from anaerobiosis to aerobic conditions
indicated that an oxidative stress was encountered and that
the cells required an alteration in redox homeostasis upon
introduction into an aerobic environment. The rapidity of this
response may play a role in sustaining growth rate and survival
that was observed when shifted to aerobic environments.
A previous report studying the effect of fatty acids in the
membrane of anaerobic and aerobic cells indicated that
anaerobically grown cells were more sensitive to H2O2
compared with aerobically grown cells but this required very
high levels of the oxidant to produce the phenotype (Steels
et al., 1994). This was an interesting observation as a later
FEMS Yeast Res 8 (2008) 1214–1222
1221
Adaptation of anaerobic yeast to hydrogen peroxide
report detected oxidative stress response enzymes at much
lower levels than aerobically grown cells, which was
hypothesized to be more of a protective mechanism in the
event of exposure to very low levels of oxygen and its radicals
(Ohmori et al., 1999). Therefore, we tested the tolerance of
anaerobically cultured cells to low and high doses of H2O2
and found that the cells were hypersensitive to very low
levels of oxidant. Because, in these experiments, anaerobically grown cells were able to induce an oxidative stress
response without affecting the viability or the kinetics when
aerated, we sought to determine whether aeration of anaerobic cells would induce an adaptive response to an additional oxidative challenge. Anaerobically grown cells have
shown adaptability to other types of redox stresses
previously (Krantz et al., 2004); therefore, it seemed likely
that adaptation to an oxidant through aeration may occur.
The adaptive response to oxidative stress in yeast is well
studied in aerobic environments and is known to involve
specific transcription factors Yap1 and Skn7 (Lee et al., 1999;
Ng et al., 2008). Yap1 activation is dependent on the sensing
protein Gpx3 (Delaunay et al., 2002) but is also known to be
activated by Gpx3-independent mechanisms (Kuge et al.,
2001; Azevedo et al., 2003). In silico identification of
transcription factors through microarray analyses identified
many transcription factors that may play a role in adapting to
the aerated environment (Higgins et al., 2003; Lai et al., 2006);
therefore, transcription factor mutants were tested for their
ability to adapt to an oxidant challenge via aeration. This study
identified that Yap1 has a major role in the adaptive process
through aeration in a Gpx3-dependent manner. Skn7 was
shown to have a minor role in the process but when deleted
together with Yap1 did not produce additive loss in adaptability. This clearly implicates Yap1 as the major transcription
factor involved in the ability to adapt to an oxidant after the
onset of aeration. Other transcription factor mutations that
were tested did not affect the adaptive response; however, it was
noted that the gcn4 strain was unable to grow under the
conditions tested. Gcn4 is a transcriptional activator of amino
acid metabolism genes (Hinnebusch, 1984) and many microarray experiments have identified its target genes to be
upregulated under anaerobic conditions (ter Linde et al.,
1999; Piper et al., 2002; Lai et al., 2006). Interestingly, in an
attempt to identify essential genes for anaerobic growth, Gcn4
was omitted (Snoek & Steensma, 2006). In contrast, we report
that the gcn4 strain did not grow under the anaerobic conditions used, which may indicate an important role for amino
acid biosynthesis for growth under anaerobic conditions.
Acknowledgements
We wish to acknowledge Nazif Alic, Cristy Gelling, Ruby Lin,
Bronwyn Robertson and Nick Matigian for their assistance in
RNA and microarray analysis, W. Scott Moye-Rowley and
FEMS Yeast Res 8 (2008) 1214–1222
Derek Jamieson for providing plasmids and Frank Estruch for
the msn2,4 strain. This work was funded by an Australian
Research Council Linkage grant and Carlton and United
Beverages. A.G.B. was funded by an Australian Postgraduate
Award and C.M.G. was funded by the Wellcome Trust.
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