Adenine auxotrophy - be aware: Some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A

W303 strain cell count mL−1 depending on OD600. Yeast strains were grown in YPD media with (100 mg L−1) and without extra adenine supplement. Cell number mL−1 was determined by hemocytometer. Vertical dashed line indicates adenine depletion during W303 ade2 cultivation in YPD media without extra adenine supplement. Approximations of third-order polynomial functions were used for visualization of cell number mL−1 data. However, it does not fully account for relationship between the variables.

W303 strain mean cell size. Cells were harvested after 27.5 h of cultivation, and their cross-section areas were measured from micrographs as described by Jorgensen et al. (2007). Cell number exceeded 300 for each strain. Means of both distributions were compared by Wilcoxon signed rank-sum test. Mean cell area of W303 ade2 and W303 ADE2 revealed to be significantly different (P < 0.0001).

Growth curves of W303 ade2 strain in SD media with different adenine content (0, 5, 10, 20, 40 and 100 mg L−1); all other nutrients were added in surplus. Each growth curve is mean of biological triplicates; error bars represent standard deviations. Statistical comparison between OD measurements during cultivation in medias of different adenine concentrations and 100 mg L−1 (positive control) was made. Asterisks below data point denote significant difference from positive control (P < 0.05).

Changes in accumulated trehalose (a) and desiccation tolerance (b) during W303 prototroph, W303 ADE2 and W303 ade2 cultivation in YPD media. Cells were desiccated in +30 °C for 10 h. Desiccation tolerance was quantified as in Calahan et al. (2011). In both (a) and (b), lines denote strain growth curves in logarithmic scale and bars trehalose (a) or survival (b). Error bars depict standard deviation from biological triplicates. Viability in first two time points (8 and 12 h) is close to zero (see b). Asterisks depict statistically significant difference (P < 0.05) between trehalose content of W303 ade2 and both W303 prototroph and W303 ADE2 strains (a). Also it depicts difference between W303 ade2 and both W303 prototroph and W303 ADE2 strains desiccation tolerance as statistically significant (P < 0.05) (b).

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RESEARCH ARTICLE

Adenine auxotrophy –be aware: some effects of adenine

auxotrophy in Saccharomyces cerevisiae strain W303-1A

Agnese Kokina, Juris Kibilds & Janis Liepins

Institute of Microbiology and Biotechnology, University of Latvia, Riga, Latvia

Correspondence: Agnese Kokina, Institute

of Microbiology and Biotechnology,

Kronvalda bldv. 4, Riga LV-1586, Latvia.

Tel./fax: +371 67034885;

e-mail: agnese.kokina@lu.lv

Received 25 September 2013; revised

24 February 2014; accepted 18 March 2014.

Final version published online 11 April 2014.

DOI: 10.1111/1567-1364.12154

Editor: Terrance Cooper

Keywords

auxotrophy; starvation; adenine; W303;

desiccation tolerance; trehalose.

Abstract

Adenine auxotrophy is a commonly used genetic marker in haploid yeast

strains. Strain W303-1A, which carries the ade2-1 mutation, is widely used in

physiological and genetic research. Yeast extract-based rich medium contains a

low level of adenine, so that adenine is often depleted before glucose. This

could affect the cell physiology of adenine auxotrophs grown in rich medium.

The aim of our study was to assess the effects of adenine auxotrophy on cell

morphology and stress physiology. Our results show that adenine depletion

halts cell division, but that culture optical density continues to increase due to

cell swelling. Accumulation of trehalose and a coincident 10-fold increase in

desiccation stress tolerance is observed in adenine auxotrophs after adenine

depletion, when compared to prototrophs. Under adenine starvation, long-term

survival of W303-1A is lower than during carbon starvation, but higher than

during leucine starvation. We observed drastic adenine-dependent changes in

cell stress physiology, suggesting that results may be biased when adenine auxo-

trophs are grown in rich media without adenine supplementation.

Introduction

Like all micro-organisms, baker’s yeast cells respond to

environmental changes and adapt their growth and prolif-

eration accordingly. A drop in nutrient availability is a

signal for the onset of nutrient limitation and impeding

starvation. Even a slight drop in the concentration of a

critical nutrient is sufﬁcient to induce alterations in cell

physiology such as the initiation of a ‘preconditioning

programme’, which prepares the cell for harsh conditions

(Smets et al., 2010).

Two types of limitations or starvations, dependent on

the nutrient, can occur: ‘natural limitations’, which sets

in when basic nutrients (carbon, phosphorous, sulphur

and nitrogen) are scarce, and ‘artiﬁcial limitations’,

which sets in when particular metabolites or metabolic

intermediates are insufﬁcient (Saldanha et al., 2004).

Additionally, depending on nutrient supply mode, dis-

tinction is made between starvation and limitation. Star-

vation for certain nutrient is deﬁned if it is absent,

whereas limitation occurs when certain nutrient is added

in scarce amounts and thus limits the growth. Starvation

is a typical phenomenon of batch cultivations, and limi-

tation is usually attributed to chemostat cultivations.

Auxotrophy is a typical example of an artiﬁcial limita-

tion. Many common laboratory yeast strains (W303,

S288C, CEN.PK and FY series) contain one or several

auxotrophic markers. Histidine, leucine, uracil, adenine

and tryptophan (his, leu, ura, ade and trp) are the most

common auxotrophic markers of Saccharomyces cerevisiae

strains used in physiology studies (Pronk, 2002; Da Silva

& Srikrishnan, 2011). Insufﬁcient concentration of an

auxotrophic agent leads to artiﬁcial limitation. Depend-

ing on the type of limitation, the yeast cells exhibit dif-

ferent responses. Cell cycle arrest and subsequent

quiescent state constitute a typical response to natural

limitations (Boer et al., 2008). However, cell cycle arrest

and quiescence do not occur for an artiﬁcial limitation

with either leucine or uracil. On the other hand, inabil-

ity to complete cell division and to halt subsequent cell

cycle leads to a decrease in viability in addition to an

observable ‘glucose wasting’ phenomenon (Boer et al.,

2008).

The adenine auxotrophic marker, ade2-1, is common

to the S. cerevisiae strain W303-1A and its derivatives.

These strains are well known for the fact that they

acquire red colouration during culture growth. This red

pigment is the oxidised form of ribosylaminoimidazole,

FEMS Yeast Res 14 (2014) 697–707 ª2014 Federation of European Microbiological Societies.

YEAST RESEARCH

an intermediate of the adenine de novo synthesis pathway.

The adenine auxotrophy-dependent red colouration is

used in white red mutant screens (Weng & Nickoloff,

1997) and synthetic lethality assays (Barbour & Xiao,

2006). In addition to ade2,ade1 is also used in white red

mutant screens, and ade8, another adenine de novo syn-

thesis pathway gene, has been used as an integration site

yielding moderate expression levels of heterologous genes

(Sadowski et al., 2007).

External adenine supplement in synthetic media is

needed to promote proliferation of adenine auxotrophs.

However, availability of adenine is sometimes ignored in

rich media (e.g. yeast extract-based media) because it is

assumed that all nutrients are present in sufﬁcient levels.

Several researchers point out that in rich media, adenine

levels vary from batch to batch and the adenine is often

depleted before exhaustion of the carbon source (VanDu-

sen et al., 1997; Zhang et al., 2003).

Besides hampering proliferation, adenine auxotrophy

might have other adverse effects on yeast physiology.

Thus far, adenine auxotrophy has been associated with a

decrease in heterologous protein expression. Interestingly,

this has been observed for both low and high external

adenine levels (VanDusen et al., 1997; Zhang et al.,

2003).

W303-1A and its derivatives have been exploited in

basic physiology research for 30 years (Carlson & Bot-

stein, 1982; Ralser et al., 2012). However, to our knowl-

edge, no research on the effects of adenine auxotrophy

on physiology of this particular strain has been per-

formed. Many physiological studies are performed in

batch mode using rich media where the effects of ade-

nine limitation can become pronounced, obfuscating the

physiological phenomenon of interest. The ﬁndings from

the studies herein can help to minimize these undesired

effects. In the present work, we report some basic cul-

ture physiology and cell morphology studies using

W303-1A batch cultivation. We ﬁnd that adenine deple-

tion has a direct impact on the cell size, trehalose con-

tent and subsequent desiccation stress tolerance. Taken

together, our results serve as a basis for new interpreta-

tions of some previous results regarding yeast stress

physiology as well as a warning against assuming that

adenine auxotrophy provides a neutral background for

physiology studies.

Materials and methods

Strains

Laboratory strains used in this study are shown in

Table 1. CEN.PK ade8 disruption was created by homolo-

gous recombination using a URA cassette and screening

for ura



mutants on 5-FOA as described in Sadowski

et al. (2007).

Growth media

YPD [10 g L

1

of yeast extract (Biolife), 20 g L

1

of pep-

tone (Biolife), 20 g L

1

of dextrose (Sigma)] was used for

yeast cell physiology studies: growth dynamics, cell mor-

phology, trehalose content and desiccation stress toler-

ance. Synthetic dextrose (SD) media [1.7 g L

1

of yeast

nitrogen base w/o amino acids and ammonium sulphate

(Difco), 5 g L

1

of (NH

)

,20gL

1

of dextrose]

supplemented with leucine (260 mg L

1

), of tryptophan

(80 mg L

1

), of uracil (100 mg L

1

), histidine

(100 mg L

1

) and adenine (100 mg L

1

) was used for

adenine titration experiments. For starvation experiments,

SD media with either adenine, leucine or glucose omitted

depending on starvation type investigated were used.

Cultivation

Yeasts were cultivated in shake ﬂasks at 180 r.p.m. in

30 °C with broth volume not exceeding 20% of the ﬂask

volume.

Morphological measurements

Optical density (OD) was measured at 600 nm with

Ultrospec 2100 pro (Amersham Biosciences), diluting

cultures below 0.3 absorbance units.

The cells dry weight was determined by harvesting bio-

mass from 20 to 40 mL of cultivation broth by centrifu-

gation, washing twice with distilled water, and drying at

Table 1. Yeast strains used in this study

Strain name in text Strain genotype Source

W303 ade2 W303-1A MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 Dr. Peter Richard

W303 ADE2 W303-1A ADE2 Dr. Arnold Kristjuhan

W303 prototroph 2832 –1B MATa can1 Dr. Frederick R. Cross

CEN.PK prototroph CEN.PK 113-1A Dr. Peter Richard

CEN.PK ADE8 CEN.PK2 MATa leu2-3/112 ura3-52 trp1-289 his3-1,MAL2-8c SUC2 Dr. Peter Richard

CEN.PK ade8 CEN.PK2 MATa leu2-3/112 ura3-52 trp1-289 his3-1,ade8,MAL2-8c SUC2 This study

FEMS Yeast Res 14 (2014) 697–707ª2014 Federation of European Microbiological Societies.

698 A. Kokina et al.

105 °C until reaching constant weight. For cell counts

and morphology assessment, cells were ﬁxed in 1% glu-

taraldehyde and then sonicated brieﬂy. Cell number was

counted by hemocytometer.

Cell size (for a sample size of n>300) was determined

by analysing micrographs with IMAGEJ software, approxi-

mating cell shape as an ellipse and calculating the area of

the acquired ellipses (Jorgensen et al., 2007). Buds, if they

exceeded half the size of the mother cell, were deﬁned as

separate cells. Budding index, deﬁned as the proportion

of cells with buds, was determined for the population

from a sample size of n>500.

Metabolite measurements

Anthrone assay was used to determine trehalose and media

glucose concentrations (Terevelyan & Harrison, 1956). For

trehalose measurements, cells were washed with distilled

water twice, disintegrated in 5% TCA with glass beads, and

then the supernatant (diluted with water when necessary)

mixed with anthrone (2 g L

1

in 75% H

)ina1:6

ratio. The mixture was heated at 100 °C for 10 min, and

absorbance at 626 nm was measured. The same procedure

was used for media glucose quantiﬁcation.

The concentration of adenine in media was determined

enzymatically following a modiﬁed protocol from Zhang

et al. (2003). More speciﬁcally, the concentration of ade-

nine in media was quantiﬁed ﬂuorometrically by hypo-

xanthine oxidase (Sigma X4500)-coupled assay using

horseraddish peroxidase (HRP; Sigma) and Amplex Ultr-

aRed dye (Molecular Probes



, ex/em 530/590 nm). The

reaction mix contained 20 lL of sample, 2 mL 0.1 M,

pH 7.5 sodium phosphate buffer, 0.02 U xanthine oxi-

dase, and 2 U HRP. Reaction mixtures were incubated at

30 °C for 30 min at which point the emission at 590 nm

(ex 530) was measured with a FluoroMax-3 (Yvon Hori-

ba) spectroﬂuorometer.

Stress tolerance assessment

Desiccation tolerance was assayed by estimating CFU

1

, before and after dehydration. One millilitre of cul-

ture at OD

600

=1 was washed with distilled water twice,

diluted serially and spotted on YPD plates. The remaining

cell suspension was centrifugated, and the pellet was left to

desiccate for 10 h at 30 °C in a desiccator and then rehy-

drated for 10 min in room temperature in distilled water.

The suspension of rehydrated cells was serially diluted and

spotted on YPD plates. The viability was calculated by

dividing the number of CFU mL

1

before and after desic-

cation, as performed in Calahan et al. (2011).

To assess starvation stress tolerance, cells were grown in

full SD media up to exponential phase, washed with dis-

tilled water, and re-suspended to OD

600

=1 in SD media

lacking either sugar, leucine or adenine with all other

broth components added in surplus. The yeasts were incu-

bated for 10 days in a rotary shaker, and samples were

taken upon inoculation (day 0) and on the 1st, 2nd, 4th,

7th and 10th day. Samples were diluted serially and plated

on YPD plates to assess viability. Undiluted sample was

ﬁxed with glutaraldehyde and later used for budding

index, cell size and count mL

1

measurements.

Statistical treatment of data

All the represented values are means from biological trip-

licates. Error bars and variation depict standard errors.

Two-tailed, two-sample unequal variance Student’s t-test

or Wilcoxon rank-sum test (for cell size comparison)

were used to compare means of physiological parameters.

P-values <0.05 were considered statistically signiﬁcant.

Results

Growth characteristics and cell morphology

The effects of adenine auxotrophy on the S. cerevisiae

strain W303-1A (from here on called W303 ade2) were

explored during cell growth in YPD (glucose content 2%)

media. A W303-1A-derived adenine prototroph (W303

ADE2) strain was used as a control. External adenine

depletion was monitored by xanthine oxidase coupled to

horseradish peroxidase assay. Also, red colouration of

W303 ade2 cells served as a signal for adenine depletion.

The red pigment in yeast cells was observed after adenine

became depleted in the media, as detected by xanthine

oxidase assay. However, adenine auxotrophs tend to accu-

mulate a vast amount of adenine and then use it upon

depletion of the external adenine pool (VanDusen et al.,

1997). The adenine synthesis pathway and subsequent red

pigment accumulation in adenine auxotrophs are induced

after adenine de novo synthesis is started, which occurs

when no free adenine remains inside the cell (Rebora

et al., 2001). Therefore, we assumed that pigment devel-

opment is a physiologically more reliable signal for ade-

nine starvation in the cell than the concentration of

adenine in the media.

We assessed growth of W303 ade2 and its correspond-

ing adenine prototroph W303 ADE2 using yeast extract

from only one producer due to known variance in ade-

nine content across different commercial suppliers

(VanDusen et al., 1997; Zhang et al., 2003). For all exper-

iments, yeast extract from the same producer and single

batch was used.

First, we compared growth dynamics of W303 adenine

auxotroph and prototroph in YPD media to determine

FEMS Yeast Res 14 (2014) 697–707 ª2014 Federation of European Microbiological Societies.

Effects of adenine auxotrophy in S. cerevisiae W303-1A 699

whether adenine content in media inﬂuences general cell

physiology.

OD measurements at 600 nm were used to monitor

growth (Fig. 1a). Throughout the exponential growth

phase (from 0 to 10 h), both strains, W303 adenine auxo-

troph and prototroph, grew similarly. The adenine content

in the media, measured by the xanthine oxidase assay, was

depleted at the same time for both strains (see Fig. 1).

Moreover, speciﬁc glucose consumption rates and biomass

yields were similar for both strains during the exponential

growth phase, before exhaustion of external adenine.

Biomass yields per substrate consumed (Y,x/s,gg

1

)

for W303 ade2 and W303 ADE2 were 0.097 and 0.099,

respectively. Speciﬁc substrate uptake rates (q) were 0.98

and 0.95 g g

1

for W303 ade2 and W303 ADE2,

respectively.

There was still some sugar left in the growth medium

after external adenine was exhausted (Fig. 1b). Surpris-

ingly, adenine depletion did not interrupt an increase in

OD for the W303 ade2 strain, a metric that suggests

uninterrupted growth. This observation conﬂicted with

the prediction that adenine exhaustion would halt prolif-

eration and an OD

600

increase (Fig. 1a). When the ratio

of dry weight to OD of the culture was analysed (Sup-

porting Information, Fig. S1), both W303 strains, ade2

and ADE2, maintained linear OD/dry weight ratios both

before and after external adenine was depleted. As

expected, W303 ade2 formed red pigment as a response

to external adenine depletion.

To resolve the discrepancy between the expected prolif-

eration cessation and the clear increase in OD for W303

ade2 in culture after adenine depletion, we compared

optical densities with the corresponding cell counts mL

1

of both cultures (Fig. 2).

There was a notable difference in cell number mL

1

when comparing the adenine auxotroph and prototroph.

While the cell number mL

1

grew steadily in adenine

prototroph, its increase ceased for the auxotroph after

adenine depletion. To show that this effect was not strain

speciﬁc, but purely adenine dependent, we added extra

adenine (100 mg L

1

) to W303 ade2 strain in YPD

media. As a result of not reaching adenine starvation

through supplementation, the behaviour of W303 ade2

cells followed the pattern of the W303 ADE2 strain.

Therefore, we concluded that an increase in OD

600

after

adenine depletion in adenine auxotrophic cultures is not

caused by cell multiplication but most likely due to

changes in the cells’ optical properties.

Two hypotheses were proposed to explain the OD

600

increase after adenine depletion: either pigment induced

by adenine auxotrophy has absorption that overlaps

600 nm or increased light scattering occurs due to

increase in cell size.

The red pigment does not have speciﬁc absorption

around 600 nm (Smirnov et al., 1967). Therefore, we

concluded that red pigment had negligible, if any, impact

on W303 ade2 OD measurements at 600 nm. Beauvoit

et al. (1993) have reported that yeast cell size affects cul-

ture suspension light scattering and attributed it to a spe-

cial case of general light scattering Mie theory.

To determine whether cell size was contributing to the

increase in OD

600

, mean cell size in W303 ade2 and

W303 ADE2 cultures was measured after 27.5 h of growth

in YPD medium. Size was measured as an elliptical

approximation of the cell’s cross-section in microphoto-

graphs. The adenine auxotrophic cells were signiﬁcantly

(P<0.0001) bigger than those of the prototrophs:

22 14 vs. 16 5lm

, respectively (Fig. 3).

(a)

(b)

Fig. 1. W303 ADE2 and W303 ade2 culture

growth (a) and glucose consumption (b) when

cultivated in YPD media. Error bars represent

standard deviation from three independent

cultivations. Dashed vertical line indicates

adenine depletion in the media.

FEMS Yeast Res 14 (2014) 697–707ª2014 Federation of European Microbiological Societies.

700 A. Kokina et al.

Based on these data, we concluded that the increase in

cell size has led to elevated turbidity of the cell suspen-

sion and, therefore, to the misleading impression of pro-

liferation of adenine auxotroph cells after adenine

depletion.

Our results indicate that growth of the W303 ade2

strain in rich medium differs signiﬁcantly from that of the

adenine prototroph when adenine is depleted. VanDusen

et al. (1997) reported on high variability of adenine con-

tent among yeast extracts of different vendors and differ-

ent batches (ranging from 0.34 up to 1.91 mg g

1

yeast

extract). The YPD media used in experiments described

here contained 13 mg L

1

of adenine.

Due to vast variability of adenine content among dif-

ferent manufacturers’ yeast extracts, and obvious adenine

insufﬁciency observed in our rich media, we decided to

determine optimal adenine concentration for the W303

ade2 strain cultivation. To ensure exact adenine concen-

trations, SD media were used. We were interested in

changes in growth rate, which, besides red pigmentation,

would indicate adenine exhaustion. We inoculated expo-

nentially growing, freshly washed W303 ade2 cells to

ﬂasks with media of different adenine content. We used

an adenine concentration of 100 mg L

1

as a positive

control (physiologically ‘safe’ after Pronk, 2002) against

which growth curves of all other adenine concentrations

(0, 5, 10, 20 and 40 mg L

1

) were compared (Fig. 4).

Results are plotted on log axis to demonstrate growth rate

changes. W303 ade2 culture growth proﬁles when culti-

vated in media with 0, 5, 10 and 20 mg L

1

adenine

diverged from 100 mg L

1

curve at different time points,

and cells started to accumulate red pigment, thus indicat-

ing depletion of external adenine. Student t-test revealed

signiﬁcant differences for OD

600

between 100 mg L

1

and

each of these cultivations. At the time points when

growth rate changed, glucose persisted in ample amounts

(20, 15.4, 14.2, 8.68 g L

1

for ﬂasks with 0, 5, 10, and

20 mg L

1

adenine supplement, respectively) further

indicating adenine depletion. No statistically signiﬁcant

Fig. 2. W303 strain cell count mL

1

depending on OD

600

. Yeast

strains were grown in YPD media with (100 mg L

1

) and without

extra adenine supplement. Cell number mL

1

was determined by

hemocytometer. Vertical dashed line indicates adenine depletion

during W303 ade2 cultivation in YPD media without extra adenine

supplement. Approximations of third-order polynomial functions were

used for visualization of cell number mL

1

data. However, it does not

fully account for relationship between the variables.

Fig. 3. W303 strain mean cell size. Cells were harvested after 27.5 h

of cultivation, and their cross-section areas were measured from

micrographs as described by Jorgensen et al. (2007). Cell number

exceeded 300 for each strain. Means of both distributions were

compared by Wilcoxon signed rank-sum test. Mean cell area of W303

ade2 and W303 ADE2 revealed to be signiﬁcantly different

(P<0.0001).

Fig. 4. Growth curves of W303 ade2 strain in SD media with

different adenine content (0, 5, 10, 20, 40 and 100 mg L

1

); all

other nutrients were added in surplus. Each growth curve is mean of

biological triplicates; error bars represent standard deviations.

Statistical comparison between OD measurements during cultivation

in medias of different adenine concentrations and 100 mg L

1

(positive control) was made. Asterisks below data point denote

signiﬁcant difference from positive control (P<0.05).

FEMS Yeast Res 14 (2014) 697–707 ª2014 Federation of European Microbiological Societies.

Effects of adenine auxotrophy in S. cerevisiae W303-1A 701

(P>0.05) differences between culture OD

600

were

observed when comparing cultures containing 40 mg L

1

with cultures of 100 mg L

1

adenine supplement.

Based on our results, we concluded that at least

40 mg L

1

supplement should satisfy uninterrupted W303

ade2 mutant growth.

Desiccation and starvation responses

Reserve carbohydrate accumulation (glycogen and treha-

lose) is reported to occur in yeast under certain limita-

tions (Lillie & Pringle, 1980; Klosinska et al., 2011).

Studies of different limitations in chemostats have

revealed that trehalose accumulation is inversely related

to the culture’s growth rate, but independent of the nat-

ure of limitation, whether it be natural or artiﬁcial (Boer

et al., 2010). Due to the observed halt of proliferation

after the onset of adenine depletion (Fig. 2), we decided

to measure trehalose accumulation in a series of W303

strains (W303 ade2, W303 ADE2, and W303 prototroph)

during cultivation in a YPD medium. A fully prototro-

phic strain was added to the analysis to assess possible

pleoitropic effects from other auxotrophies present in

W303 ade2 and W303 ADE2 cells. Cells were sampled at

exponential growth phase, shortly before and after

exhaustion of adenine, after exhaustion of glucose, and

during stationary phase. Both (W303 ADE2 and W303

prototroph) strains had consumed all the glucose after

13 h of growth. At the same time, we observed accumula-

tion of red pigment and cessation of W303 ade2 growth.

Glucose measurements revealed that there was still

8gL

1

glucose left in the media. Trehalose content in

exponential phase cells, of all three strains, was close to

zero. After adenine exhaustion, W303 ade2 started to

accumulate trehalose and its content increased with time.

After glucose exhaustion, prototrophic strains began to

accumulate trehalose as well, but at a slower rate. Inter-

estingly, trehalose content continues to increase with time

in a fully prototrophic strain but not in W303 ADE2

(Fig. 5a). Trehalose content of adenine-starved W303

ade2 cells differed signiﬁcantly (P<0.05) from W303

prototroph and W303 ADE2.

Traditionally, the increase in trehalose content has been

linked to elevated tolerance to stress (e.g. desiccation).

We assessed desiccation stress tolerance in cells sampled

during various stages of culture growth, both adenine suf-

ﬁcient and adenine starved (Fig. 5b). All cell cultures

sampled during exponential phase showed low desiccation

stress tolerance, but desiccation tolerance of W303 ade2

cells increased sharply after adenine depletion. Desicca-

tion tolerance over time roughly corresponded to the pat-

tern of trehalose accumulation, and for adenine-starved

W303 ade2 cells, desiccation tolerance differed signiﬁ-

cantly (P<0.05) from W303 prototroph and W303

ADE2.

A distinction has previously been made between starva-

tion for natural and artiﬁcial nutrients (Saldanha et al.,

2004). Cells starved for natural (C, P, N) nutrients sur-

vive for longer periods than cells under artiﬁcial starva-

tions and do so by arresting the cell cycle. In contrast,

auxotrophically starved cells have elevated glucose con-

sumption rate and reduced survival and do not arrest

their cell cycle (Brauer et al., 2008). To place adenine

starvation in the landscape of natural and artiﬁcial starva-

tions, W303 ade2 cells were incubated in synthetic media

lacking either leucine, adenine or carbon source. While

carbon-starved cells clearly showed high and stable sur-

vival rates when compared to both auxotrophies, survival

of leucine and adenine differed signiﬁcantly (P<0.05)

when measured at day 4, 7 and 10. Adenine starvation

(a)

(b)

Fig. 5. Changes in accumulated trehalose (a) and desiccation

tolerance (b) during W303 prototroph, W303 ADE2 and W303 ade2

cultivation in YPD media. Cells were desiccated in +30 °C for 10 h.

Desiccation tolerance was quantiﬁed as in Calahan et al. (2011). In

both (a) and (b), lines denote strain growth curves in logarithmic scale

and bars trehalose (a) or survival (b). Error bars depict standard

deviation from biological triplicates. Viability in ﬁrst two time points (8

and 12 h) is close to zero (see b). Asterisks depict statistically

signiﬁcant difference (P<0.05) between trehalose content of W303

ade2and both W303 prototroph and W303 ADE2 strains (a). Also it

depicts difference between W303 ade2and both W303 prototroph

and W303 ADE2 strains desiccation tolerance as statistically

signiﬁcant (P<0.05) (b).

FEMS Yeast Res 14 (2014) 697–707ª2014 Federation of European Microbiological Societies.

702 A. Kokina et al.

led to higher culture viability than leucine starvation

except for ﬁrst 2 days where survival did not differ signif-

icantly between leucine and adenine starvation (Fig. 6a).

To ﬁnd out whether survival of adenine- and leucine-

starved cultures can be explained by differences in the

cells’ ability to arrest their cell cycle, we estimated the

budding index of the starved cultures. The number of

cells with small buds was counted, and the relative frac-

tion for all cells calculated. Presence of a small bud indi-

cates that the given cell is in the beginning of S phase

and is not arrested (Smets et al., 2010). Typically, a large

percentage of cells are in a budded state while starving

for leucine or uracil (Brauer et al., 2008). Adenine-starved

W303 ade2 cells showed a smaller percentage of budded

cells. Differences between leucine and adenine starvations

were established in the ﬁrst 2 days of starvation and

remained relatively unchanged through the remainder of

the experiment (Fig. 6b). When comparing the viability

and budding index results, we noticed that higher mortal-

ity during starvation corresponds to a higher percentage

of budded cells. That conﬁrmed the possible role of cell

cycle arrest in viability during auxotrophic starvation.

Although exponentially growing, double-washed cells

were used for SD adenine- and leucine-deﬁcient media

inocula, a two- to fourfold increase in OD of the culture

was observed during the ﬁrst days of cultivation

(Fig. S2a). This might be explained either by cell multi-

plication due to accumulated resources within the cells or

by cell size increase. We estimated cell number mL

1

during cultivation (Fig. S2b). Almost no increase in cell

numbers was seen. When using the real cell density (cell

number mL

1

) instead of OD

600

measurements, viability

curves showed no statistically signiﬁcant (P<0.05) dif-

ference in cell survival between adenine- and leucine-

starved cultures, during the ﬁrst 2 days of starvation

(Fig. S2c).

Because differences in cell size and cell number per

optical unit were noted during W303 ade2 and W303

ADE2 cultivation in YPD medium, we expected a similar

effect during long-term adenine or leucine starvation.

Mean cell size of the culture increased for both cultures,

albeit to a different degree (Fig. 7). The increase in the

mean cell size for leucine-starved cultures stopped after

2 days, whereas it continued to increase in adenine-

starved culture. These differences are statistically signiﬁ-

cant for each day of cultivation (P<0.001). When the

distribution of the frequencies of cell size was plotted

(Fig. S3), the distributions were shown to widen with

each starvation day. While some of the starved cells

slowly lost viability and retained their size, living cells

became increasingly larger. It seems plausible that the

increase in cell size is caused by metabolite (e.g. treha-

lose) accumulation.

On the basis of our results, we conclude that adenine

auxotrophy is distinctively different from leucine auxotro-

phy. Although cell viability drops over time relative to

carbon starvation, adenine starvation exhibits characteris-

tics similar to those of other natural starvations: higher

survival rates and ability to arrest cell cycle more efﬁ-

ciently when starved in comparison with artiﬁcial starva-

tions such as leucine.

Discussion

Auxotrophy is a common property of haploid laboratory

strains. Traditionally, amino acid and purine/pyrimidine

auxotrophic markers are used in yeast strain genetic

(a)

(b)

Fig. 6. Survival rate (a) and budding index (b) during prolonged

W303 ade2 auxotrophic (leucine or adenine) or carbon source

starvation. Cells were grown in full SD media up to exponential

phase, washed with distilled water and re-suspended to OD600 =1

in SD media lacking sugar, leucine or adenine; all other broth

components were added in surplus. Error bars represent standard

deviation from three independent cultivations. Viability in (a) depicted

as percentage of CFU from OD600 =1 in the beginning of

experiment. Budding index is calculated as ratio of cell number with

small bud against total cell number. Asterisks depict statistically

signiﬁcant difference (P<0.05) between adenine and leucine and

carbon starvations.

FEMS Yeast Res 14 (2014) 697–707 ª2014 Federation of European Microbiological Societies.

Effects of adenine auxotrophy in S. cerevisiae W303-1A 703

engineering. Leucine, tryptophan, adenine, uracil, methio-

nine, and histidine auxotrophies are typical markers and

targets for complementation with plasmids or integration

constructs (Pronk, 2002; Da Silva & Srikrishnan, 2011).

However, questions have been raised regarding how this

common strain property affects the general physiology of

cells (M€

ulleder et al., 2012; Liu et al., 2013). Problems

with auxotrophic strains might arise for two reasons:

depletion of the auxotrophic agent in the growth medium

may occur with great regularity if the concentration of

the agent is insufﬁcient and there may exist pleiotropic

interactions between multiple auxotrophies in the same

strain. Several protocols deﬁne different amounts of

auxotrophic agents needed in synthetic media, and yeast

cell physiologists have raised concerns that the amounts

usually used might not be sufﬁcient for uninterrupted cell

growth (Pronk, 2002). However, rich media have been

considered safe in respect to auxotrophies, and additional

amino acids or nucleotides are seldom added.

Our results show that additional adenine (at least

40 mg L

1

or more), even to the rich media, should be

added to avoid adenine starvation. Concerns are raised

regarding growth attenuation effects of auxotrophic sup-

plements, if added in excess (M€ulleder et al., 2012). Our

results on synthetic media (Fig. 4) show no statistically

signiﬁcant differences in growth if 40 or 100 mg L

1

ade-

nine is added. Furthermore, no difference between growth

parameters of W303 prototroph and W303 ade2 on YPD

with extra 100 mg L

1

adenine supplement is seen

(µ=0.45 h

1

for both strains). Similarly, VanDusen

et al. (1997) and Zhang et al. (2003) reported on

sufﬁcient adenine concentration to be 50–80 mg L

1

Auxotrophic markers can have pleiotropic effects on

S. cerevisiae physiology. There are many examples of the

effects of tryptophan, methionine and histidine autotro-

phy on yeast physiology. For example, any gene deletion

in the tryptophan biosynthesis pathway (trp1-5 genes)

leads to decreased growth in the presence of rapamycin,

caffeine and SDS. Notably, wild-type characteristics are

not regained after the respective gene complementation

(Gonz

alez et al., 2008). Also, depending on the length of

the HIS3 gene deletion (200 bp or 1 kbp) used to gener-

ate the histidine auxotrophy, various levels of respiration

deﬁciency at 37 °C can be observed (Young & Court,

2008). Criticism has also been raised regarding the use of

methionine auxotrophs because the need for methionine

supplement masks the effects of other gene deletions as

seen in zwf1 strains (Thomas et al., 1991; Pronk, 2002).

In contrast to the above auxotrophies exhibiting pleio-

tropic impact on physiology, our results on adenine aux-

otrophy show purely adenine-dependent effects. When

ample amount of adenine is available in the media, ade-

nine auxotrophs and prototrophs are physiologically

indistinguishable: autotroph and prototroph cell size, OD

and dry weight ratio, biomass yield, glucose consumption,

and desiccation tolerance are similar. On the other hand,

when adenine concentration in medium is limiting, cell

morphology and physiology change signiﬁcantly. Addi-

tionally, adenine-dependent effect is not strain or speciﬁ-

cally ADE2 gene dependent. We repeated desiccation

tolerance experiments with strains of CEN.PK series: full

CEN.PK prototroph, CEN.PK ADE8 (CEN.PK2 MATa

leu2-3/112 ura3-52 trp1-289 his3-1,MAL2-8c SUC2) and

CEN.PK ade8 deletion (made on CEN.PK ADE8 back-

ground). Growth rate, trehalose accumulation and desic-

cation tolerance were measured. Results follow the same

pattern as in W303 –the adenine auxotroph accumulates

trehalose and shows signiﬁcantly (P<0.05) elevated des-

iccation tolerance when adenine becomes depleted (results

are shown in Fig. S4). From this, we conclude that the

observed phenomenon is not strain or ade2 gene speciﬁc;

instead, it relates to adenine auxotrophy generally.

When comparing fully prototrophic strains with their

respective his,leu,trp,ura auxotrophs, a decrease in tre-

halose accumulation and desiccation tolerance can be

seen; however, differences are not statistically signiﬁcant

(P>0.05). Some or even all of these common auxotro-

phies could decrease desiccation resistance in prototrophs.

To fully understand the phenotypical interplay between

all auxotrophic markers, each auxotrophy alone and its

combination with the others should be tested in other-

wise prototrophic strains. Some interplay between ade-

nine, histidine and tryptophan auxotrophies could take

Fig. 7. W303 ade2 culture mean cell size dynamics during prolonged

adenine or leucine starvation. Cell cross-section areas were measured

from micrographs as described by Jorgensen et al. (2007). Cell

number exceeded 300 for each data point. Asterisks depict

statistically signiﬁcant difference (P<0.0001) between cell sizes of

adenine and leucine starvations, as determined by Wilcoxon rank-sum

test.

FEMS Yeast Res 14 (2014) 697–707ª2014 Federation of European Microbiological Societies.

704 A. Kokina et al.

place as they share some common elements in their bio-

synthesis pathway. Moreover, multiple stress-related phe-

notypic traits of tryptophan auxotrophs have been

described before (Gonz

alez et al., 2008). We did segregant

analyses of crosses between ade

trp



and ade



trp

strains

and found that increased desiccation stress tolerance and

trehalose accumulation after adenine depletion occurs in

ade



segregants independently of trp gene functionality

(data not shown). However, adenine auxotrophy is usu-

ally accompanied by other four auxotrophic markers in

W303 series strains or through ade8 disruption by an

integration vector. Statistically signiﬁcant differences

among adenine auxotrophs and prototrophs that we

observed in two strain backgrounds, W303 and CEN.PK,

indicate that the observed effects are adenine auxotrophy

speciﬁc and thus should be carefully considered when

doing physiological studies with those strains.

We explored this phenomenon further to determine

the extent of biases in yeast physiology that can be pro-

duced due to insufﬁcient adenine in the medium. Mea-

surements of OD, a common indicator of cell growth

used in microbiology (Madrid & Felice, 2005), can be

misleading in adenine auxotrophs due to the swelling of

cells –OD

600

increases even after cell proliferation has

stopped. One of the reasons why ade markers are widely

used in genetic research is because the accumulation of

red pigment is a convenient visual marker used when dis-

tinguishing segregants. The same pigment is autoﬂuores-

cent and hinders cell visualization (Weisman et al., 1987),

so usually care is taken to use adenine-enriched media

when growing cells for visualization studies. On the other

hand, stress physiology research is quite often performed

on adenine auxotrophs after exponential growth phase in

rich media, without any additional supplementation

(Carrasco et al., 2001; Petrezselyova et al., 2010). In our

opinion, increased stress tolerance of stationary-phase

adenine auxotrophs in rich media is due to adenine

depletion and not because of other strain characteristics.

Desiccation is a multifactorial stress that challenges

cells with hyperosmolarity, hyperoxidation, hyperionicity

and protein misfolding/aggregation during dehydration

and rehydration (Chakrabortee et al., 2007; Franc

ßaet al.,

2007). Trehalose is a widely discussed storage carbohy-

drate, which accompanies various stress conditions (Crowe

et al., 1998). Still, whether trehalose is an important desic-

cation stress protector or just a metabolite that accumu-

lates during slow growth remains unclear (Paalman et al.,

2003; Ratnakumar & Tunnacliffe, 2006). Although treha-

lose accumulation in W303 ade2 coincides with the

increase in desiccation tolerance, adenine prototroph

strain does not show the same relationship between treha-

lose accumulation and desiccation tolerance. This indi-

cates that trehalose accumulation could serve as a signal

for elevated desiccation tolerance, but it might not be a

prerequisite for it. Previously, rate of trehalose accumula-

tion has been attributed to the growth rate (Paalman

et al., 2003). Adenine auxotroph cells indeed accumulate

far greater amounts of trehalose after they cease to prolif-

erate, and dynamic trehalose levels that change over time

in arrested cells indicate that there may be additional reg-

ulatory mechanisms.

Trehalose levels in cells are determined as an outcome

of dynamic equilibrium of trehalose synthesis and hydro-

lysis (Hohman & Mager, 2003). The activity of trehalase

and trehalose synthase is regulated by the cAMP-PKA

pathway, which is upregulated in the presence of glucose

(Winderickx et al., 1996). A great increase in trehalose lev-

els in adenine-starved cells could indicate downregulation

of cAMP-PKA pathway and consistent downregulation of

trehalase activity and upregulation of trehalose synthase

complex activity. Research shows that yeast cells, when

starved for carbon, nitrogen or phosphorous, upregulate

both sides of the trehalose metabolism –synthesis and

hydrolysis. However, trehalose accumulates only in nitro-

gen-starved cells (Klosinska et al., 2011). We have mea-

sured trehalase activity in ade8 deletion strain in CEN.PK

background, and it shows the same tendency –trehalose

accumulation is accompanied by elevated trehalase activity

during adenine starvation (data not shown). Additional

research is needed to clarify dynamics of trehalose accu-

mulation during adenine starvation to determine whether

they are similar to those observed in nitrogen starvation.

The recent work of Welch et al. (2013) elucidates the

role of TOR and RAS pathway in acquiring desiccation

stress resistance. These two pathways regulate cell growth

rate by monitoring external carbon and nitrogen supplies

(Smets et al., 2010). Here, we show that adenine deple-

tion does stop cell division and increases cell viability

after desiccation, indicating a possible role for these two

signalling pathways in physiological changes observed

during adenine starvation. The increase in desiccation tol-

erance in response to carbon, nitrogen and phosphorus

starvation has already been reported (Welch et al., 2013).

Although adenine and uracil are nucleotides, different

physiological responses to starvation can be observed for

them. It is possible that either yeast cells sense external

adenine levels or, as adenine becomes limited, the cells

sense limited or imbalanced adenylate levels intracellu-

larly. Yet there are no reported transcriptional response

mechanisms to disturbed nucleotide balance (Ljungdahl

& Daignan-Fornier, 2012). Little is known about adenyl-

ate levels in cells undergoing adenine starvation, so fur-

ther studies are required to elucidate possible cell

response mechanisms. Existence of speciﬁc transcriptional

response leading to a quiescent state for yeast is under

debate (Klosinska et al., 2011).

FEMS Yeast Res 14 (2014) 697–707 ª2014 Federation of European Microbiological Societies.

Effects of adenine auxotrophy in S. cerevisiae W303-1A 705

To summarize our ﬁndings, the physiological state of

W303-1A changes dramatically after adenine is exhausted

in YPD media. Cell proliferation ceases while cells

increase in size and accumulate trehalose. Prominent

increase in desiccation stress tolerance follows. Trehalose

accumulation and elevated desiccation tolerance imply

that adenine auxotrophs change their carbon ﬂow and

internal signalling after adenine depletion. In prolonged

starvation experiments, adenine-deﬁcient cells show

increased ability to arrest cell cycle and are viable for

longer period of time compared with cells starved for leu-

cine. These facts distinguish adenine starvation from

other auxotrophies described thus far. To avoid unwanted

phenotypic changes due to adenine depletion in rich

media, we suggest adding extra adenine for ade



if cell

physiology will be studied after exponential growth phase,

to avoid adenine exhaustion before glucose depletion.

Acknowledgements

J.L. and A.K. were supported by ESF project Nr. 2009/

0138/1DP/1.1.2.1.2/09/IPIA/VIAA/004. J.K. was supported

by ERAF, contract no. L-KC-11-0005, project Nr. KC/

2.1.2.1.1/10/01/006. We thank Prof. U. Kalnenieks for the

valuable discussions and Dr. M. I. Pena for critical read-

ing of the manuscript.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. W303 adenine auxotroph and prototroph dry

weight dependence on optical density measurements

when cultivated in YPD medium.

Fig. S2. Optical density (a), cell count mL

1

(b) and rela-

tive survival rate (c) during W303 ade2 adenine (dia-

monds) and leucine (squares) starvation.

Fig. S3. Cell size distribution during adenine (upper

panel) and leucine starvation (lower panel).

Fig. S4. Changes in accumulated trehalose (a) and desic-

cation tolerance (b) during CEN.PK prototroph, CEN.PK

ADE2 and CEN.PK ade2 cultivation in YPD media.

FEMS Yeast Res 14 (2014) 697–707 ª2014 Federation of European Microbiological Societies.

Effects of adenine auxotrophy in S. cerevisiae W303-1A 707

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