Analytica Chimica Acta 495 (2003) 217–224
Use of ATP measurements by bioluminescence to quantify
yeast’s sensitivity against a killer toxin
Sandrine Alfenore a,∗ , Marie-Line Délia b ,
Pierre Strehaiano b
a
b
Equipe “Génie Microbiologique”, Laboratoire Biotechnologies-Bioprocédés, INSA, UMR CNRS 5504, UMR INRA 792,
135 Avenue de Rangueil, 31077 Toulouse Cedex, France
Equipe “Fermentations + Bioréacteurs”, Laboratoire de Génie Chimique, INP-ENSIACET, UMR CNRS 5503, BP 1301,
5 rue Paulin Talabot, 31106 Toulouse Cedex 1, France
Received 8 April 2003; received in revised form 29 July 2003; accepted 11 August 2003
Abstract
An original method, based on ATP measurements by bioluminescence, is described for quantifying the killer activity
induced by a killer strain in a liquid medium. The aim was to propose a more rapid and selective technique directly linked
to cell response to the killer damages. The sensitivity degree of strains plays an important part in these “killer-sensitive”
interactions. Until now, there are few quantitative method was accurate and selective enough to rank the strains depending on
this criterion. In the first step, the thought process leading to the new quantitative method for killer activity is presented. The
originality of the method is based on the measurement of the initial velocity of ATP release (Vi ), induced by the action of the
killer protein on sensitive cells. This criterion (Vi ) was correlated to the measurement of the killer activity in liquid medium:
when 0 < Vi < 0.17 ␮mol l−1 h−1 , the killer activity (%) is directly proportional to Vi ; when Vi > 0.17 ␮mol l−1 h−1 , the
killer activity remained constant (85 ± 3%). Then, this method was used to classify some commercial yeasts (four sensitive
or neutral strains and four killer strains) depending on either their intrinsic sensitivity to a killer toxin or their killer power
against a sensitive strain chosen as a reference.
© 2003 Elsevier B.V. All rights reserved.
Keywords: ATP measurement; Bioluminescence; Killer effect quantification; Killer toxin; Saccharomyces cerevisiae
1. Introduction
The killer effect has an important influence on the
balance between different populations of yeasts, especially in winemaking. Its mechanism is well known
and widely reported in literature but there are still few
kinetic studies. Nevertheless, a dynamic characteri∗ Corresponding author. Tel.: +33-5-61-55-94-21;
fax: +33-5-61-55-94-00.
E-mail addresses: [email protected],
[email protected] (S. Alfenore).
zation would provide essential data to understand the
mechanism better and improve the control of industrial processes [1,2]. Quantifying the killer activity
in a liquid medium requires reliable experimental
techniques. All the existing quantitative methods to
evaluate the killer activity in a liquid medium are
based on the measurements of both viability and
growth perturbations due to the toxin action. The action mode of the K1 killer toxin on sensitive cells is
well known: first, the protein links to the receptors of
the sensitive cell wall, and secondly it creates pores
in the plasma membrane [3–5]. Then, the membrane
0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2003.08.023
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S. Alfenore et al. / Analytica Chimica Acta 495 (2003) 217–224
damages induce different phenomena leading finally
to the death of the target cells. The leak of intracellular
components (including ATP) to the extracellular
medium is one of the perturbations due to the killer
toxin. Young [6] indicated the close similarities between different killer toxins within the genus Saccharomyces, especially for K1 and K2 toxins. More
recently, Franken et al. (1998) showed that the cells
damaged by the K2 toxin present the same aspect
as those affected by the K1 toxin. The sensitive
cells shrink, which suggests the efflux of cytosolic
components through pores and the alteration of the
plasma membrane permeability [7]. Along the same
lines, discussing the difference in the primary nucleic
acid sequence encoding for the K1 and K2 toxins,
Bussey et al. [8] mentioned the similarity of these two
toxins.
Bioluminescence is a well known technique to
measure the ATP concentrations. This kind of measurement is rapid and presents a low threshold of
detection: around 10−11 mol ATP l−1 [9] and, with
our apparatus (LUCY, Prodemat SA), the threshold
of detection is 10−13 mol ATP l−1 (data given by the
constructor). Usually, this technique is used to detect
the appearance of a possible microbial contamination.
It has been frequently carried out to control food
safety or area asepsis in various industrial fields such
as dairy production [10], beverage making [11,12] or
poultry and meat industries [13].
In this paper, bioluminescence was used to develop
an original method to quantify the killer activity. The
aim was to propose a more rapid and selective technique directly linked to cell response to the damages
induces by the killer phenomenon. Then, ATP measurements were used to classify enological strains depending on either their intrinsic sensitivity degree to
a killer toxin or their killer power against a sensitive
reference yeast.
Table 1
List of the tested strains (S. cerevisiae) (commercial name, phenotype)
Microorganism
(genus, species, commercial name)
Phenotype
(from agar method)
S. cerevisiae A
S. cerevisiae B
S. cerevisiae C
S. cerevisiae D
S. cerevisiae E
S. cerevisiae K1
S. cerevisiae S6
S. cerevisiae 522D
S cerevisiae BC
S. cerevisiae CEG
S. cerevisiae 3079RI
Killer
Killer
Killer
Killer
Killer
Killer
Sensitive
Sensitive
Sensitive
Sensitive
Neutral
supplier. The killer strains, referenced with code
letters, were all K2 toxin producers.
2.2. Phenotype characterization
2. Materials and methods
Killer, neutral and sensitive phenotypes, given initially by the yeast supplier, were confirmed by an agar
diffusion method on Petri dishes adapted from the one
proposed by Woods and Bevan [14] (results shown in
Table 1).
An YED agar medium (glucose: 20 g l−1 ; yeast
extract: 10 g l−1 ; agar: 20 g l−1 ; initial pH 4.5) was
seeded with a target strain (25 × 106 viable cells l−1 ).
The killer phenotype was always determined against
S.c. S6 sensitive cells. Colonies of the tested strains
were replicated on the agar medium surface. After a
48 h incubation at 30 ◦ C, the colonies surrounded by
a clear area were identified as killer strains.
The neutral or sensitive character was observed using the tested strains as target cells (mixed with YED
agar). The S.c. K1 killer strain was replicated on the
agar surface. After incubation (48 h at 30 ◦ C), if a clear
area surrounded the S.c. K1 colonies, the target cells
were sensitive. Neutral strains showed no reaction and
grew normally.
2.1. Strains
2.3. Media and chemicals
Different strains of Saccharomyces genus were
used. All of them were enological yeasts provided
by Lallemand (Montreal, Canada) (except the killer
strain E). The phenotypes were specified by the yeast
A minimal liquid medium (glucose: 50 g l−1 ;
(NH4 )2 SO4 : 5 g l−1 ; K2 HPO4 : 2 g l−1 ; MgSO4 ·7H2 O:
0.4 g l−1 ; yeast extract: 1 g l−1 ; initial pH 4) was used
for all the cultures.
S. Alfenore et al. / Analytica Chimica Acta 495 (2003) 217–224
219
3. Experimental
3.1. Killer toxin production
To prepare the toxin solutions, each killer strain
(A–E and K1) was cultivated in a bioreactor (volume: 10 l). The same culture conditions were applied
(minimal medium at 25 ◦ C and stirred at 250 rpm,
under non-strict anaerobiosis, 15 h culture) in order
to compare their toxic activity. After a 15 h culture,
the prefermented medium containing the extracellular type K2 toxin, was filtered with Sartorius modules made of cellulose acetate (0.8–0.65 ␮m pore size,
0.45–0.2 ␮m pore size), homogenized in a glass tank
and filtered with a 0.2 ␮m pore diameter filter before
storage in sterile bottles. Half of the filtered prefermented medium was denatured by a thermal treatment to inactivate the killer protein (10 min, 121 ◦ C,
1 bar). This denatured toxin was used as a reference
whereas the active one was used to quantify the killer
activity.
3.2. Quantifying the killer activity
In order to prove the relevance of the bioluminescent method to quantify the killer activity, this
new technique was correlated to the one proposed by
Ramon-Portugal et al. [15]. This method was carried
out in a liquid medium and the action of the killer
toxin was directly measured on a growing sensitive
population. The experimental procedure is described
in Fig. 1. The sensitive strain was cultivated simultaneously either with the active killer toxin (test
culture) or with the same killer toxin solution, denatured by a thermal treatment (reference culture).
After 10 h culture, the total biomass dry weight (DW
in g l−1 ) was measured in each culture, using a gravimetric method (filtration of a known volume of cell
suspension on an acetate membrane (0.45 ␮m pore
size), first vacuum dried and weighted). The cell viability (Viab) was also determined in each culture
by microscopic counting after methylene blue staining; the viability was defined as the ratio between
the viable cells (non-colored cells) and the total
population.
In this case, the killer activity is defined as the reduction of the sensitive viable population induced by
the action of the killer toxin (Eq. (1)):
Fig. 1. Experimental procedure for quantifying killer activity
in a liquid medium (adapted from the method proposed by
Ramon-Portugal et al. [15]).
Killer activity (%)
(DWRef ×ViabRef ) − (DWTest ×ViabTest )
= 100 ×
DWRef ×ViabRef
(1)
3.3. ATP measurements
To determine ATP concentrations, an enzymatic
luciferin–luciferase system, extracted from lucile
abdomens (Photinus pyralis) [10] was used. A luminescent reaction occurred between ATP and the
luciferin–luciferase complex. The energy of the emitted light was quantified by a luminometer (LUCY,
Prodemat SA) in RLU (relative light unit). The luminescent substrate used was luciferin (C␦ life), stored
at −18 ◦ C.
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A calibration curve was carried out in a range of
10−8 to 10−4 mol l−1 extracellular ATP. The ATP standards were prepared using adenosine tri-phosphate
(Sigma) diluted in a minimum medium (pH 4.0).
Studies on these standards have shown that there is no
degradation of the ATP at this pH. So, extracellular
ATP concentrations were correlated to RLU measurements corrected by the RLU value of the background noise. The correlation plots: −log10 [ATP] vs.
log10 RLUc. Standard deviation was determined from
10 experiments with luciferin from different batches
and various ATP standards. The variation of the calibration curve coefficients was ±3% for the slope
value and ±2.1% for the ordinate at zero.
NB :
RLUc = RLU corrected = RLU measured
− RLU(background noise)
3.4. Sample preparation for ATP measurements
Culture samples (50 ␮l) were mixed with 2.5 ml of
buffer solution (diluted Biofax A solution, C␦ life) and
homogenized. A 10 ␮l volume of luciferin was added
to 200 ␮l of the previous mixture. After homogenization, RLU was measured; the luminometer response
has a parabolic form, three integrations of 10 s per
sample were done and only the maximum value was
taken into account.
3.5. Flow cytometry
Two dyes were used: fluorescein diacetate (FDA)
and propidium iodide (PI). Their specificity made it
possible to characterize the physiological state of the
cells. FDA is specific of metabolic activity. It enters
the cell and is reduced by the cytosol esterases leading
to a green fluorescence. PI is specific for membrane
integrity. When the membrane is damaged, the dye
enters the cell and a red fluorescence is induced. So,
according to the observed fluorescences, three different physiological states should be distinguished:
viable cells which have an esterase activity and an intact membrane (green fluorescence), dead cells which
have no more metabolic activity and a damaged membrane (red fluorescence) and “injured” or “damaged”
cells which continue to have an esterase activity although the membrane is damaged (both green and red
fluorescences).
3.6. Analytical procedure
Culture sample (1 ml) was mixed with 0.5 ml of
FDA solution (Sigma reagent diluted to 5 mg ml−1
with filtered acetone and diluted again to 25 ␮g ml−1
in PBS solution, phosphate buffered saline; Sigma).
The mixture was incubated for 20 min at room temperature. Then, 0.5 ml of PI solution (concentration:
0.5 mg ml−1 , commercial solution, Boehringer) was
added. After 10 min at 0 ◦ C, the sample was analyzed
by the flow cytometer which was a Beckman-coulter
analyzer (Elite model). The flow cytometer used an
argon laser (λ = 488 nm), enabling molecule excitation. The fluorescent-emitted beams were collected
at the appropriate wavelengths (respectively, 525 and
630 nm for FDA and PI emissions). Given that for each
experiment more than 5000 cells were counted, the
error on the count by flow cytometry (FC) was below
1% [16].
4. Results and discussion
4.1. ATP measurements: the new quantitative
method for killer activity
In the first part, a measurement criterion was chosen
and used to correlate the measured ATP concentrations
and the killer activity obtained by a reference method.
Then, in the second part, the new method was used
to evaluate the degree of toxicity and sensitivity of
different enological strains.
4.1.1. Correlation between the ATP release and the
killer effect
First, the suitability of ATP release to represent the
damages due to the toxin was verified. So the concentration of released ATP was compared to the ratio of
affected cells determined by FC.
Kurzweilova and Sigler [17] showed that the remaining viability depends on the volume of added
toxic solution. Likewise, Ramon-Portugal et al. [15]
found a linear correlation between the decrease of viable yeasts and the volume of the medium prefermented by Saccharomyces cerevisiae K1. Sensitive
cells of S. cerevisiae S6 were incubated in presence of
a killer toxin (medium prefermented by the killer strain
S. cerevisiae A). The initial microbial concentration
S. Alfenore et al. / Analytica Chimica Acta 495 (2003) 217–224
221
Fig. 2. Evolution of the percentage of affected cells (determined by FC) and the released ATP (in ␮mol l−1 ) during the killer toxin action
(couple S.c. S6/killer strain A).
was between 20 × 106 and 25 × 106 cells ml−1 (viability: 95–100%), the prefermented medium represented
1/3 of the total culture volume. The assay was run under the conditions presented in Fig. 1.
The influence of the killer toxin on the sensitive population was observed in the first hours of
incubation. Indeed, according to literature, many authors agreed on the fact that the changes (membrane
breakdown, ATP release, etc.) induced by the killer
protein are already effective in 2 h [4,5,18,19]. Our
observations confirmed this statement (Fig. 2). At the
beginning of the incubation, dead cells represented
less than 4% of the total population and some “damaged cells” were also present. After only a 30 min
contact with the toxin, the “injured” cell amount rose
significantly whereas the dead cell concentration remained unchanged. This result showed how quickly
the membrane is damaged. Mortality was induced
in a second step. After 1 h, 58% of the population
is affected, 20% is dead and 38% is damaged. The
percentage of affected cells reached a 75% maximum
value (2.5 h) and remained around 70% up to 5 h of
incubation. Therefore, a 2 h incubation is enough to
obtain a significant response of the sensitive strain.
Fig. 2 clearly demonstrates that released ATP due
to the killer toxin action gives a good representation
of the killer effect. Indeed, the evolution of released
ATP was similar to the one of the affected population
(dead and damaged cells). So, in order to quantify the
killer effect, we were interested in the ATP leak from
the sensitive cells.
4.1.2. Released ATP as a new quantitative criterion
for killer activity
The evolution of ATP concentrations in the medium
were followed for a few hours (Fig. 3) for three target strains in cultivation with the active or denatured
toxin: S.c. S6, 522D and 3079 RI, respectively, sensitive, sensitive and neutral strains. Fig. 3 shows that
ATP concentration was not altered in the medium during the first 2 h of incubation. Furthermore, during this
short period, it was also possible to observe the differences of the killer toxin action on each strain. Using the method based on viability determination, it is
difficult to quantify the toxic effect after only 2 h because the difference in viable cells between the test
and reference cultures was not always significant. So,
a longer time of incubation was required for this test.
On the other hand, Fig. 3 clearly demonstrates that the
ATP releases were very quick, which shortened the
measurement. Moreover, the maximum amount of released ATP and the incubation time to reach this maximum were different depending on the sensitivity of
the target strain (Table 2).
Based on these observations, the chosen criterion to
quantify the killer phenomenon was the initial velocity
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Fig. 3. Evolution of the released ATP per million of cells during the killer test for three different strains (S.c. S6, sensitive; S.c. 522D,
sensitive; S.c. 3079 RI, neutral).
of ATP release estimated in the first 2 h (Vi ). Furthermore, the use of a velocity term is a classical way to
express a protein activity in enzymology.
So, the ATP measurements were carried out at the
beginning of the incubation and after 2 h. The amount
of released ATP due to the action of the toxin action
was observed by the difference between the extracellular ATP concentrations in the test and reference culture. Taking into account the extracellular ATP in the
reference ensures that the observed phenomena are
solely induced by the action of the killer protein.
Then, to validate the method, the initial velocity of
ATP release (Vi ) was compared to the toxicity test proposed by Ramon-Portugal et al. [15]. Several assays
were carried out with the same killer toxic solution
against various sensitive strains.
Vi was estimated after 2 h, and the killer activity
was determined after 10 h. Two areas of correlation
were found between the two methods: when Vi value
was between 0 and 0.17 ␮mol l−1 h−1 , Vi was directly
proportional to the killer activity and the following
equation was obtained (Eq. (2)):
Killer activity (%) = 474.5Vi (␮mol l−1 h−1 ),
r2 = 0.993
(2)
when Vi was higher than 0.17 ␮mol l−1 h−1 , the killer
activity remained constant (85 ± 3%).
This value indicates a saturation threshold of the
technique based on the mortality estimation. In the
range of the sensitive cell concentrations used in the
killer test, it was impossible to observe a total mortality
of the target cells. According to the growth of the
population during the incubation, a part of the sensitive
cells was still alive and so, the mortality induced by the
killer protein did not reach 100%. The bioluminescent
method seems more accurate when the detected killer
activity is high.
The measurement of ATP leak is a good way to estimate the killer phenomenon, it is precise with a wide
Table 2
Sensitivity and released ATP concentration for two sensitive strains (S.c. S6 and S.c. 522D) against the killer strain A
Strains
Released ATP maximum
concentration (10−12 mol/106 cells)
Incubation time for the
maximum ATP release (h)
Sensitivity (%) (reference
method: [15])
S.c. S6
S.c. 522D
29.0
18.5
3
5
87.1 ± 7.8
10.9 ± 1.0
S. Alfenore et al. / Analytica Chimica Acta 495 (2003) 217–224
range of responses. When all the parameters (temperature, pH, initial sensitive population) were the same,
except the strain used, it was possible to evaluate the
intrinsic sensitivity of the target population against a
given killer toxin. The higher is the Vi value obtained,
the higher is the sensitivity of the target strain or the
killer power of the killer yeast.
4.2. Application field: hierarchization of yeasts
according to either their sensitivity or killer degree
The bioluminescent test was used to compare and
classify the killer power of some killer strains (A–E)
against sensitive (S6, BC and CEG) and neutral
(3079RI) ones. The phenotypes were mentioned in
Table 1.
All the prefermented media were prepared in the
same way: 15 h of cultivation before filtration. During
the tests, the sensitive cell concentration was 25 ×
106 cells ml−1 . The background noise never exceeded
10−10 mol ATP l−1 .
The higher is the Vi value registered, the higher
the sensitivity of the target strain. Fig. 4 shows the
obtained responses to the different tests.
S. cerevisiae S6 clearly exhibited the highest sensitivity. This strain was always affected. So was S.c. BC,
but with weaker responses. CEG, which was expected
to be as sensitive, did not confirm this phenotype. Indeed, it was only affected by C and D. According to
223
the S.c. S6 response, C and D seemed to have the
highest “killer” power. Moreover, the ATP leaks were
very weak; after 2 h, the measured extracellular ATP
concentrations were 4.54×10−9 mol l−1 h−1 (CEG/D)
and 6.88 × 10−10 mol l−1 h−1 (CEG/C). As the sensitive character was not significant, this strain should be
considered as neutral. For 3079 RI, registered as neutral, it did not react with A–C and E; D which is the
“strongest” killer induced a very weak ATP leak.
Concerning the overall sensitivity, the classification is
(S.c.)S6 > BC > CEG ≈ 3079RI
Next, to rank the strength of the killer power, S6 (the
most sensitive strain) was chosen as a reference. The
killer tests were run at the same time using the same
starter of target strain. Five killer strains were tested.
Some significant differences were observed between
the responses, Vi varied from 0.10 ␮mol l−1 h−1 to less
than 0.02 ␮mol l−1 h−1 . The kinetic characteristics of
the killer strains were rather equal (growth rate, maximum biomass concentration, etc.). They excreted the
same kind of killer protein: the K2 protein (data provided by Lallemand). As all the assays were run under the same conditions, these differences should be
induced by different toxin concentrations. The killer
cell concentrations before filtration were equivalent
(between 100 × 106 and 120 × 106 viable cells ml−1 )
and it was previously shown that the toxin production is closely linked to growth [15], so, it seems that
each killer strain has a specific excretion rate of protein leading to a different toxin amount in the prefermented medium.
So, from the “strongest” killer to the weakest one,
the strains were ranked as follows:
D>C>B>A>E
All these results point out that the sensitivity or the
killer power of a strain strongly depends on the other
strain(s) brought together.
Acknowledgements
Fig. 4. Vi measurements (␮mol l−1 h−1 ) for ordering target strains
depending on their sensitivity towards a killer toxin secreted by
five different killer strains.
Special thanks to M. CASSAR and the INSERM
Unit 395 of Toulouse Purpan Hospital (Cytometry service of P. Sabatier University, Toulouse) where all the
flow cytometry assays were carried out.
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