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Use of ATP measurements by bioluminescence to quantify yeast’s sensitivity against a killer toxin
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
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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.
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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
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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
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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
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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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References [1] C. Rozières, F. Raginel, C. Sanchez, P. Strehaiano, R.F.Œ. 119 (1989) 37. [2] S. Michalcakova, P. Sulo, E. Slavikova, Acta Biotechnol. 13 (1993) 341. [3] H. Bussey, D. Sherman, Biochim. Biophys. Acta 298 (1973) 868. [4] P. De la Peña, F. Barros, S. Gascón, P.S. Lazó, S. Ramos, Biochem. Biophys. Res. Commun. 96 (1980) 544. [5] P. De la Peña, F. Barros, S. Gascón, P.S. Lazó, S. Ramos, J. Biol. Chem. 256 (1981) 10420. [6] T.W. Young, in: A.H. Rose, J.S. Harrison (Eds.), The Yeasts, vol. 2, Physiology and Biochemistry of the Yeasts, Academic Press, London, 1993, p. 131. [7] D.B. Franken, M. Ariatti, I.S. Pretorius, A.S. Gupthar, Antonie van Leeuwenhoek 73 (1998) 263. [8] H. Bussey, C. Boone, H. Zhu, T. Vernet, M. Whiteway, D.Y. Thomas, Experientia 46 (1990) 193.
[9] P.E. Stanley, J. Biolumin. Chemilumin. 4 (1989) 375. [10] M. Pernelle, La Technique Laitière. 980 (1983) 34. [11] J. Beguin, C. Cuinier, F. Guyot, A. Bruetschy, J.Y. Morard, R.F.Œ. 198 (1998) 26. [12] L. Lurton, B. Cao-Thanh, in: XXIIIème Congrès Mondial de la Vigne et du Vin, II-Œnologie, Lisbonne, Portugal, 1998, pp. 109–116. [13] M.W. Griffiths, Food Technol. 50 (1996) 62. [14] D.R. Woods, E.A. Bevan, J. Gen. Microbiol. 51 (1968) 115. [15] F. Ramon-Portugal, M.L. Délia, G. Schneider, P. Strehaiano, Biotechnol. Tech. 8 (1994) 797. [16] G. Nebe Von Caron, R.A. Badley, J. Microsc. 179 (1995) 55. [17] H. Kurzweilova, K. Sigler, Experientia 51 (1995) 26. [18] H. Bussey, Nature New Biol. 235 (1972) 73. [19] H. Bussey, N. Skipper, Antimicrob. Agents Chemother. 2 (1976) 352.
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
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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.
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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
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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
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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
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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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