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Effect of cinnamic acid on the growth and on plasma membrane H+–ATPase activity of Saccharomyces cerevisiae
International Journal of Food Microbiology 50 (1999) 173–179 www.elsevier.nl / locate / ijfoodmicro
Effect of cinnamic acid on the growth and on plasma membrane H 1 –ATPase activity of Saccharomyces cerevisiae * ´ Alexandra Chambel, Cristina A. Viegas, Isabel Sa-Correia ´ ´ ´ Centro de Engenharia Biologica e Quımica , Instituto Superior Tecnico , Av. Rovisco Pais, 1049 -001 Lisboa, Portugal Received 4 January 1999; received in revised form 13 April 1999; accepted 14 June 1999
Abstract Cinnamic acid and cinnamic acid derivatives occur in plants and fruits, providing a natural protection against infections by pathogenic microorganisms. They may also inhibit wine fermentation and other fruit juice fermentations by Saccharomyces cerevisiae and raise difficulties in the biological treatment of waste water from some food industries. In the present work, it is shown that cells of S. cerevisiae YPH499 grown at pH 4 and 308C, in the presence of concentrations of cinnamic acid (20 or 35 mg / l) that reduce the maximum specific growth rate by 46 or 53%, respectively, exhibit a more active plasma membrane H 1 –ATPase than cells grown in its absence. This stimulatory effect was detected by assaying, during yeast growth in absence or presence of cinnamic acid, both the plasma membrane ATPase activity in crude membrane extracts and its action as a proton-pump by comparing extracellular acidification as a function of culture cell density. The lag-phase of approximately 8 h observed during cultivation in the presence of 20 mg / l cinnamic acid of yeast cells previously grown in its absence was eliminated by growing the inoculum in medium supplemented with the same concentration of cinnamic acid. These cinnamic acid adapted cells exhibited a more active plasma membrane H 1 –ATPase and this phenomenon may be due to and / or be among the mechanisms underlying the adaptative response to this toxic acid in yeast. 1999 Elsevier Science B.V. All rights reserved. Keywords: Saccharomyces cerevisiae; Plasma membrane H 1 –ATPase; Cinnamic acid
1. Introduction Aromatic antimicrobial agents, such as benzoic acid, have been widely applied as preservatives in food industry (Brown and Booth, 1991; Davidson, *Corresponding author. Tel.: 1 351-1-8417233; fax: 1 351-18480072. ´ E-mail address: [email protected] (I. Sa-Correia)
1997). Chemically related to benzoic acid is cinnamic acid and its naturally occurring derivatives. Cinnamic acid and cinnamic acid derivatives occur in plants and fruits, providing a natural protection against infections by pathogenic microorganisms (Mazza and Miniati, 1993; Davidson, 1997). They may also inhibit wine fermentation and other fruit juice fermentations by Saccharomyces cerevisiae and raise difficulties in the biological treatment of waste
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00100-2
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A. Chambel et al. / International Journal of Food Microbiology 50 (1999) 173 – 179
water from some food industries (Baranowski et al., 1980; Hamdi, 1993; Davidson, 1997). Despite the deleterious effects that cinnamic acid derivatives may have in the performance of several bioprocesses, little attention has been given to the study of the mechanisms underlying their toxicity and tolerance in microorganisms, particularly in yeast. The lipophilicity and the ionization constant of substituted benzoic and cinnamic acids were found to influence the level of inhibition of Listeria monocytogenes growth; the higher the lipophilicity and the higher the pKa (corresponding to higher proportion of the liposoluble undissociated weak acids present, at a particular extracellular pH), the higher the antimicrobial activity observed (Ramos-Nino et al., 1996). This indicates that partitioning is required for the compound to penetrate to their target sites or that the lipophilic part of the molecule is directly involved in inhibition at the target site itself (RamosNino et al., 1996). Consistently, it was found that cinnamic compounds uncouple the energy transducing cytoplasmic membrane in bacteria and this effect on the bioenergetic status of the membrane may contribute to their antimicrobial action (Mirzoeva et al., 1997). In fact, it is expected that these compounds, like other lipophilic weak acids, may lead to the stimulation of the passive influx of protons across the plasma membrane by causing the non-specific ´ increase of membrane permeability (Sa-Correia et al., 1989). Their antimicrobial activity may also rely on the dissociation of their liposoluble acidic form in the cytoplasm (their pKa’s are in the range 3.9–4.5 (Ramos-Nino et al., 1996), significantly below the approximately neutral pH of the cytosol) leading to additional acidification of the cell interior (Brown ´ and Booth, 1991; Viegas and Sa-Correia, 1991, 1995; Carmelo et al., 1997). Several growth inhibitors that lead to the permeabilization of yeast plasma membrane induce the stimulation of plasma membrane H 1 –ATPase activity of cells grown under mild stress compared with ´ cells grown in their absence (Rosa and Sa-Correia, ´ 1991, 1992; Viegas and Sa-Correia, 1991; Alexandre et al., 1996; Carmelo et al., 1997; Viegas et al., 1994, 1995, 1998; Fernandes et al., 1998). The H 1 – ATPase in the plasma membrane of yeasts creates the proton-motive force that drives transmembranar secondary transport of solutes and is implicated in pH homeostasis (Goffeau and Slayman, 1981; Ser-
rano, 1991). The in vivo activation of this membrane enzyme, which plays an essential role in yeast physiology, has been considered a response that presumably helps the cells to counteract the stressinduced dissipation of the proton-motive force across the plasma membrane and the intracellular acidification occuring in the presence of stressors that affect ´ plasma membrane organization (Rosa and Sa-Cor´ reia, 1991, 1992,; Viegas and Sa-Correia, 1991; Alexandre et al., 1996; Carmelo et al., 1997; Viegas et al., 1994, 1995, 1998; Fernandes et al., 1998). In the present work, we report that S. cerevisiae YPH499 cells grown, at pH 4 and 308C, in the presence of growth-inhibitory concentrations of cinnamic acid (20 and 35 mg / l), exhibited a more active plasma membrane H 1 –ATPase (approximately, 1.2 to 2-fold, depending on growth phase) than cells grown in its absence. This stimulatory effect was detected not only by assaying ATPase activity in crude membrane extracts (the methodology that has been used in our laboratory to prove ATPase activa´ tion induced by other stresses (Rosa and Sa-Correia, ´ 1991, 1992; Viegas and Sa-Correia, 1991; Carmelo et al., 1997; Viegas et al., 1995, 1998; Fernandes et al., 1998)), but also based on its action as a protonpump. Results suggesting the critical role on the duration of cinnamic acid-induced latency of yeast cells adaptation to cinnamic acid, are also shown.
2. Materials and methods
2.1. Strain and growth conditions Saccharomyces cerevisiae YPH499, previously examined concerning yeast response to ethanol, organic acids, acid pH, supra-optimal temperatures and copper, was also used in the present work (Viegas et al., 1994, 1995; Monteiro et al., 1994; Carmelo et al., 1996, 1997; Fernandes et al., 1998). Yeast cells were batch-cultured at 308C with orbital agitation (150 rpm) in 250 ml Erlenmeyer flasks containing 150 ml of liquid medium. Growth medium composition was: 30 g glucose (Merck, Germany), 6.7 g Yeast Nitrogen Base w / o amino acids (Difco, Detroit, USA), 40 mg adenine (Sigma, St. Louis, USA) and a mixture of amino acids to complement strain auxotrophies (Viegas et al., 1994), per liter of distilled water (pH 4.0760.01).
A. Chambel et al. / International Journal of Food Microbiology 50 (1999) 173 – 179
Absolute ethanol and / or ethanolic solutions of cinnamic acid (Sigma, St. Louis, USA) were added to this medium to give a final concentration of 0.5% (v / v) of ethanol and increasing concentrations of cinnamic acid up to 35 mg / l. The ethanol concentration used allowed cinnamic acid solubilization in the growth medium without affecting yeast growth kinetics (results not shown). Cell growth was monitored by measuring culture optical density at 640 nm (OD 640 nm ). For most of the growth experiments carried out, media were inoculated with cells pregrown in medium without cinnamic acid supplementation until late-exponential phase (OD 640 nm 5 1.7060.01). For the adaptation experiments, cells used as inoculum were cultivated in medium containing 20 mg / l cinnamic acid until the standardized OD 640 nm of 1.7060.01. For all the growth experiments, initial OD 640 nm was equal to 0.2060.01.
2.2. Assessment of extracellular acidification during yeast growth The pH of the supernatant obtained by centrifugation, at 9000 3 g for 5 min, of culture samples (3 ml) taken at suitable times during yeast growth in medium unsupplemented or supplemented with 20 or 35 mg / l of cinnamic acid was measured, by potentiometry, using a pH microelectrode attached to a pH meter (Metrohm E516, Metrohm Herisau, Switzerland).
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prepared after cells disruption with glass beads (0.5 mm; Sigma, St. Louis, USA) as previously described ´ (Viegas and Sa-Correia, 1991). Plasma membrane ATPase activity was assayed in the crude membrane suspensions using 1.6 mM of Mg ? ATP as substrate [2mM of ATP, disodium salt (Sigma, St. Louis, USA) and 10 mM of MgSO 4 ? 7H 2 O (Merck, ger´ many)], as previously described (Viegas and SaCorreia, 1991; Viegas et al., 1995). Plasma membrane ATPase specific activity (mU / mg) was calculated by linear regression from the slope of released Pi versus time (up to 8 min) and was expressed as nanomole of Pi released per min (mU) and per mg of protein. Protein concentration in the crude membrane suspensions was determined by the method of Bradford (Bradford, 1976), using bovine serum albumine (Sigma, St. Louis, USA) as the standard.
3. Results
3.1. Inhibition of yeast growth by cinnamic acid The addition of increasing concentrations of cinnamic acid (up to 35 mg / l) to the growth medium, at pH 4, led to the gradual reduction of the maximum specific growth rate of S. cerevisiae YPH499 (Fig. 1). A lag-phase, with a duration of 4 to 11 h, was necessary for yeast cells, cultivated under non-stressing conditions, to adapt to the deleterious effects of concentrations of total cinnamic acid in the range 15
2.3. Specific activity of plasma membrane H 1 – ATPase in yeast cells grown in the presence or absence of cinnamic acid Yeast cells were harvested by centrifugation at 9000 3 g for 5 min at 48C, at suitable times during growth in medium unsupplemented or supplemented with 20 or 35 mg / l of cinnamic acid. After centrifugation, cell pellets were resuspended in their supernatants to a standardized cell density of 20 mg (dry wt.) per ml and, after the addition of Tris (Sigma, St. Louis, USA), EDTA (Merck, Germany) and dithiothreitol (Sigma, St. Louis, USA) to final concentrations of 100, 5 and 2 mM, respectively, cell suspensions were rapidly frozen (2708C) and stored until used. Cell suspensions were thawed at room temperature and crude membrane suspensions were
Fig. 1. Effect of increasing concentrations of cinnamic acid on the growth curve of S. cerevisiae YPH499, at pH 4 and 308C. Total cinnamic acid concentrations were: 0 (s), 10 (^), 15 (h), 20 (d), 25 (j) and 35 (m) mg / l. Results are representative of the many growth experiments carried out.
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3.2. Increase of extracellular acidification by yeast cultivation with cinnamic acid
Fig. 2. Comparison of the growth curves of S. cerevisiae YPH499 in medium supplemented with 20 mg / l total cinnamic acid, at pH 4 and 308C, when cells used as inoculum were cultivated either in medium supplemented with this same concentration of cinnamic acid (h) or in medium without cinnamic acid (d). Growth curve in the absence of cinnamic acid is also shown (s).
to 35 mg / l, being the duration of lag-phase dependent on the severity of acid stress (Fig. 1). The lag-phase of approximately 8 h observed during cultivation in the presence of 20 mg / l cinnamic acid of yeast cells previously grown in its absence was eliminated by growing the inoculum in medium supplemented with the same concentration of cinnamic acid (Fig. 2).
During exponential growth of S. cerevisiae YPH499 in the absence or presence of cinnamic acid, broth acidification occurred (Fig. 3a and b). This continued decrease of culture pH is mainly due to the activity of plasma-membrane-bound-H 1 -pump (Sigler and Hofer, 1991). Due to the inhibition of yeast growth kinetics and global metabolic rate by cinnamic acid, the decrease of broth pH was slower during cultivation in the presence of cinnamic acid compared with the unstressed cultivation (Fig. 3a). However, for identical cell concentration (associated to culture OD 640 nm ), extracellular acidification by cinnamic acid-grown-cells was clearly above the values associated to the unstressed cells (Fig. 3b). These results suggest that proton-pumping activity is stimulated in cinnamic acid-adapted cells.
3.3. In vivo activation by cinnamic acid of plasma membrane ATPase Consistent with the stimulation of extracellular acidification by yeast cells cultivated with 20 and 35 mg / l cinnamic acid, at pH 4 (Fig. 3b), the specific activity of plasma membrane ATPase was higher in cinnamic acid-grown cells (Fig. 4). A peak of
Fig. 3. Decrease of broth extracellular pH during exponential growth at 308C of S. cerevisiae YPH499 in media (pH 4) supplemented with 20 (d) or 35 (m) mg / l of total cinnamic acid or in unsupplemented medium (s), as a function of (a) the incubation time or (b) culture OD 640 nm . Results are representative of at least three independent growth experiments.
A. Chambel et al. / International Journal of Food Microbiology 50 (1999) 173 – 179
Fig. 4. Specific activity of plasma membrane ATPase assayed in crude membrane suspensions prepared from cells of S. cerevisiae YPH499 harvested during exponential growth carried out at 308C in media (pH 4) supplemented with 20 (d) or 35 (m) mg / l of total cinnamic acid or in unsupplemented medium (s), at the indicated values of culture OD 640 nm . Results are the mean of plasma membrane ATPase specific activity values from at least two independent growth experiments.
maximal plasma membrane ATPase activity was observed at mid-exponential phase and the level of cinnamic acid-induced activation of this ATPase was also maximal (2-fold) at this phase of growth (Fig. 4). A similar growth phase-dependence of plasma membrane H 1 –ATPase specific activity and activation was previously reported for the same yeast strain / growth medium in absence or presence of sub-critical levels of several stressors (Monteiro et al., 1994; Viegas et al., 1994, 1995; Fernandes et al., 1998).
4. Discussion In the present work, it is reported the activation of plasma-membrane-bound-H 1 –ATPase due to yeast cultivation with growth-inhibitory concentrations of cinnamic acid. This stimulatory effect was detected by both assaying the plasma membrane ATPase activity and its action as a proton-pump by comparing broth acidification, during stressed and unstressed cultivations. Although the production of CO 2 and organic acids during yeast growth may also contribute to the overall process of extracellular acidification, the decrease of culture pH is mainly the result of the action of plasma-membrane-bound-H 1 translocating ATPase (Sigler and Hofer, 1991). As
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indicated by results herein shown, broth acidification as a function of culture cell density can actually be used as the first indication of the possible modification of activity of this H 1 –ATPase. The in vivo activation of plasma membrane H 1 – ATPase in response to growth with cinnamic acid inhibitory concentrations allows enhanced catalysed H 1 -efflux and thus may help the cell to counteract the dissipation of the transmembranar H 1 -motive force and the intracellular acidification expected to occur in cinnamic acid-stressed cells. Since the ability of yeast cells to grow in the presence of lipophilic acids, at low pH, reflects their capacity to maintain control over internal pH and transmembranar H 1 -motive force, the reported phenomenon may be among the mechanisms involved in the acquisition of tolerance in cinnamic acid-adapted cells, as previously suggested by Holyoak et al. (1996) and Viegas et al. (1998) for sorbic acid- and octanoic acid-adaptations, respectively. Significantly, cinnamic acid-adapted cells, which exhibited a more active plasma membrane H 1 –ATPase, resumed exponential growth in cinnamic acid-supplemented medium without a detectable period of latency that was otherwise significant for inoculum cells grown under non-stressed conditions. These results clearly point out the critical role of the physiology of cells used as inoculum in the duration of cinnamic acidinduced latency. Interestingly, our laboratory recently reported the stimulation of plasma membrane ATPase activity during octanoic acid-induced latency of unadapted yeast cells, reaching maximal values when cells eventually recovered and entered exponential growth (Viegas et al., 1998). The molecular mechanisms underlying the stimulation of plasma membrane H 1 –ATPase caused by cinnamic acid stress were not addressed in the present work. It is possible that the adaptative modification of plasma-membrane-bound-ATPaselipid-environment in cinnamic acid-adapted cells may be among the mechanisms involved in this phenomenon, as suggested by Alexandre et al. (1996) and Fernandes et al. (1998) for decanoic acid and Cu 21 -induced ATPase activations, respectively. Whether involved in cinnamic acid-adaptation, the stimulation of plasma membrane H 1 –ATPase activity is just one of the mechanisms that may underly yeast adaptative response to this particular stress. Other mechanisms may include the increased cellular
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buffering capacity of cinnamic acid-grown cells due ´ to lower intracellular volume (Viegas and Sa-Correia, 1995), the more favorable plasma membrane lipid composition (Alexandre et al., 1996), and the induction of the active expulsion of the respective anion out of the cells (Henriques et al., 1997; Piper et al., 1998).
Acknowledgements This work was supported by JNICT / FCT, FEDER and PRAXIS XXI Programme (grants: PRAXIS-2 / 2.1 / BIO / 20 / 94 and PBIC / C / BIO / 2031 / 95, and a M.Sc. scholarship (BM / 3106 / 92-IF) to A.C.).
References Alexandre, H., Mathieu, B., Charpentier, C., 1996. Alteration in membrane fluidity and lipid composition, and modulation of H 1 -ATPase activity in Saccharomyces cerevisiae caused by decanoic acid. Microbiology 142, 469–475. Baranowski, J.D., Davidson, P.M., Nagel, C.W., Nranen, A.L., 1980. Inhibition of Saccharomyces cerevisiae by naturally occuring hydroxycinnamates. J. Food Sci. 45, 592–594. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protin–dye binding. Anal. Biochem. 72, 248–254. Brown, M.H., Booth, I.R., 1991. Acidulants and low pH. In: Russell, N.J., Gould, G.W. (Eds.), Food Preservatives, Blackie, Glasgow, pp. 22–43. ´ Carmelo, V., Bogaerts, P., Sa-Correia, I., 1996. Activity of plasma membrane H 1 –ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at optimal and low pH. Arch. Microbiol. 166, 315–320. ´ Carmelo, V., Santos, H., Sa-Correia, I., 1997. Effect of extracellular acidification on the activity of plasma membrane ATPase and on the cytosolic and vacuolar pH of Saccharomyces cerevisiae. Biochim. Biophys. Acta 1325, 63–70. Davidson, P.M., 1997. Chemical preservatives and natural antimicrobial compounds. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology: Fundamentals and Frontiers, ASM Press, Washington D.C, pp. 520–556. ´ Fernandes, A.R., Peixoto, F.P., Sa-Correia, I., 1998. Activation of 1 the H –ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress. Arch. Microbiol. 171, 6–12. Goffeau, A., Slayman, C.W., 1981. The proton-translocating ATPase of the fungal plasma membrane. Biochim. Biophys. Acta 639, 197–223. Hamdi, M., 1993. Future prospects and constraints of olive mill wastewaters use and treatment: a review. Bioprocess Eng. 8, 209–214.
Henriques, M., Quintas, C., Loureiro-Dias, M.C., 1997. Extrusion of benzoic acid in Saccharomyces cerevisiae by an energydependent mechanism. Microbiology 143, 1877–1883. Holyoak, C.D., Stratford, M., McMullin, Z., Cole, M.B., Crimmins, K., Brown, A.J.P., Coote, P.J., 1996. Activity of the plasma membrane H 1 –ATPase and optimal glycolitic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid. Appl. Environ. Microbiol. 62, 3158–3164. Mazza, G., Miniati, E., 1993. Grapes. In: Anthocyanins in Fruits, Vegetables and Grains, CRC Press, pp. 149–200. Mirzoeva, O.K., Grishanin, R.N., Calder, P.C., 1997. Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and mobility of bacteria. Microbiol. Res. 152, 239–246. ´ Monteiro, G.A., Supply, P., Goffeau, A., Sa-Correia, I., 1994. The in vivo activation of Saccharomyces cerevisiae plasma membrane H 1 –ATPase by ethanol depends on the expression of the PMA1 gene, but not of the PMA2 gene. Yeast 10, 1439–1446. ´ Y., Thompson, S., Pandjaitan, R., Holyoak, C., Piper, P., Mahe, Egner, R., Muhlbauer, M., Coote, P., Kuchler, K., 1998. The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast. EMBO J. 17, 4257–4265. Ramos-Nino, M.E., Clifford, M.N., Adams, M.R., 1996. Quantitative structure activity relationship for the effect of benzoic acids, cinnamic acids and benzaldehydes on Listeria monocytogenes. J. Appl. Microbiol. 80, 303–310. ´ Rosa, M.F., Sa-Correia, I., 1991. In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae. Appl. Environ. Microbiol. 57, 830–835. ´ Rosa, M.F., Sa-Correia, I., 1992. Ethanol tolerance and activity of plasma membrane ATPase in Kluyveromyces marxianus and Saccharomyces cerevisiae. Enzyme Microb. Technol. 14, 23– 27. ´ Sa-Correia, I., Salgueiro, S.P., Viegas, C.A., Novais, J.M., 1989. Leakage induced by ethanol and octanoic and decanoic acids in Saccharomyces cerevisiae. Yeast 5 (Suppl.), S123–S127. Serrano, R., 1991. In: Broach, J.R., Pringle, J.R., Jones, E.W. (Eds.), The Molecular and Cellular Biology of the Yeast saccaromyces, Transport across yeast vacuolar and plasma membranes, Vol. vol. 1, CSHL Press, pp. 523–586. Sigler, K., Hofer, M., 1991. Mechanisms of acid extrusion in yeast. Biochim. Biophys. Acta 1071, 375–391. ´ Viegas, C.A., Sa-Correia, I., 1991. Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid. J. Gen. Microbiol. 137, 645–651. ´ Viegas, C.A., Sa-Correia, I., 1995. Toxicity of octanoic acid in Saccharomyces cerevisiae at temperatures between 8.5 and 308C. Enzyme Microb. Technol. 17, 826–831. Viegas, C.A., Supply, P., Capieux, E., van Dyck, L., Goffeau, A., 1 ´ Sa-Correia, I., 1994. Regulation of the expression of the H – ATPase genes PMA1 and PMA2 during growth and effects of octanoic acid in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1217, 74–80. ˜ P., Nunes, A.G., Sa-Correia, ´ Viegas, C.A., Sebastiao, I., 1995. Activation of plasma membrane H 1 –ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at supraoptimal temperatures. Appl. Environ. Microbiol. 61, 1904–1909.
A. Chambel et al. / International Journal of Food Microbiology 50 (1999) 173 – 179 ´ Viegas, C.A., Almeida, P.F., Cavaco, M., Sa-Correia, I., 1998. The H 1 –ATPase in the plasma membrane of Saccharomyces cerevisiae is activated during growth latency in octanoic acid-
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supplemented medium accompanying the decrease in intracellular pH and cell viability. Appl. Environ. Microbiol. 64, 779–783.
Effect of cinnamic acid on the growth and on plasma membrane H 1 –ATPase activity of Saccharomyces cerevisiae * ´ Alexandra Chambel, Cristina A. Viegas, Isabel Sa-Correia ´ ´ ´ Centro de Engenharia Biologica e Quımica , Instituto Superior Tecnico , Av. Rovisco Pais, 1049 -001 Lisboa, Portugal Received 4 January 1999; received in revised form 13 April 1999; accepted 14 June 1999
Abstract Cinnamic acid and cinnamic acid derivatives occur in plants and fruits, providing a natural protection against infections by pathogenic microorganisms. They may also inhibit wine fermentation and other fruit juice fermentations by Saccharomyces cerevisiae and raise difficulties in the biological treatment of waste water from some food industries. In the present work, it is shown that cells of S. cerevisiae YPH499 grown at pH 4 and 308C, in the presence of concentrations of cinnamic acid (20 or 35 mg / l) that reduce the maximum specific growth rate by 46 or 53%, respectively, exhibit a more active plasma membrane H 1 –ATPase than cells grown in its absence. This stimulatory effect was detected by assaying, during yeast growth in absence or presence of cinnamic acid, both the plasma membrane ATPase activity in crude membrane extracts and its action as a proton-pump by comparing extracellular acidification as a function of culture cell density. The lag-phase of approximately 8 h observed during cultivation in the presence of 20 mg / l cinnamic acid of yeast cells previously grown in its absence was eliminated by growing the inoculum in medium supplemented with the same concentration of cinnamic acid. These cinnamic acid adapted cells exhibited a more active plasma membrane H 1 –ATPase and this phenomenon may be due to and / or be among the mechanisms underlying the adaptative response to this toxic acid in yeast. 1999 Elsevier Science B.V. All rights reserved. Keywords: Saccharomyces cerevisiae; Plasma membrane H 1 –ATPase; Cinnamic acid
1. Introduction Aromatic antimicrobial agents, such as benzoic acid, have been widely applied as preservatives in food industry (Brown and Booth, 1991; Davidson, *Corresponding author. Tel.: 1 351-1-8417233; fax: 1 351-18480072. ´ E-mail address: [email protected] (I. Sa-Correia)
1997). Chemically related to benzoic acid is cinnamic acid and its naturally occurring derivatives. Cinnamic acid and cinnamic acid derivatives occur in plants and fruits, providing a natural protection against infections by pathogenic microorganisms (Mazza and Miniati, 1993; Davidson, 1997). They may also inhibit wine fermentation and other fruit juice fermentations by Saccharomyces cerevisiae and raise difficulties in the biological treatment of waste
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00100-2
174
A. Chambel et al. / International Journal of Food Microbiology 50 (1999) 173 – 179
water from some food industries (Baranowski et al., 1980; Hamdi, 1993; Davidson, 1997). Despite the deleterious effects that cinnamic acid derivatives may have in the performance of several bioprocesses, little attention has been given to the study of the mechanisms underlying their toxicity and tolerance in microorganisms, particularly in yeast. The lipophilicity and the ionization constant of substituted benzoic and cinnamic acids were found to influence the level of inhibition of Listeria monocytogenes growth; the higher the lipophilicity and the higher the pKa (corresponding to higher proportion of the liposoluble undissociated weak acids present, at a particular extracellular pH), the higher the antimicrobial activity observed (Ramos-Nino et al., 1996). This indicates that partitioning is required for the compound to penetrate to their target sites or that the lipophilic part of the molecule is directly involved in inhibition at the target site itself (RamosNino et al., 1996). Consistently, it was found that cinnamic compounds uncouple the energy transducing cytoplasmic membrane in bacteria and this effect on the bioenergetic status of the membrane may contribute to their antimicrobial action (Mirzoeva et al., 1997). In fact, it is expected that these compounds, like other lipophilic weak acids, may lead to the stimulation of the passive influx of protons across the plasma membrane by causing the non-specific ´ increase of membrane permeability (Sa-Correia et al., 1989). Their antimicrobial activity may also rely on the dissociation of their liposoluble acidic form in the cytoplasm (their pKa’s are in the range 3.9–4.5 (Ramos-Nino et al., 1996), significantly below the approximately neutral pH of the cytosol) leading to additional acidification of the cell interior (Brown ´ and Booth, 1991; Viegas and Sa-Correia, 1991, 1995; Carmelo et al., 1997). Several growth inhibitors that lead to the permeabilization of yeast plasma membrane induce the stimulation of plasma membrane H 1 –ATPase activity of cells grown under mild stress compared with ´ cells grown in their absence (Rosa and Sa-Correia, ´ 1991, 1992; Viegas and Sa-Correia, 1991; Alexandre et al., 1996; Carmelo et al., 1997; Viegas et al., 1994, 1995, 1998; Fernandes et al., 1998). The H 1 – ATPase in the plasma membrane of yeasts creates the proton-motive force that drives transmembranar secondary transport of solutes and is implicated in pH homeostasis (Goffeau and Slayman, 1981; Ser-
rano, 1991). The in vivo activation of this membrane enzyme, which plays an essential role in yeast physiology, has been considered a response that presumably helps the cells to counteract the stressinduced dissipation of the proton-motive force across the plasma membrane and the intracellular acidification occuring in the presence of stressors that affect ´ plasma membrane organization (Rosa and Sa-Cor´ reia, 1991, 1992,; Viegas and Sa-Correia, 1991; Alexandre et al., 1996; Carmelo et al., 1997; Viegas et al., 1994, 1995, 1998; Fernandes et al., 1998). In the present work, we report that S. cerevisiae YPH499 cells grown, at pH 4 and 308C, in the presence of growth-inhibitory concentrations of cinnamic acid (20 and 35 mg / l), exhibited a more active plasma membrane H 1 –ATPase (approximately, 1.2 to 2-fold, depending on growth phase) than cells grown in its absence. This stimulatory effect was detected not only by assaying ATPase activity in crude membrane extracts (the methodology that has been used in our laboratory to prove ATPase activa´ tion induced by other stresses (Rosa and Sa-Correia, ´ 1991, 1992; Viegas and Sa-Correia, 1991; Carmelo et al., 1997; Viegas et al., 1995, 1998; Fernandes et al., 1998)), but also based on its action as a protonpump. Results suggesting the critical role on the duration of cinnamic acid-induced latency of yeast cells adaptation to cinnamic acid, are also shown.
2. Materials and methods
2.1. Strain and growth conditions Saccharomyces cerevisiae YPH499, previously examined concerning yeast response to ethanol, organic acids, acid pH, supra-optimal temperatures and copper, was also used in the present work (Viegas et al., 1994, 1995; Monteiro et al., 1994; Carmelo et al., 1996, 1997; Fernandes et al., 1998). Yeast cells were batch-cultured at 308C with orbital agitation (150 rpm) in 250 ml Erlenmeyer flasks containing 150 ml of liquid medium. Growth medium composition was: 30 g glucose (Merck, Germany), 6.7 g Yeast Nitrogen Base w / o amino acids (Difco, Detroit, USA), 40 mg adenine (Sigma, St. Louis, USA) and a mixture of amino acids to complement strain auxotrophies (Viegas et al., 1994), per liter of distilled water (pH 4.0760.01).
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Absolute ethanol and / or ethanolic solutions of cinnamic acid (Sigma, St. Louis, USA) were added to this medium to give a final concentration of 0.5% (v / v) of ethanol and increasing concentrations of cinnamic acid up to 35 mg / l. The ethanol concentration used allowed cinnamic acid solubilization in the growth medium without affecting yeast growth kinetics (results not shown). Cell growth was monitored by measuring culture optical density at 640 nm (OD 640 nm ). For most of the growth experiments carried out, media were inoculated with cells pregrown in medium without cinnamic acid supplementation until late-exponential phase (OD 640 nm 5 1.7060.01). For the adaptation experiments, cells used as inoculum were cultivated in medium containing 20 mg / l cinnamic acid until the standardized OD 640 nm of 1.7060.01. For all the growth experiments, initial OD 640 nm was equal to 0.2060.01.
2.2. Assessment of extracellular acidification during yeast growth The pH of the supernatant obtained by centrifugation, at 9000 3 g for 5 min, of culture samples (3 ml) taken at suitable times during yeast growth in medium unsupplemented or supplemented with 20 or 35 mg / l of cinnamic acid was measured, by potentiometry, using a pH microelectrode attached to a pH meter (Metrohm E516, Metrohm Herisau, Switzerland).
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prepared after cells disruption with glass beads (0.5 mm; Sigma, St. Louis, USA) as previously described ´ (Viegas and Sa-Correia, 1991). Plasma membrane ATPase activity was assayed in the crude membrane suspensions using 1.6 mM of Mg ? ATP as substrate [2mM of ATP, disodium salt (Sigma, St. Louis, USA) and 10 mM of MgSO 4 ? 7H 2 O (Merck, ger´ many)], as previously described (Viegas and SaCorreia, 1991; Viegas et al., 1995). Plasma membrane ATPase specific activity (mU / mg) was calculated by linear regression from the slope of released Pi versus time (up to 8 min) and was expressed as nanomole of Pi released per min (mU) and per mg of protein. Protein concentration in the crude membrane suspensions was determined by the method of Bradford (Bradford, 1976), using bovine serum albumine (Sigma, St. Louis, USA) as the standard.
3. Results
3.1. Inhibition of yeast growth by cinnamic acid The addition of increasing concentrations of cinnamic acid (up to 35 mg / l) to the growth medium, at pH 4, led to the gradual reduction of the maximum specific growth rate of S. cerevisiae YPH499 (Fig. 1). A lag-phase, with a duration of 4 to 11 h, was necessary for yeast cells, cultivated under non-stressing conditions, to adapt to the deleterious effects of concentrations of total cinnamic acid in the range 15
2.3. Specific activity of plasma membrane H 1 – ATPase in yeast cells grown in the presence or absence of cinnamic acid Yeast cells were harvested by centrifugation at 9000 3 g for 5 min at 48C, at suitable times during growth in medium unsupplemented or supplemented with 20 or 35 mg / l of cinnamic acid. After centrifugation, cell pellets were resuspended in their supernatants to a standardized cell density of 20 mg (dry wt.) per ml and, after the addition of Tris (Sigma, St. Louis, USA), EDTA (Merck, Germany) and dithiothreitol (Sigma, St. Louis, USA) to final concentrations of 100, 5 and 2 mM, respectively, cell suspensions were rapidly frozen (2708C) and stored until used. Cell suspensions were thawed at room temperature and crude membrane suspensions were
Fig. 1. Effect of increasing concentrations of cinnamic acid on the growth curve of S. cerevisiae YPH499, at pH 4 and 308C. Total cinnamic acid concentrations were: 0 (s), 10 (^), 15 (h), 20 (d), 25 (j) and 35 (m) mg / l. Results are representative of the many growth experiments carried out.
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3.2. Increase of extracellular acidification by yeast cultivation with cinnamic acid
Fig. 2. Comparison of the growth curves of S. cerevisiae YPH499 in medium supplemented with 20 mg / l total cinnamic acid, at pH 4 and 308C, when cells used as inoculum were cultivated either in medium supplemented with this same concentration of cinnamic acid (h) or in medium without cinnamic acid (d). Growth curve in the absence of cinnamic acid is also shown (s).
to 35 mg / l, being the duration of lag-phase dependent on the severity of acid stress (Fig. 1). The lag-phase of approximately 8 h observed during cultivation in the presence of 20 mg / l cinnamic acid of yeast cells previously grown in its absence was eliminated by growing the inoculum in medium supplemented with the same concentration of cinnamic acid (Fig. 2).
During exponential growth of S. cerevisiae YPH499 in the absence or presence of cinnamic acid, broth acidification occurred (Fig. 3a and b). This continued decrease of culture pH is mainly due to the activity of plasma-membrane-bound-H 1 -pump (Sigler and Hofer, 1991). Due to the inhibition of yeast growth kinetics and global metabolic rate by cinnamic acid, the decrease of broth pH was slower during cultivation in the presence of cinnamic acid compared with the unstressed cultivation (Fig. 3a). However, for identical cell concentration (associated to culture OD 640 nm ), extracellular acidification by cinnamic acid-grown-cells was clearly above the values associated to the unstressed cells (Fig. 3b). These results suggest that proton-pumping activity is stimulated in cinnamic acid-adapted cells.
3.3. In vivo activation by cinnamic acid of plasma membrane ATPase Consistent with the stimulation of extracellular acidification by yeast cells cultivated with 20 and 35 mg / l cinnamic acid, at pH 4 (Fig. 3b), the specific activity of plasma membrane ATPase was higher in cinnamic acid-grown cells (Fig. 4). A peak of
Fig. 3. Decrease of broth extracellular pH during exponential growth at 308C of S. cerevisiae YPH499 in media (pH 4) supplemented with 20 (d) or 35 (m) mg / l of total cinnamic acid or in unsupplemented medium (s), as a function of (a) the incubation time or (b) culture OD 640 nm . Results are representative of at least three independent growth experiments.
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Fig. 4. Specific activity of plasma membrane ATPase assayed in crude membrane suspensions prepared from cells of S. cerevisiae YPH499 harvested during exponential growth carried out at 308C in media (pH 4) supplemented with 20 (d) or 35 (m) mg / l of total cinnamic acid or in unsupplemented medium (s), at the indicated values of culture OD 640 nm . Results are the mean of plasma membrane ATPase specific activity values from at least two independent growth experiments.
maximal plasma membrane ATPase activity was observed at mid-exponential phase and the level of cinnamic acid-induced activation of this ATPase was also maximal (2-fold) at this phase of growth (Fig. 4). A similar growth phase-dependence of plasma membrane H 1 –ATPase specific activity and activation was previously reported for the same yeast strain / growth medium in absence or presence of sub-critical levels of several stressors (Monteiro et al., 1994; Viegas et al., 1994, 1995; Fernandes et al., 1998).
4. Discussion In the present work, it is reported the activation of plasma-membrane-bound-H 1 –ATPase due to yeast cultivation with growth-inhibitory concentrations of cinnamic acid. This stimulatory effect was detected by both assaying the plasma membrane ATPase activity and its action as a proton-pump by comparing broth acidification, during stressed and unstressed cultivations. Although the production of CO 2 and organic acids during yeast growth may also contribute to the overall process of extracellular acidification, the decrease of culture pH is mainly the result of the action of plasma-membrane-bound-H 1 translocating ATPase (Sigler and Hofer, 1991). As
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indicated by results herein shown, broth acidification as a function of culture cell density can actually be used as the first indication of the possible modification of activity of this H 1 –ATPase. The in vivo activation of plasma membrane H 1 – ATPase in response to growth with cinnamic acid inhibitory concentrations allows enhanced catalysed H 1 -efflux and thus may help the cell to counteract the dissipation of the transmembranar H 1 -motive force and the intracellular acidification expected to occur in cinnamic acid-stressed cells. Since the ability of yeast cells to grow in the presence of lipophilic acids, at low pH, reflects their capacity to maintain control over internal pH and transmembranar H 1 -motive force, the reported phenomenon may be among the mechanisms involved in the acquisition of tolerance in cinnamic acid-adapted cells, as previously suggested by Holyoak et al. (1996) and Viegas et al. (1998) for sorbic acid- and octanoic acid-adaptations, respectively. Significantly, cinnamic acid-adapted cells, which exhibited a more active plasma membrane H 1 –ATPase, resumed exponential growth in cinnamic acid-supplemented medium without a detectable period of latency that was otherwise significant for inoculum cells grown under non-stressed conditions. These results clearly point out the critical role of the physiology of cells used as inoculum in the duration of cinnamic acidinduced latency. Interestingly, our laboratory recently reported the stimulation of plasma membrane ATPase activity during octanoic acid-induced latency of unadapted yeast cells, reaching maximal values when cells eventually recovered and entered exponential growth (Viegas et al., 1998). The molecular mechanisms underlying the stimulation of plasma membrane H 1 –ATPase caused by cinnamic acid stress were not addressed in the present work. It is possible that the adaptative modification of plasma-membrane-bound-ATPaselipid-environment in cinnamic acid-adapted cells may be among the mechanisms involved in this phenomenon, as suggested by Alexandre et al. (1996) and Fernandes et al. (1998) for decanoic acid and Cu 21 -induced ATPase activations, respectively. Whether involved in cinnamic acid-adaptation, the stimulation of plasma membrane H 1 –ATPase activity is just one of the mechanisms that may underly yeast adaptative response to this particular stress. Other mechanisms may include the increased cellular
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buffering capacity of cinnamic acid-grown cells due ´ to lower intracellular volume (Viegas and Sa-Correia, 1995), the more favorable plasma membrane lipid composition (Alexandre et al., 1996), and the induction of the active expulsion of the respective anion out of the cells (Henriques et al., 1997; Piper et al., 1998).
Acknowledgements This work was supported by JNICT / FCT, FEDER and PRAXIS XXI Programme (grants: PRAXIS-2 / 2.1 / BIO / 20 / 94 and PBIC / C / BIO / 2031 / 95, and a M.Sc. scholarship (BM / 3106 / 92-IF) to A.C.).
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