Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, UK1
Author for correspondence: Peter Piper. Tel: +44 207 679 2212. Fax: +44 207 679 7193. e-mail: piper{at}bsm.bioc.ucl.ac.uk
Keywords: Saccharomyces cerevisiae, Zygosaccharomyces bailii, organic acid stress, oxidative stress, food spoilage
a Present address: Departamento de Quimica y Biologia, Facultad de Ciencias, Universidad de Atacama, Capiapo, Chile.
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Overview |
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Although weak acid adaptation probably evolved to facilitate growth at low pH in the presence of weak organic acids, it poses problems for the food industry as it leads to substantial increases in resistance to the major organic acid food preservatives. As a result, it is often necessary to use these preservatives at millimolar rather than micromolar levels in order to prevent yeast spoilage of low pH foods and beverages. This review summarizes the current knowledge of the mechanisms of weak acid resistance in S. cerevisiae and Zygosaccharomyces bailii, two important food spoilage yeasts. Both organisms are able to maintain lower intracellular levels of weak acid than would be expected on the basis of a free equilibration across the cell membrane. Nevertheless, it is unlikely they achieve this by identical strategies. S. cerevisiae expends considerable energy in actively extruding acid from the cell, high levels of a specific ATP binding cassette (ABC) transporter (Pdr12) being induced in its plasma membrane in order to catalyse this efflux. Z. bailii, in contrast, does not show major changes to its plasma membrane protein composition, but may place more reliance instead on limiting the initial diffusional entry of the acid to the cells. Z. bailii, unlike S. cerevisiae, can also catalyse oxidative degradation of two of the most commonly used food preservatives, sorbate and benzoate.
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The problems posed by weak acid resistance |
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The conditions imposed by many preserved food materials [low water activity (aw); low pH; the presence of high preservative levels, carbon dioxide or ethanol; or the absence of oxygen] do not represent the ideal environments for microbial growth. Yeasts and fungi pose a major spoilage threat for many materials preserved at low pH, low aw and/or with high levels of preservative (Deak, 1991 ; Fleet, 1992
). Nevertheless, it is only a tiny fraction of the very many characterized yeast species that are of major importance in this regard and which are therefore classified as spoilage yeasts (see Steels et al., 2000
, and references cited therein). They include a number of Zygosaccharomyces, as well as some isolates of S. cerevisiae. Often Zygosaccharomyces rouxii is considered the most important Zygosaccharomyces from the standpoint of an ability to grow at very low aw, while Z. bailii and Zygosaccharomyces lentus are the most important from the standpoint of weak acid preservative resistance. The latter yeasts can sometimes grow in the presence of the highest levels of those acids allowed in food preservation, at pH values below the pKa values of these acids (Thomas & Davenport, 1985
; Deak, 1991
; Fleet, 1992
; Steels et al., 1999
, 2000
). Z. bailii has also attracted attention as a spoilage agent of wine, a property that accrues from its high ethanol tolerance (Kalathenos et al., 1995
) and ability to metabolize acetic acid in the presence of the complex mixtures of sugars often found in wine fermentations (Sousa et al., 1996
, 1998
).
In any Z. bailii culture, the individual cells differ very considerably in their sorbate resistance, a small fraction being remarkably resistant (Steels et al., 2000 ). The minimum sorbate levels needed to inhibit the growth of Z. bailii therefore increase with the size of the inoculum, making it difficult to place numerical values on the weak acid resistances of this organism, or the extents to which these resistances exceed those of S. cerevisiae. Nevertheless, S. cerevisiae isolated from instances of food spoilage can frequently adapt to levels of sorbate and benzoate only slightly lower than the levels inhibitory to Z. bailii and Z. lentus.
Apart from a number of studies on the uptake and utilization of acetate by Z. bailii (Sousa et al., 1996 , 1998
), comparatively little is known about how Z. bailii and Z. lentus acquire their remarkable weak acid resistances. Considerably more is known about weak acid adaptation in S. cerevisiae, as this is a species amenable to genetic analysis. This article will therefore focus mainly on S. cerevisiae, where the acquisition of weak organic acid resistance appears to involve a stress response quite distinct from other, more widely studied, stress responses, such as those induced by osmostress or heat shock. A picture is emerging of an adaptation that acts to limit accumulation of the weak organic acid in the cells of the adapted yeast. Resistance to sulphite, a non-organic weak acid preservative (Pilkington & Rose, 1988
), is not discussed.
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Weak acid stress in S. cerevisiae |
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Sorbate- and benzoate-stressed S. cerevisiae and Z. bailii are, in addition, experiencing a very severe energy (ATP) depletion (Warth & Nickerson, 1991 ; Piper et al., 1997
). At least in S. cerevisiae this energy crisis is partly caused by strong inhibitory effects of sorbate and benzoate on glycolysis, an inhibition exerted mainly at the phosphofructokinase (Pfk) reaction (Krebs et al., 1983
; Pearce et al., 2001
). The trehalose accumulation with sorbate treatment of S. cerevisiae is probably in response to this Pfk inhibition (Cheng et al., 1999
). In the presence of oxygen, this energy crisis is exacerbated still further by the severe influences of the more lipophilic weak acid preservatives on membrane transport processes and energy coupling. The associated mitochondrial electron transport chain dysfunction increases free radical formation, causing sorbate- and benzoate-treated S. cerevisiae to suffer an excessively high endogenous production of superoxide free radicals (Piper, 1999
).
Generally it is in low pH cultures that these effects of weak organic acids are most apparent. At neutral pH, residues of acetic, sorbic or benzoic acids are essentially completely dissociated. As such, they pose a much smaller threat and may even provide a potential carbon source. At neutral pH though, high sorbate levels still exert some inhibitory effects on S. cerevisiae (Stratford & Anslow, 1996 ) and a strong transcriptional response to sorbate is still apparent (Martinez-Pastor et al., 1996
; Piper et al., 1998
).
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Weak acid adaptation in S. cerevisiae |
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Our interest in weak acid stress was originally generated by the chance discovery that these acids act as inhibitors of yet another stress response, the heat-shock response (Cheng & Piper, 1994 ). We subsequently discovered that an alternative stress response was being induced, a response leading to strong induction of two plasma membrane proteins, Pdr12 and Hsp30 (the latter so called because it is also a heat-shock protein) (Panaretou & Piper, 1992
; Piper et al., 1997
, 1998
). Pdr12 is the product of one of the 31 ABC transporter genes in the S. cerevisiae genome (Bauer et al., 1999
). So strong is its weak acid induction that it becomes one of the most abundant plasma membrane proteins in adapted cells (Fig. 2
).
|
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Pdr12 induction is important for S. cerevisiae to adapt to growth in the presence of weak organic acids |
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The ability of Pdr12 to give resistance to short-chain alkanols, compounds whose toxic effects are thought to be due mainly to their ability to dissolve in membranes (Weber & de Bont, 1996 ), suggests that this ABC transporter may bind acid anions or alcohol molecules actually incorporated in the inner leaflet of the plasma membrane (rather than dissolved in the cytosol as shown in Fig. 1b
). One can surmise that Pdr12 then transports these to the opposite (periplasmic) side of the membrane, in order to release them into the aqueous phase of the periplasm. Such active efflux may be able to lower the intracellular level of the acid anion or alcohol, on the basis that the polar groups on these carboxylate anion or alcohol substrates will slow their diffusion back across the cell membrane and consequent re-equilibration between the extracellular and intracellular milieu.
It is possible to visualize Pdr12 activity in vivo as the ability of cells to catalyse an active extrusion of fluorescein. Experimentally this involves loading the cells with fluorescein diacetate, then observing the energy-dependent efflux of the fluorescent weak acid produced by the actions of intracellular esterases on this fluorescein diacetate. Such fluorescein extrusion is competitively inhibited by the presence of weak organic acid preservatives (Holyoak et al., 1999 ). Even though wild-type cells cultured at pH 4·5 in the absence of weak acids contain Pdr12 in their membranes (Piper et al., 1998
), they do not display this active fluorescein efflux (Holyoak et al., 2000
). Pdr12 may not therefore be an active transporter in the absence of weak acid stress. In contrast, pH 4·5 cultures of cmk1 cells do display an active efflux of fluorescein, even though their Pdr12 levels are no higher than in CMK1+ cultures (Holyoak et al., 2000
). This indicates that the Cmk1 protein kinase maintains the Pdr12 transporter in an inactive state until there is the need for a catalysed acid efflux.
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The plasma membrane H+-ATPase in weak acid adaptation |
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According to the model in Fig. 1(a), acid influx will act to dissipate the pH, though not the charge (Z) component of the electrochemical potential at the plasma membrane. The same would also occur with the addition of a classical uncoupler such as 2,4-dinitrophenol (the effects of sorbate and 2,4-dinitrophenol on S. cerevisiae being remarkable in their similarity; Stratford & Anslow, 1996
). The extents to which increased H+-ATPase activity alone can counteract any intracellular acidification that accompanies weak acid influx (Fig. 1
) may be limited, since there is a finite limit to the extent that H+-ATPase action can enhance the charge component (Z) of the electrochemical potential (Z
pH). One way to avoid this problem is to ensure the movement of a charge that compensates for the charge on a H+-ATPase-extruded proton. Anion (XCOO-) exit from the cell, as through a membrane pore, could satisfy this requirement and could be driven by the membrane potential. However, the movement of this compensating charge in adapted S. cerevisiae cells is also satisfied with Pdr12-transporter-catalysed extrusion of an acid anion (Fig. 1b
). There might thus be two benefits of catalysed anion extrusion in organic-acid-stressed S. cerevisiae: (i) a lowering of intracellular acid levels; and (ii) movement of a charge that balances the charge on a H+-ATPase-extruded proton, thereby facilitating higher levels of catalysed proton extrusion. The combined actions of H+-ATPase and Pdr12 may therefore be needed for acid-stressed S. cerevisiae to restore homeostasis to the point where growth can resume (Fig. 1b
). This is undoubtedly extremely expensive in terms of energy consumption, at least 2 ATPs being consumed for each weak acid molecule that enters the cell (Fig. 1b
). This very high energy requirement of counteracting weak acid stress is reflected in the dramatic reductions in biomass yield for cultures grown in the presence of this stress (Warth, 1988
; Viegas & Sa Correia, 1991
; Verduyn et al., 1992
; Stratford & Anslow, 1996
; Piper et al., 1997
).
What is the role played by the other weak-acid-induced plasma membrane protein, Hsp30? Cells of the hsp30 mutant are considerably less sensitive to sorbate than pdr12 cells, revealing Hsp30 to be less important for weak acid resistance than Pdr12. With the loss of Hsp30 the sorbate activation of the H+-ATPase is enhanced, indicating that Hsp30 acts in some manner to limit the activation of H+-ATPase by stress (Piper et al., 1997 ). Also hsp30 mutant cells take longer to adapt to weak acid stress and, when stressed, have abnormally low ATP levels that might be a reflection of their excessive H+-ATPase activity. We have therefore suggested that Hsp30 might serve an energy conservation function in stressed cells (Piper et al., 1997
).
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Multiple plasma membrane transporters influence weak acid resistance |
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The sorbate resistance of S. cerevisiae is elevated with the loss of Pdr1, a transcription factor that regulates a number of the genes for ABC transporters (though not it appears PDR12) (Piper et al., 1998 ). A number of the Pdr1 target genes, including genes for ABC transporters other than Pdr12, may therefore counteract weak acid resistance. Thus weak acid adaptation, while it involves inducing Pdr12, may also require the downregulation of other plasma membrane transporters. The mRNA for Pdr5, a major determinant of drug resistance in yeast (Bauer et al., 1999
), has been observed to disappear in response to sorbate stress (Piper et al., 1998
).
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What is the PDR12-inducing signal? |
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What is the signalling mechanism leading to PDR12 induction? This is not known, but it appears not to require MAP kinase signalling pathways or Ca2+/calmodulin signalling mediated via calcineurin, since none of the appropriate mutants affect sorbate sensitivity (unpublished observations). Conceivably the inducing signal could simply be a high intracellular level of acid anion, acting through a direct binding to the relevant transcription factor.
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How do yeasts avoid a futile cycle of diffusional entry and active extrusion of organic acids? |
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The influences of oxygen on weak acid resistance |
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Z. bailii is a petite-negative yeast. Aerobic, weak-acid-stressed Z. bailii does not therefore segregate respiratory-deficient cells. Instead it is able to oxidatively degrade sorbate and benzoate and use these compounds as sole carbon source (Mollapour & Piper, 2001 ). In contrast, S. cerevisiae is unable to utilize benzoate as it lacks a benzoate 4-hydroxylase (Mollapour & Piper, 2001
). We recently isolated a small Z. bailii gene (ZbYME2) that, when heterologously expressed in S. cerevisiae, confers the ability to catabolize benzoate, sorbate and phenylalanine (Mollapour & Piper, 2001
). ZbYME2 encodes a protein with high (74%) sequence similarity to the N-terminal, mitochondrial matrix domain of the S. cerevisiae Yme2p (Leonhard et al., 2000
) and the product of ZbYME2 expressed heterologously in S. cerevisiae is also mitochondrial (unpublished data). Probably, therefore, ZbYME2 confers a broad-specificity monooxygenase function with benzoate 4-hydroxylase activity in the mitochondrion of Z. bailii, a function that may have been lost by its S. cerevisiae homologue, YME2.
We recently deleted the two ZbYME2 gene copies in the Z. bailii genome. The resulting mutant lacks any ability to utilize benzoate or sorbate as carbon sources and is, in addition, more sensitive to benzoate and sorbate inhibition on pH 4·5 glucose plates (Mollapour & Piper, 2001 ). ZbYME2 therefore contributes to the weak acid resistance of Z. bailii, probably as a mitochondrial monooxygenase that facilitates preservative degradation.
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Z. bailii and S. cerevisiae may not use identical strategies for acquiring weak organic acid resistance |
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Z. bailii might also have developed much more efficient ways of changing its cell envelope so as to limit the diffusional entry of the acid. This, in turn, will dramatically reduce any need for active extrusion of protons and acid anions. Indications that this may be the case come from observations that Z. bailii is much more resistant than S. cerevisiae to any short-term decrease in pHi induced by acetate (Arneborg et al., 2000 ). Also the trehalose induction of sorbate-stressed S. cerevisiae, symptomatic of glycolysis becoming inhibited at the Pfk step, is not observed with Z. bailii (Cheng et al., 1999
). Instead, conditions of sorbate and benzoate stress that lead to Pfk inhibition in S. cerevisiae tend to stimulate, rather than inhibit, Z. bailii glycolytic flux (Cole, 1987
) and lead to no induction of trehalose (Cheng et al., 1999
). However, it is unclear if this represents the glycolytic flux of Z. bailii being more resistant to acid inhibition, or a reduced permeation of the acid into Z. bailii cells relative to the cells of S. cerevisiae. When the benzoate sensitivities of several different yeast species were compared, these were, to a rough approximation, inversely proportional to rates of diffusional entry of propionate into the cells (Warth, 1989
). Reducing diffusional entry of the acid into the cells is therefore probably a key mechanism of resistance.
The strategy adopted by S. cerevisiae, whereby resistance is conferred largely through high levels of proton and acid extrusion (see Fig. 1b), potentially has one fundamental flaw. Active (mainly Pdr12-catalysed) anion extrusion at the plasma membrane can only export the weak acid as far as the periplasm (Fig. 1b
). From there, undissociated acid can possibly just as readily diffuse back into the cell as out through the cell wall. Ensuring that the initial diffusion of the acid through the cell wall or membrane is much more restricted, so that lower amounts initially reach the periplasm or cytosol, should be a much better strategy for achieving resistance. It is conceivable that Z. bailii puts more reliance on the latter strategy and that therein lies the secret of its extreme weak acid resistance. This would explain why acid-adapting Z. bailii has no apparent need for any dramatic induction of a weak acid transporter (Fig. 2
) and suffers no reductions in pHi with acetate challenge.
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ACKNOWLEDGEMENTS |
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