Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid

The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.     

Glucose respiration and fermentation in Zygosaccharomyces bailii and Saccharomyces cerevisiae express different sensitivity patterns to ethanol and acetic acid

In the yeast Zygosaccharomyces bailii ISA 1307, respiration and fermentation ofglucose were exponentially inhibited by ethanol, both processes displaying similar sensitivity tothe alcohol. Moreover, the degree of inhibition on fermentation was of the same magnitude as thatreported for Saccharomyces cerevisiae. Acetic acid also inhibited these two metabolicprocesses in Z. bailii , with the kinetics of inhibition again being exponential. However,inhibition of fermentation was much less pronounced than in S. cerevisiae. The valuesestimated with Z. bailii for the minimum inhibitory concentration of acetic acid rangedfrom 100 to 240 mmol l⁻¹ total acetic acid compared with values of near zeroreported for S. cerevisiae. The inhibitory effects of acetic acid on Z. bailii were notsignificantly potentiated by ethanol.     

Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments.

Cells of Zygosaccharomyces bailii ISA 1307 grown in a medium with acetic acid, ethanol, or glycerol as the sole carbon and energy source transported acetic acid by a saturable transport system. This system accepted propionic and formic acids but not lactic, sorbic, and benzoic acids. When the carbon source was glucose or fructose, the cells displayed activity of a mediated transport system specific for acetic acid, apparently not being able to recognize other monocarboxylic acids. In both types of cells, ethanol inhibited the transport of labelled acetic acid. The inhibition was noncompetitive, and the dependence of the maximum transport rate on the ethanol concentration was found to be exponential. These results reinforced the belief that, under the referenced growth conditions, the acid entered the cells mainly through a transporter protein. The simple diffusion of the undissociated acid appeared to contribute, with a relatively low weight, to the overall acid uptake. It was concluded that in Z. bailii, ethanol plays a protective role against the possible negative effects of acetic acid by inhibiting its transport and accumulation. Thus, the intracellular concentration of the acid could be maintained at levels lower than those expected if the acid entered the cells only by simple diffusion.     

Caffeine induces macroautophagy and confers a cytocidal effect on food spoilage yeast in combination with benzoic acid

Weak organic acids are an important class of food preservatives that are particularly efficacious towards yeast and fungal spoilage. While acids with small aliphatic chains appear to function by acidification of the cytosol and are required at high concentrations to inhibit growth, more hydrophobic organic acids such as sorbic and benzoic acid have been suggested to function by perturbing membrane dynamics and are growth-inhibitory at much lower concentrations. We previously demonstrated that benzoic acid has selective effects on membrane trafficking in Saccharomyces cerevisiae. Benzoic acid selectively blocks macroautophagy in S. cerevisiae while acetic acid does not, and sorbic acid does so to a lesser extent. Indeed, while both benzoic acid and nitrogen starvation are cytostatic when assayed separately, the combination of these treatments is cytocidal, because macroautophagy is essential for survival during nitrogen starvation. In this report, we demonstrate that Zygosaccharomyces bailii, a food spoilage yeast with relatively high resistance to weak acid stress, also exhibits a cytocidal response to the combination of benzoic acid and nitrogen starvation. In addition, we show that nitrogen starvation can be replaced by caffeine supplementation. Caffeine induces a starvation response that includes the induction of macroautophagy, and the combination of caffeine and benzoic acid is cytocidal, as predicted from the nitrogen starvation data.     

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

A TRPA1-dependent mechanism for the pungent sensation of weak acids

Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO(2), through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca(2+) microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca(2+)-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.     

Mechanism of Resistance of Saccharomyces bailii to Benzoic, Sorbic and Other Weak Acids Used as Food Preservatives

Saccharomyces bailii grows in the presence of high concentrations of sorbic, benzoic and other short-chain monocarboxylic acids commonly used as preservatives. Starved cells concentrate these acids intracellularly, approximately as expected from the pH of the ceil and the p K _a of the acid. On addition of glucose, the intracellular content of preservative is considerably reduced. The glucose effect is sensitive to metabolic inhibitors, and anaerobic respiration is stimulated by the preservatives. The ability to maintain a low intracellular concentration of any of the preservatives tested is induced by growth in the presence of sorbic or benzoic acid and less effectively by butyric or acetic acid. Both induced and uninduced cells are permeable to benzoic and butyric acids. Benzoate and sorbate are not metabolized at a rate significant with respect to the permeation rate. Resistance to these preservatives apparently results primarily from an inducible, energy requiring system which transports preservative from the cell.     

酱香型白酒酿造拜耳接合酵母生理代谢特征及其与地衣芽孢杆菌相互作用

【目的】拜耳接合酵母(Zygosaccharomyces bailii)是酱香型白酒酿造过程优势菌株。通过研究拜耳接合酵母酿造相关生理代谢特征及其与酿造环境中其它功能菌株的相互作用,探索其在白酒酿造过程中的贡献。【方法】从酱香酒酿造中筛选一株性能优良的拜耳接合酵母,比较其与模式菌株(Z.bailii,ATCC 58445)的生理代谢特征。通过组合发酵研究其与产酱香特征风味细菌地衣芽孢杆菌的相互作用。【结果】从酱香型酒醅中筛选得到一株性状优良的拜耳接合酵母(Z.bailii 15),可耐受p H 2.0和37°C高温及8%酒精浓度(体积比),较模式株更适应酿造环境,酒精产量(33.58 g/L)也远高于模式株(19.04 g/L),且与酱香型白酒酿酒酵母MT1(34.29 g/L)相当。该菌可产多种风味物质,与模式株相比,独特产生法呢醇、十二醇、2-壬醇、2-乙基己醇、癸酸、月桂酸、辛酸、辛酸乙酯、苯乙酮、4-叔丁基苯酚。共培养体系中,30°C条件下地衣芽孢杆菌对拜耳接合酵母生长影响不大,而在37°C地衣芽孢杆菌抑制拜耳接合酵母生长。此外,地衣芽孢杆菌对拜耳接合酵母乙醇转化率有促进作用,共培养体系风味物质种类及含量也都受到很大影响。【结论】拜耳接合酵母在酱香型白酒酿造体系中产酒精、产风味方面表现优异,对酱香型白酒生产具有重要贡献。     

Adaptive response to acetic acid in the highly resistant yeast species Zygosaccharomyces bailii revealed by quantitative proteomics

Zygosaccharomyces bailii is the most tolerant yeast species to acetic acid-induced toxicity, being able to grow in the presence of concentrations of this food preservative close to the legal limits. For this reason, Z. bailii is the most important microbial contaminant of acidic food products but the mechanisms behind this intrinsic resistance to acetic acid are very poorly characterized. To gain insights into the adaptive response and tolerance to acetic acid in Z. bailii, we explored an expression proteomics approach, based on quantitative 2DE, to identify alterations occurring in the protein content in response to sudden exposure or balanced growth in the presence of an inhibitory but nonlethal concentration of this weak acid. A coordinate increase in the content of proteins involved in cellular metabolism, in particular, in carbohydrate metabolism (Mdh1p, Aco1p, Cit1p, Idh2p, and Lpd1p) and energy generation (Atp1p and Atp2p), as well as in general and oxidative stress response (Sod2p, Dak2p, Omp2p) was registered. Results reinforce the concept that glucose and acetic acid are coconsumed in Z. bailii, with acetate being channeled into the tricarboxylic acid cycle. When acetic acid is the sole carbon source, results suggest the activation of gluconeogenic and pentose phosphate pathways, based on the increased content of several proteins of these pathways after glucose exhaustion.     

Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2)

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.     

Distinct Effects of Sorbic Acid and Acetic Acid on the Electrophysiology and Metabolism of Bacillus subtilis

Sorbic acid and acetic acid are among the weak organic acid preservatives most commonly used to improve the microbiological stability of foods. They have similar pK_a values, but sorbic acid is a far more potent preservative. Weak organic acids are most effective at low pH. Under these circumstances, they are assumed to diffuse across the membrane as neutral undissociated acids. We show here that the level of initial intracellular acidification depends on the concentration of undissociated acid and less on the nature of the acid. Recovery of the internal pH depends on the presence of an energy source, but acidification of the cytosol causes a decrease in glucose flux. Furthermore, sorbic acid is a more potent uncoupler of the membrane potential than acetic acid. Together these effects may also slow the rate of ATP synthesis significantly and may thus (partially) explain sorbic acid''s effectiveness.     

Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii

Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.     

Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine

The most important factors affecting microbial growth in an alcoholic beverage are the ethanol content and the low pH. The effectiveness of different organic acids in conjunction with ethanol concentration in controlling growth of yeasts was determined for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces lambicus, Pichia anomala, Zygosaccharomyces bailii, Saccharomycodes ludwigii and Kluyveromyces thermotolerans in malt extract broth (MEB). The results are summarized as undissociated concentrations of different acids required to inhibit growth of yeasts in MEB containing 10% (v/v) ethanol. About half the amount of undissociated malic or tartaric acid is necessary for inhibition of the yeasts, compared with acetic and lactic acid but the concentrations of acid necessary to inhibit growth were generally very high and unrealistic in wines for controlling growth of most of the yeasts tested. All the yeasts tested were able to grow in acidified non- or low alcoholic beverages but, at higher ethanol concentrations, Sacch. cerevisiae and Zygosacch. bailii have the greatest spoilage potential.     

Weak acid and alkali stress regulate phosphatidylinositol bisphosphate synthesis in Saccharomyces cerevisiae

Weak organic acids are used as food preservatives to inhibit the growth of spoilage yeasts, including Saccharomyces cerevisiae. Long-term adaptation to weak acids requires the increased expression of the ATP-binding cassette transporter Pdr12p, which catalyses the active efflux of the weak acids from the cytosol; however, very little is known about the signalling events immediately following application of weak acid stress. We have investigated the effects of weak acids on two stress-responsive signalling molecules, PtdIns(3,5)P2 and PtdIns(4,5)P2, which in S. cerevisiae are synthesized by Fab1p and Mss4p respectively. At low extracellular pH, benzoic acid, sorbic acid and acetic acid all cause a transient reduction in PtdIns(3,5)P2 accumulation and a more persistent rise in PtdIns(4,5)P2 levels. The increase in PtdIns(4,5)P2 levels is accompanied by a reorganization of the actin cytoskeleton. However, changes in PtdInsP2 levels are independent of weak acid-induced Pdr12p expression. In contrast, changing the extracellular medium to alkaline pH provokes a prolonged and substantial rise in PtdIns(3,5)P2 levels. As PtdIns(3,5)P2 synthesis is required for correct vacuole acidification, it is possible that levels of this molecule are modulated to maintain intracellular pH homoeostasis in response to weak acid and alkali stresses. In conclusion, we have expanded the repertoire of stress responses that affect PtdInsP2 levels to include weak acid and alkali stresses.     

Reduction of volatile acidity of wines by selected yeast strains

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose-low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose-high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.     

Killer toxin-secreting double-stranded RNA mycoviruses in the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii.

Killer toxin-secreting strains of the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii were shown to contain linear double-stranded RNAs (dsRNAs) that persist within the cytoplasm of the infected host cell as encapsidated virus-like particles. In both yeasts, L- and M-dsRNAs were associated with 85-kDa major capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was shown to be encapsidated by a 35-kDa coat protein. Although Northern (RNA) blot hybridizations indicated that L-dsRNA from Z. bailii is a LA species, additional peptide maps of the purified 85-kDa capsid from Z. bailii and the 88- and 80-kDa major coat proteins from K1 and K28 killer viruses of Saccharomyces cerevisiae revealed distinctly different patterns of peptides. Electron microscopy of purified Z. bailii viruses (ZbV) identified icosahedral particles 40 nm in diameter which were undistinguishable from the S. cerevisiae killer viruses. We demonstrated that purified ZbVs are sufficient to confer the Z. bailii killer phenotype on transfected spheroplasts of a S. cerevisiae nonkiller strain and that the resulting transfectants secreted even more killer toxin that the original ZbV donor strain did. Curing experiments with ZbV-transfected S. cerevisiae strains indicated that the M-dsRNA satellite from Z. bailii contains the genetic information for toxin production, whereas expression of toxin immunity might be dependent on Z-dsRNA, which resembles a new dsRNA replicon in yeasts that is not dependent on an LA helper virus to be stably maintained and replicated within the cell.     

Zygosaccharomyces kombuchaensis: the physiology of a new species related to the spoilage yeasts Zygosaccharomyces lentus and Zygosaccharomyces bailii

Zygosaccharomyces kombuchaensis was recently discovered in the 'tea fungus' used to make fermented tea. Z. kombuchaensis was shown by ribosomal DNA sequencing to be a novel species, and a close relative of Zygosaccharomyces lentus, from which it could not be distinguished by conventional physiological tests. Z. lentus was originally established as a new taxon by growth at 4 degrees C, sensitivity for heat and oxidative stress, and lack of growth in aerobic shaken culture at temperatures above 25 degrees C. Subsequent analysis of Z. kombuchaensis reveals that this species shares these unusual characteristics, confirming its close genealogical relationship to Z. lentus. Detailed physiological data from a number of Z. kombuchaensis and Z. lentus strains clearly demonstrate that these two species can in fact be distinguished from one another based on their differing resistance/sensitivity to the food preservatives benzoic acid and sorbic acid. The spoilage yeasts Zygosaccharomyces bailii and Z. lentus are resistant to both acetic acid and sorbic acid, whereas Z. kombuchaensis is resistant to acetic acid but sensitive to sorbic acid. This would indicate that Z. kombuchaensis strains lack the mechanism for resistance to sorbic acid, but possess the means of resistance to acetic acid. This observation would therefore suggest that these two resistance mechanisms are different, and that in all probability acetic and sorbic acids inhibit yeast growth by different modes of action. Z. kombuchaensis strains were also sensitive to benzoic acid, again suggesting inhibition dissimilar from that to acetic acid.     

Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to sulphite

Sulphite inhibited growth of all four yeasts studied, Zygosaccharomyces bailii NCYC 563 being most sensitive and Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S. cerevisiae than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S. cerevisiae TC8 and Z. bailii NCYC 563, but to a lesser extent of S. cerevisiae NCYC 431 and Z. bailii NCYC 1427, was inhibited when mid exponential-phase cultures were supplemented with 1.0 or 2.0 mM-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.     

Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (Delta(pH)), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.