The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin*

The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin.

Killer phenomena are widespread in yeasts. Killer toxins are proteins or glycoproteins that are lethal to sensitive strains of the same species and a different variety of other yeast genera. In this line, attention has focused mainly on the characterization of killer toxins from Saccharomyces cerevisiae (K1, K2, and K28) followed more recently by the investigation of yeasts such as Kluyveromyces lactis, Zygosaccharomyces bailii, Hanseniaspora uvarum, Pichia membranifaciens, Debaryomyces hansenii, Schwanniomyces occidentalis, etc. (1)(2)(3)(4)(5)(6)(7).
P. membranifaciens CYC 1106 is a strain originally isolated from fermenting olive brines with pronounced killer activity against a variety of yeast species (8) and fungi (9). Once the protein nature of the toxin produced was established, the secreted protein was purified from the supernatant of growing cultures of P. membranifaciens in the early stationary phase. Previous biochemical studies on the PMKT 2 mechanism of killing of sensitive yeast cells indicated that PMKT is an 18-kDa protein that interacts with the (1 3 6)-␤-D-glucans of the cell wall of sensitive yeasts (6,10). Recently the killing mechanism of this killer toxin has been elucidated (10). Regardless of certain possible additional effects, the killer toxin of P. membranifaciens CYC 1106 acts by disrupting plasma membrane electrochemical gradients. The death of sensitive cells in the presence of killer toxin is characterized by a leakage of com-mon physiological ions through non-regulated ion channels in the plasma membrane causing a discharge of cellular membrane potential and changes in ionic homeostasis in a way comparable to that of certain killer toxins (K1) (11). Non-selective channel formation has been suggested to be the cytotoxic mechanism of action of PMKT (10).
Yeasts must cope with different adverse environmental conditions, including heat shock, oxidative stress, high osmolarity, extreme pH values, nutrient availability, and toxins from plants, fungi, or bacteria as well as heavy metals and different xenobiotics. Yeasts have therefore adapted to growth under these conditions by developing a variety of protective mechanisms ranging from general stress responses to highly specific regulatory pathways. A variety of changes in the environment activate multiple mitogen-activated protein kinase cascades, which convert these signals into appropriate metabolic responses (12,13). Five mitogen-activated protein kinase cascades that coordinate intracellular activities in response to rapidly changing environmental conditions have been characterized (14). Because of our knowledge about its sequenced genome and the availability of powerful genetic tools, S. cerevisiae has been a particularly valuable model system for studying such responses. Of particular significance for this unicellular eukaryote is its response to changes in the osmolarity of the medium. Yeast cells are able to detect and respond to changes in osmolarity by two independent osmosensors: Sln1p and Sho1p (15)(16)(17). These membrane-bound proteins activate the so-called high osmolarity glycerol (HOG) mitogenactivated protein kinase pathway, and this results in a rise in the intracellular glycerol concentration to adapt the cellular osmotic pressure and to prevent the loss of water (18 -21). These survival mechanisms must operate within the first seconds after a sudden osmotic shift because water loss or uptake occurs very fast (22). The stimulation of glycerol synthesis is achieved by increasing the transcription of genes such as GPD1 (encoding glycerol-3-phosphate dehydrogenase), GPP2 (glycerol-3-phosphate phosphatase), HSP12 (heat shock protein), CTT1 (cytosolic catalase T), YAP4 (yeast activator protein), STL1 (encoding a member of the hexose transporter family of the major facilitator superfamily), etc. (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Regardless of their effects on osmolarity, high concentrations of certain ions, such as lithium or sodium, may be toxic for yeast cells because they block several metabolic reactions (34). In this case, the transcriptional activation of the ENA1 gene, which encodes a P-type ATPase, is required for the efflux of sodium ions (35,36). Two plasma membrane proteins, Psr1p and Psr2p, are essential for an efficient sodium ion stress response through transcriptional activation of ENA1 (37). Apart from the HOG-dependent induction during sodium ion stress, ENA1 transcription is also induced by another independent pathway mediated by the calcium/calmodulin-dependent phosphatase calcineurin (38 -40).
The aim of this study was to determine the global gene expression responses of S. cerevisiae to the killer toxin produced by P. membranifaciens CYC 1106 with a view to gaining insight into the mechanisms and processes underlying the killing of sensitive yeast cells. Here for the first time we report evidence that the transcriptional response of S. cerevisiae to the presence of PMKT is very similar to the response of cells undergoing adaptation to ionic or osmotic changes in the cellular environment. Western analysis of the signaling through the HOG pathway revealed a Hog1p phosphorylation in response to PMKT. The analysis of arrayed ORFs was complemented and extended by the fact that a high proportion of deletion mutants of genes whose expression was observed to be affected by PMKT had altered sensitivity phenotypes.
Killer Toxin Assay-We assayed for killer toxin sensitivity with a diffusion test using 6-mm-diameter antibiotic assay AA Whatman paper discs on buffered YMA-MB seeded with the selected S. cerevisiae mutant strains in parallel with the wild-type strain. Incubation was carried out at 20°C because killer factor is rapidly inactivated at temperatures above 25°C. The diameter of the inhibition zone was used as a measure of sensitivity to the killer toxin. For each mutant, sensitivity was tested up to three times, and the results were compared with those from the wild-type strain ((mutant inhibition area)/(wild-type inhibition area) ϫ 100).
Measurement of Cell Death-S. cerevisiae BY4743 cells were grown to logarithmic phase in buffered YMB medium, collected, and subsequently resuspended in the same medium containing killer activity (1,205 AU/ml). The final cell concentration was 10 6 cells/ml. A control with heat-inactivated (5 min at 75°C) killer toxin was run in parallel. Aliquots were taken periodically, and additional 10-fold dilutions were made serially to a final dilution of 10 Ϫ4 . Four volumes of 50 l each were used for plating on YMA medium. The colonies were counted after 48 h of growth at 30°C.
Experimental Design and RNA Isolation-For RNA isolation, time course experiments of the killing process, using S. cerevisiae BY4743, were performed as three independent biological repeats. Approximately 200 ml of asynchronously grown cells were cultured in YMB medium (buffered with 100 mM sodium citrate-phosphate, pH 4.0) at 20°C, shaking at 125 rpm until an A 600 of 0.5 was reached. Ten milliliters of cells were harvested by centrifugation (14,000 rpm for 1 min at 4°C) and snap-frozen in liquid nitrogen (at this point cells were collected for determination of expression profiles under basal conditions). Immediately afterward the rest of the culture was exposed to the killer toxin (1,205 AU/ml), and samples of 10 ml each were collected at 0, 15,30,45,60,90, and 120 min. Then samples for RNA extraction were harvested and frozen as above. Total RNA was extracted using the hot acid phenol method (42).
Microarray Hybridization, Scanning, and Data Acquisition-Based on the results on cell death, measured by plating, reverse transcription (Superscript, Invitrogen) was performed for 40 g using total RNA extracted from cells after 45 min of exposure to PMKT. After the Cy3and Cy5-dCTP-labeled cDNAs had been hybridized onto glass DNA microarrays containing 100% of all known and predicted S. cerevisiae genes, 40 l of the mixed cDNA solution were allowed to hybridize to the microarrays at 62°C for 12 h (Agilent hybridization chamber, Agilent Technologies, Palo Alto, CA). When the hybridization process was completed, the microarrays were washed with buffer A (0.1% SDS, 0.6ϫ SSC) at 50°C followed by a 5-min wash in buffer B (0.03ϫ SSC) at 20°C. Microarrays were scanned with Agilent scanner G2565BA (Agilent Technologies), and microarray images were analyzed with Agilent Feature Extraction software, version 7.5 (43,44). If expression ratios were Ͼϩ3.0 (up-regulated) or Ͻ-2.0 (down-regulated) in four different arrays, the corresponding genes were then considered to be expressed differentially, and the average ratio from the change in expression was calculated and treated as the true ratio between the different arrays. Only genes with constant expression ratios between different experiments were considered. It must be taken into account that a large proportion of the genes (those between ϩ3.0-fold induction and Ϫ2.0-fold repression) responded, according to our results, in an apparently random manner. It is known that when a stimulus occurs the nature of regulated transcripts changes over time, suggesting that different functions need to be activated at different time points. The origin of this variability is unknown but may be due to events that affect cells at different moments of the killing process but not simultaneously for the whole PMKT-treated yeast population. Other possibilities are small differences in toxin purity (high performance liquid chromatographytested), media composition, or cell age. In light of this, we decided to confirm these results with quantitative real time PCR under the assumption that the constantly expressed genes were the main ones involved in the underlying process of killing.
Real Time Quantitative PCR-Total RNA was obtained from cells at a time point of 45 min and then treated with DNase I (Sigma). cDNA was synthesized from samples of 1.5 g of total RNA making use of the random primer procedure and the RETRoscript kit (Ambion, Austin, TX) following the manufacturer's instructions. Q-PCR was performed on an ABI PRISM 7900 HT (Applied Biosystems, Warrington, UK) device using the SYBR Green PCR Master Mix (Applied Biosystems) and specific oligonucleotide primers at a concentration of 300 nM. Matching oligonucleotide primers were designed using the Primer Express software (PerkinElmer-Applied Biosystems) and Oligo software (Oligo Analizer) and synthesized by Sigma-Genosys (Sigma-Genosys, Ltd). Amplification of cDNA was performed over 40 cycles. The first cycle was performed at 95°C for 10 min. Cycles 2-40 were performed at 95°C for 15 s followed by 60°C for 1 min. Each assay was performed in quadruplicate. For normalization of cDNA loading, all samples were run in parallel with a housekeeping gene (IPP1, inorganic pyrophosphatase). The specificity of PCR amplifications from the different sets of oligonucleotide primers was examined routinely by agarose gel electrophoresis.
Northern Blot Analysis of the PMKT-dependent Genes-According to the results from microarrays, confirmed by Q-PCR, some osmoresponsive genes (GPD1 and GPP2) as well as CTT1 and HSP12, two general stress-responsive genes; PDR12 and TRK2, two genes highly induced at low pH; and ENA1, a gene known to be induced under high salt conditions, were studied by Northern blotting (32). To determine the extent of induction or repression of gene expression, all signals were compared with IPP1, which encodes inorganic pyrophosphatase, whose expression is not affected by osmotic stress (33). Total RNA was isolated from cells (BY4743 and hog1⌬) at the selected time points (0, 15, 30, 45, 60, 90, and 120 min), separated by formaldehyde gels, and transferred onto nylon membranes (Hybond XL, Amersham Biosciences). Probes were generated by PCR from chromosomal DNA of BY4743. PCR probes of GPD1, GPP2, CTT1, HSP12, PDR12, TRK2, ENA1, and IPP1 were radiolabeled by random priming with [␣-32 P]ATP (MegaPrime, Amersham Biosciences), G-50-purified, and hybridized overnight at 65°C in hybridization buffer (7% (w/v) SDS, 0.25 M sodium phosphate buffer, pH 7.5, 1 mM EDTA, and 1% (w/v) bovine serum albumin). The blots were washed (20 mM sodium phosphate, pH 7.5, 0.1% (w/v) SDS, and 1 mM EDTA), and the signal was detected either by exposure to radiosensitive film (Biomax MR, Eastman Kodak Co.). Values were normalized by comparison with IPP1 signals.
Western Blotting of Hog1p and Hog1p Phosphorylation-Separation of total soluble protein (30 g of total protein), isolated as described previously (45,46), was accomplished on 12% polyacrylamide gels (Bio-Rad), whereas subsequent Western blotting of Hog1p and phosphorylated Hog1p was performed according to standard procedures on polyvinylidene difluoride membranes (Bio-Rad). Briefly total soluble protein was isolated from S. cerevisiae BY4743 strain grown in buffered YMB medium after 0, 5, 10, 15, 30, 45, and 60 min of exposure to PMKT. Dual phosphorylation of Hog1p was determined using an anti-dually phosphorylated (Thr-174 and Tyr-176) p38 antibody (New England Biolabs, Beverly, MA). Hog1p was detected using an anti-C-terminal Hog1p antibody (Yc20, catalog number sc-6815, Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was visualized using an ECL kit (Amersham Biosciences) after the binding of a horseradish peroxidaseconjugated secondary antibody (Sigma).
Glycerol Determination-Cells (BY4743 and hog1⌬) grown overnight in YMB medium (buffered with 100 mM sodium citrate-phosphate, pH 4.0) at 20°C were diluted to an A 600 of 0.3 and grown for 4 h at 20°C. Cells were then subjected to PMKT (1,205 AU/ml). At time points (0, 0.5, 1, 1.5, 2, 2.5, 3, and 4 h) and in triplicate, 10-ml samples were taken for glycerol and dry weight determinations. Intracellular glycerol was determined by filtration with glass microfiber filters (Whatman GF/C), measuring the glycerol released by boiling the filters for 10 min. Dry weight was determined by drying filters with cells at 80°C for 16 h. To determine the glycerol concentration of the extracellular medium during PMKT treatment, 1-ml samples of the treated cultures were centrifuged, and the supernatant was used for glycerol assays. Glycerol was determined with a glycerol measurement kit (catalog number 148270, Roche Applied Science).

Genome-wide Gene Expression
Profiles-To examine the gene expression response to PMKT a time course study of the killing process was carried out. After exposure of asynchronously grown cells to PMKT at times between 0 and 8 h (not shown), cell death was observed to begin after 1 h. The cell death rate in the presence of PMKT (1,205 AU/ml) was 0.22 h Ϫ1 . From these death kinetics, a PMKT dosage of 1,205 AU/ml and sampling times of 0 min (untreated control) and 45 min (treated cells) after exposure to PMKT were chosen for mRNA isolation and subsequent microarray studies and Q-PCR.
Genome-wide gene expression profiles of S. cerevisiae cultures grown asynchronously were examined after exposure to PMKT using wholegenome microarrays and Q-PCR. Similar results were observed for mRNA expression levels using both techniques. Validation of microarray results with Q-PCR was done to verify that array data were not the result of problems inherent to the array technology. The grouped distribution of genes that were either up-or down-regulated after exposure to PMKT (TABLES ONE and TWO) provided information pertinent to the killing mechanism of PMKT and to the response of S. cerevisiae to the toxin at the molecular level. The mRNA level of a total of 99 genes was at least 3-fold higher after the addition of PMKT (TABLE ONE). Forty-seven genes were observed to exhibit 2-fold or higher repression levels after 45 min of exposure to PMKT (TABLE TWO).
In our results, the functional group of up-regulated genes with the highest representation (31 genes) was the group of genes related to signal transduction, gene expression, transcription, and RNA processing, showing that there is an important underlying cellular response to PMKT. In this group of genes TFS1, NAM8, YAP1, KKQ8, SCH9, STE20, PBS2, MED2, SRB4, HAL9, and GAL11 had the highest expression levels. Between them, YAP1, XBP1, SCH9, and HAL9 were directly related to cell stress or salt tolerance.
Heat shock proteins (HSPs) were found to be up-regulated in response to PMKT (HSP12, HSP104, HSP82, HSP26, DDR2, SSA2, SSB2, and SSA1). Furthermore we found a severe induction of CTT1 expression in the presence of PMKT, showing that a general stress response occurred in the presence of the toxin (TABLE ONE and Fig. 1). PMKT also induced YAP1, which specifically mediates oxidative stress responses and appears to be responsible for the rapid establishment of a transient transcriptional response.
Northern Blots-Subsequent time course studies of the expression of some induced genes using Northern blot ( Fig. 1) revealed that the process of killing occurred after the shift in mRNA synthesis had taken place, showing that changes in mRNA levels in response to PMKT occurred 30 -45 min before the process of killing could be observed quantitatively by plating (not shown). Then mRNA levels did not decrease with time to restore the initial expression levels in a process probably related to cell death (with cells unable to regulate their mRNA levels) or to a permanent stimulation of the remaining active cells. The hog1⌬ mutant had a similar response to PMKT, but the amount of mRNA of many of the induced genes were severalfold lower in comparison with the wildtype strain (ENA1, 18-fold lower; GPP2, 9-fold; TRK2, GPD1, and HSP12, 5-fold; CTT1, 4-fold; and PDR12, 2.5-fold).
Glycerol Production and Leakage-Our results showed that treatment with PMKT induces glycerol synthesis, and this process was indeed observed in the first 30 min after toxin addition. The same result was observed in the hog1⌬ mutant, which is hypersensitive to PMKT, The Transcriptional Response to PMKT DECEMBER 23, 2005 • VOLUME 280 • NUMBER 51

Genes induced by more than 3-fold after a PMKT exposure
Genes are sorted into functional categories. PMKT activity against deletion mutants of these genes is shown. WT, wild type; RT, real time; PAS, Per-ARNT-Sim; MAP, mitogen-activated protein; GABA, ␥ -aminobutyric acid; ABC, ATP-binding cassette; SRB, suppressor of RNA polymerase B; MIP, major intrinsic protein.   that was also observed to accumulate glycerol but less so than the wildtype strain (Fig. 2). Thus, the intracellular amount of glycerol was observed to decrease during PMKT treatment.
Hog1p Phosphorylation-HOG pathway signaling, which is manifested by the phosphorylation of Hog1p, was rapidly observed when cells were shifted to PMKT exposure. The phosphorylation of Hog1p was observed to occur in the first 10 min of exposure to PMKT, and only the first samples (0 and 5 min) failed to show a phospho-Hog1p signal. During the rest of the experiment the phospho-Hog1p signal was observed, pointing to permanent activation of the signaling pathway (Fig. 3).
Yeast Deletion Mutants and Sensitivity to PMKT-As may be seen in TABLE ONE, there is a clear relationship between the level of induction of up-regulated genes and the presence of hypersensitive phenotypes in the mutants studied. In contrast, the particular case of hog1⌬ mutant constructed for this study, found to be hypersensitive (180%) but with a very low up-regulation of its expression (0.4 -0.8-fold expression), was very significant. Deletion mutants of genes known to be involved in a response to osmotic stress (YAP1, HAL9, GPP1, GPP2, GPD1, PSR1, IST1, NHA1, ENA1, SAT2, TRK2, HSP12, CTT1, and CSF1) were highly hypersensitive to PMKT, and the glycerol synthesis-related mutants formed the group with the highest levels of hypersensitivity. On the other hand, there was no clear relationship between the deletion mutants obtained for down-regulated genes and the degree of toxin sensitivity (TABLE TWO). In general terms, a marked tendency to obtain PMKT-resistant mutants (47%) and wild-type phenotypes (36%) was observed when repressed genes were deleted.

DISCUSSION
The response of the yeast S. cerevisiae to PMKT was studied by genomic expression profiling. Yeast genome scale DNA microarrays have been used to study global responses to chemical agents, antifungal agents, and environmental stresses (25, 29, 49 -51). The gene expression response to PMKT was examined taking into account that the killing process of sensitive cells started 1 h after toxin addition in a process that supposedly involves several steps of the toxin mechanism of action. Because the binding of PMKT to cell wall receptors, (136)-␤-D-glucans, occurs in the first 2-3 min after toxin addition (6), the observed lag phase, which is necessary for changes in cell viability to be observed, probably involves several events (Fig. 4): adhesion to membrane receptors; formation of ion channels in the plasma membrane (10); metabolic and genetic changes, such as the dissipation of transmembrane electrochemical gradients (9) or the induction of stress response elements; and the formation of large membrane pores that allow the passage of large molecules either simultaneously to or sequentially with the formation of ion channels (10,11,52).
Correlation with Known Yeast Stress Responses-Comparison of the genes up-regulated in response to PMKT (TABLE ONE) and those induced by osmotic stress (33) suggests that, with some exceptions, the genes induced in both cases would be similar. Therefore, the majority of PMKT-induced genes must encode proteins known or predicted to be involved in the osmotic response, thus indicating an underlying response to intracellular osmotic changes in response to the toxin. PMKT-induced genes include genes such us CTT1, HSP12, GPD1, GPP2, TRK2, PDR12, ENA1, YAP1, XBP1, SCH9, HAL9, and STL1 that are found to be directly related to cell stress or salt tolerance. Similarities were also found with the response of S. cerevisiae to polyene antibiotics such as amphotericin B and nystatin (51).
Under environmental stress or treatment with certain polyene antibiotics it is very common to observe a reduction in the expression levels of some ribosomal proteins (29,51,53). This reduction is thought to be involved in the maintenance of a basal level of protein synthesis and in energy utilization to be redirected for increased expression of the genes involved in protective responses under these conditions. Our results indicated that mitochondrial ribosomal protein gene levels were not severely repressed and that nuclear ribosomal protein genes were not highly repressed (less than 2-fold repression). This difference in the genomic response to PMKT must be taken into account when the mechanism of killing is discussed because such differences between environmental stress and the action of polyene antibiotics could differentiate the underlying mechanisms of stress caused by different toxins, antibiotics, heavy metals, radiation, etc. These differences could plausibly be attributed to different observations over time of two similar processes, and hence another possibility is that there would be a different description of a succession of biochemical categories that are progressively up-regulated. This is justified because early stress responses usually affect mainly nucleotide and protein biosynthetic pathways and are different from later responses, which include intracellular protein and metabolite transport activities and increased energy consumption for metabolic and ion homeostasis. Transcription after prolonged stress also exemplifies ascending functions in cell rescue, aging (cell death), and defense-related roles.
Many induced genes during the response to PMKT were observed in the category of ion, sugar, amino acid, or multidrug transporters (TABLE ONE). Among the ion transporters known to play a role in sodium detoxification, several transcripts were up-regulated (47,48). It  is known that the plasma membrane is the target of some killer toxins such as K1 (3,11,54), and the results of previous investigations exploring PMKT are consistent with the idea that the effect induced by the toxin lies in an alteration of the permeability of the yeast membrane (10). The leakage of micronutrients and ions induced by the formation of unregulated channels could generate an increased gene expression of transmembrane transporters such us ENA1, TRK2, PHO89, ZRT1, and PDR12 and compensate to some extent the deleterious effects of PMKT over a short period of time. During this time increased gene expression could allow efficient uptake of ions or nutrients from the extracellular environment to counteract the leakage caused by PMKT (10). ENA1/PMR2 encodes a plasma membrane Na ϩ export pump required for growth in the presence of Na ϩ or Li ϩ (35). The expression of ENA1 is controlled in a complex way by sodium stress, glucose repression, and osmotic stress. The monovalent cations K ϩ and Na ϩ share a common uptake system in S. cerevisiae. When challenged with growth-inhibitory concentrations of Na ϩ , yeast cells change the kinetic properties of the K ϩ transport system, increasing its affinity for K ϩ to control the influx of Na ϩ . This response is dependent on a functional Trk high affinity potassium transporter encoded by the TRK1 and TRK2 genes (55). In addition, Na ϩ induces the expression of ENA1, a gene encoding a P-type ATPase that mediates Na ϩ efflux, to restore low cytosolic Na ϩ levels. In addition to the HOG-dependent induction occurring during sodium stress, ENA1 is also up-regulated by another independent pathway mediated by the calcium/calmodulin-dependent phosphatase calcineurin (32, 38, 40, 56). The calcineurin pathway responds mainly to challenges in the ionic environment. High salinity  -dependent genes (HSP12, CTT1,  TRK2, GPP2, ENA1, PDR12, GPD1, and ENA1) after a treatment with PMKT tested in wild-type S. cerevisiae BY4743 and the hog1⌬ mutant. A, Northern blotting. B, quantification. f, HSP12; •, CTT1; OE, TRK2; , GPP2; ࡗ, ENA1; ϩ, PDR12; ϫ, GPD1. The highest relative mRNA level in the wild type was set to 100.   The Transcriptional Response to PMKT DECEMBER 23, 2005 • VOLUME 280 • NUMBER 51 represents a stress for organisms because the excess of sodium or other monovalent cations imbalances the osmotic potential and generates a water deficit, and the influx of sodium may lead to metabolic toxicity (57). ENA1, its transcriptional activator HAL9, and TRK2 were significantly induced after PMKT exposure, showing that PMKT generates an ionic or an osmotic stress in sensitive yeast cells. Additional genes involved in ionic stress and detoxification were up-regulated in the presence of PMKT, namely NHA1, IST1, TOK1, PSR1, PHO84, PHO89, PHO87, ZRT1, and CTR3. Synthesis and Accumulation of Compatible Osmolytes-The behavior of many transcripts in our analyses correlated with known biochemical hyperosmotic stress responses. Glycerol, a compatible osmolyte known to accumulate rapidly in response to stress in yeast, accumulated transiently, and transcripts in the glycerol biosynthetic pathway increased (58). The transcripts of enzymes leading to glycerol production, GPD1 and GPP2, were increased during PMKT treatment ( Fig. 1  and TABLE ONE). These observations agree with the fact that glycerol was observed to increase in response to PMKT (Fig. 2). Glycerol is produced in yeast by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-phosphate followed by dephosphorylation. The first step is catalyzed by NAD-dependent glycerol-3phosphate dehydrogenase, which is encoded by the two isogenes GPD1 and GPD2. GPD1 is induced by hyperosmotic stress, whereas the expression of GPD2 is relatively unaffected by hyperosmotic stress (59). Mutants defective for GPD1 have an osmosensitive phenotype (27), and the double mutant gpd1⌬ gpd2⌬ is even more osmosensitive. The severity of these phenotypes is roughly proportional to the reduction in the cellular glycerol level (24,60). Thus, the synthesis of glycerol is required for growth in a high osmolarity environment. According to Pagé et al. (61), Hog1 mutants show hypersensitivity to K1 killer toxin, but the wild-type phenotypes of gpd1 gpd2 double deletion mutants show that the mechanism of killing by K1 is not related to the accumulation of glycerol.
After the initial increment of the glycerol content, the intracellular amount of glycerol was observed to decrease in the presence of PMKT (Fig. 2). This observation is in accordance with the fact that the toxin enhances membrane permeability (10). When by different mechanisms a cell is de-energized, any ion or metabolite accumulated in the cell against its concentration gradient will tend toward equilibrium, and a flow will be observed (i.e. K ϩ , ATP, and glycerol). This is consistent with the notion that the initial intracellular acidification of sensitive cells was observed simultaneously to the glycerol efflux 30 -60 min after toxin addition and is in agreement with the fact that PMKT generates the outflow of ions such as potassium (Fig. 4). Another possibility would be that the presence of the killer toxin might generate channels through which glycerol could pass. This possibility, however, was not taken into account because of previous results obtained with patch clamp techniques suggesting that PMKT generates unregulated ionic channels (10).
Similarly to glycerol, trehalose metabolism genes, GLK1, PGM2, TSL1, TPS1, and NTH1, were up-regulated during PMKT activity. Trehalose is involved in yeast stress responses as an osmoprotectant, although it does not accumulate at osmotically significant concentrations in salt-stressed yeast (62,63). PGM2, TPS1, and the regulatory factor encoded by TSL1 catalyze trehalose biosynthesis, whereas Nth1p (trehalase) leads to trehalose degradation and the formation of glucose (63). Completion of this cycle seems to be indicated by the up-regulated transcripts for the kinase GLK1. The presence of high transcript amounts for Nth1p and Glk1p may explain why the osmoprotectant trehalose does not accumulate during salt stress. Trehalose synthesis and degradation, in combination with glycerol production, play a key metabolic role in protection against high salinity; this notion, also based FIGURE 4. Proposed mechanism of action of PMKT and transcriptional response of S. cerevisiae. The process begins with the binding of the toxin to a primary receptor, the ␤-D-(136)-glucans of the cell wall. Then the toxin interacts, directly or indirectly, with the plasma membrane through an unknown mechanism, leading to disruption of cellular homeostasis. The presence of a secondary membrane (R2) receptor is postulated. The toxin induces the passage of physiological ions (H ϩ , K ϩ , and Na ϩ ) and small metabolites (e.g. glycerol) through the plasma membrane. These events are detected by the sensitive cell in an undefined manner that elicits the activation of a transcriptional response through the phosphorylation of Hog1p. General stress genes (CTT1, HSP12, etc.) and genes such us GPD1 and GPP2 that induce the synthesis of glycerol are up-regulated to counteract the effects of the toxin. Glycerol and intracellular ions are lost from sensitive cells leading to cell death. Highlighted by boxes are the level of induction of some induced genes. DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; FBP, fructose-1,6-bisphosphate; MG, methylglyoxal; CoQ, coenzyme Q; S-LG, S-D-lactoylglutathione; GSH, glutathione. on gene expression changes, has been proposed previously (62) and confirms the fact that PMKT generates a HOG response very similar to that obtained with a hyperosmotic stress.
To validate the data obtained from microarrays and Q-PCR that revealed the involvement of many HOG-regulated genes, Hog1p phosphorylation was examined in a S. cerevisiae sensitive strain (Fig. 3). Phosphorylation of Hog1p was rapidly observed when cells were shifted to PMKT exposure. The phospho-Hog1p signal was observed between 10 min after toxin addition and during the rest of the experiment, showing a permanent activation of the HOG signaling pathway. This result could be attributed to the permanent loss of glycerol (Fig. 2) from the sensitive cells due to the activity of the killer toxin and is confirmed by the observations made previously by different groups, who observed that mutants that are unable to produce or retain the osmolyte glycerol show strongly enhanced and sustained Hog1p phosphorylation (64). It has also been suggested that intracellular turgor pressure, caused by the glycerol level, leads to deactivation of the HOG pathway. This finding indicates that the kinetics of Hog1p phosphorylation and the subsequent induction and repression of genes are direct effects of PMKT. Taken together, the data also indicate the intricate interdependence of the PMKT mechanism of action, HOG signaling, and intracellular glycerol in the stress response of yeast sensitive to PMKT. Furthermore the observations from the Northern blotting experiments (Fig. 1) in which a permanent induction of mRNA synthesis was observed could reinforce the thought that the toxin generates a permanent stimulation of the cell because intracellular and extracellular ions are flowing through the plasma membrane simultaneously with some intracellular low molecular weight metabolites such as glycerol (Fig. 2). Finally the disruption of homeostasis across the plasma membrane could well lead to an increased mortality of the poisoned cells that, according to previously reported results, would not affect the physical integrity of the plasma membrane during the initial stages of the action of PMKT (10).
General Stress Defense Response-Whole-genome expression analysis has shown that the expression of more than 10% of S. cerevisiae genes is affected by several apparently unrelated stress conditions and that the exposure of yeasts to one type of stress strongly increases their capacity to resist another, different type of stress (25). The HOG signaling pathway of the yeast S. cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the mitogen-activated protein kinase kinase and of the mitogen-activated protein kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1 or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements. These genes are induced by different stimuli such as oxidative stress, nutrient starvation, heat shock, and hyperosmotic shock. The percentage of up-regulated ORFs related to the defense response to PMKT was 16%. HSPs, which have been reported to be up-regulated in a variety of stressful conditions such as oxidative stress, methyl methanesulfonate, and heat shock (53), were found to be up-regulated in response to PMKT (HSP12,  HSP104, HSP82, HSP26, DDR2, SSA2, SSB2, and SSA1). The groups of HSPs induced during PMKT exposure were different from those induced in osmotic stress (65), indicating that different sets of HSPs could have different functional targets in the responses to osmotic stress or PMKT.
Our results also presented in TABLE ONE and Fig. 1 showed that CTT1 (cytosolic catalase T) expression was increased with a PMKT treatment. HOG1-dependent osmotic induction of transcription of the CTT1 gene occurs rapidly after an increase in osmolarity (66). Consistent with a role of stress response elements in the induction of stress resistance, a number of other stress protein genes (e.g. HSP104) are regulated like CTT1. Furthermore catalase T has been shown to be important for viability under severe osmotic stress, and heat shock has been demonstrated to provide cross-protection against osmotic stress (67). Furthermore PMKT also induced YAP1, which specifically mediates oxidative stress responses and appears to be responsible for the rapid establishment of a transient transcriptional response. Yap1p, a relevant transcription factor that plays a major role in this response, binds, in vivo, promoters of genes that are not automatically up-regulated. It has been proposed that Yap1p nuclear localization and DNA binding are necessary, but not sufficient, to elicit the specificity of the chemical stress response (68).
Yeast Deletion Mutants-The S. cerevisiae gene deletion library offers a powerful tool for the assignment of new functions to sequenced genes. Because PMKT is an antimycotic protein with unknown targets in yeast, it was of interest to identify genes contributing to PMKT sensitivity and resistance that might lead to further insight into the cellular mechanism of PMKT-mediated toxicity. We screened a library of single gene deletions for hypersensitivity to PMKT. The genes whose expression levels were seen to be regulated in response to PMKT were selected, and the deletion mutants for those genes were obtained. Deletion of up-regulated genes generated hypersensitive phenotypes to PMKT. Several mutants of genes related with a osmotic stress response (YAP1, HAL9, GPP1, GPP2, GPD1, PSR1, IST1, NHA1, ENA1, SAT2, TRK2, HSP12, CTT1, and CSF1) were observed to be highly hypersensitive to PMKT, and the group of genes related to glycerol synthesis had the highest levels of hypersensitivity. With a general reading of these results we may assume that yeast cells respond to PMKT by increasing the expression of some genes that could counteract the effects of the toxin to some extent over a short period of time. Taking into account the fact that the mutations of down-regulated genes were observed to generate PMKTresistant mutants or wild-type phenotypes, down-regulated genes could be involved in the mechanism of cell defense and rescue but in an indirect manner.
Regardless of certain possible additional effects, the results reported here indicate that PMKT generates a serious challenge for cell survival by disrupting plasma membrane electrochemical gradients, inducing a general stress response in a way comparable to that of some stimuli like hyperosmotic shock. The observed transcriptional response is consistent with the idea that the effect induced by PMKT lies in an alteration to the ionic homeostasis of the sensitive yeast cell. As such, the PMKTinduced stress response requires the simultaneous expression of several components of the sensitive yeast genome. In addition to producing a proper response to the stimulus of PMKT stress, the HOG pathway could play an important role in the response to PMKT. The mechanism of HOG-mediated response to PMKT is as yet unknown but will prove to be an exciting area of study in the future. Further studies are currently in progress to gain additional information about the properties of P. membranifaciens killer toxin in the hope of contributing to the understanding of a killer toxin with promising antifungal properties.