Hog1 Mitogen-Activated Protein Kinase Plays Conserved and Distinct Roles in the Osmotolerant Yeast Torulaspora delbrueckii

ABSTRACT Torulaspora delbrueckii has emerged during evolution as one of the most osmotolerant yeasts. However, the molecular mechanisms underlying this unusual stress resistance are poorly understood. In this study, we have characterized the functional role of the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in mediating the osmotic stress response, among others, in T. delbrueckii. We show that the T. delbrueckii Hog1p homologue TdHog1p is phosphorylated after cell transfer to NaCl- or sorbitol-containing medium. However, TdHog1p plays a minor role in tolerance to conditions of moderate osmotic stress, a trait related mainly with the osmotic balance. In consonance with this, the absence of TdHog1p produced only a weak defect in the timing of the osmostress-induced glycerol and GPD1 mRNA overaccumulation. Tdhog1Δ mutants also failed to display aberrant morphology changes in response to osmotic stress. Furthermore, our data indicate that the T. delbrueckii HOG pathway has evolved to respond to specific environmental conditions and to play a pivotal role in the stress cross-protection mechanism.

The ability of Saccharomyces cerevisiae to withstand increasing osmotic pressures is critical for its survival in natural habitats and is an essential trait in many biotechnological processes. Variation in ambient osmolarity is a constant phenomenon occurring in almost all steps from yeast biomass production to downstream applications in which cells have to grow and perform optimal fermentation under hostile conditions (51). However, the success of this industrial microorganism is based on its capacity to transform carbohydrates rapidly into ethanol and CO 2 rather than its unusual resistance to environmental stresses. Yeasts other than S. cerevisiae, such as Zygosaccharomyces rouxii, Debaryomyces hansenii, and Torulaspora delbrueckii, display much higher resistance to adverse conditions and in particular to osmotic stress. This characteristic has made these yeasts potential models to explore and understand the mechanisms underlying osmotic tolerance in eukaryotic cells. However, little is known about the molecular basis of this phenotype, and, in most cases, the characterization of signaling pathways, transcription factors, and gene targets in these non-Saccharomyces species remains elusive.
Stress responses in S. cerevisiae have been widely researched. Yeast cells exposed to an osmostressing environment show a particular transcription profile. Thus, over 250 to 400 genes, covering a wide variety of physiological functions, are up-regulated after different conditions of osmotic shock (16,24,54). Stimulated expression of these genes appears to depend mainly on well-characterized molecular signaling pathways: the cyclic AMP-activated protein kinase A pathway (61), the high-osmolarity glycerol (HOG) pathway (66), one of the five mitogen-activated protein kinase (MAPK) pathways known in S. cerevisiae (28), and the calcineurin/Crz1p pathway, which is specifically required for adaptation to high-salt conditions (55).
The HOG pathway consists of two discrete signaling branches composed of a putative osmosensor coupled to a MAPK cascade, some of which can lead to the phosphorylation and activation of the core MAPK Hog1p, the orthologue to mammalian p38 and fission yeast Sty1p stress-activated protein kinase (22). Osmostress-induced phosphorylation of Hog1p triggers its nuclear accumulation (21,52) and the later induction of many osmostress-responsive genes (49,54), among them GPD1 (53), the gene encoding the main enzyme that produces the compatible osmolyte glycerol in S. cerevisiae (2). Although the correlation between Hog1p activation, enhanced glycerol production, and osmotic resistance in S. cerevisiae is well established, it is unclear whether the MAPK pathway plays a similar role in other yeasts, particularly in highly osmotolerant species.
MAPKs homologous to S. cerevisiae Hog1p have been identified in different yeast species such as Schizosaccharomyces pombe (46), Candida albicans (57), Z. rouxii (37), and D. hansenii (9) as well as in multicellular fungi including Aspergillus nidulans (31), Neurospora crassa (70), and the human pathogen Cryptococcus neoformans (8). Although only a few of them have been studied in detail, it appears that Hog1p orthologues share conserved but different roles in response to a variety of environmental cues. Whereas inactivation of MAPK in S. cerevisiae has dramatic effects on growth under hyperosmotic conditions, hog1⌬ null mutants from Z. rouxii can grow as well as the parental strain in the presence of 2 M NaCl (37). Similarly, disruption of HOG1 homologues from C. neoformans (8) or A. nidulans (39) has a weak effect or no effects on growth at high osmolyte concentrations. Moreover, in some species, the HOG pathway appears to have evolved to respond to additional extracellular stimuli and carry out different cellular roles in a niche-dependent way. Examples include cell-to-cell signaling and virulence in C. albicans (5,59) or C. neoformans (8), fungicide resistance in N. crassa (70), or methylglyoxal tolerance in S. cerevisiae (1). However, it is not yet clear whether this niche-specific evolution also applies to highly osmotolerant species.
In this study, we have isolated the Hog1p homologue of the yeast T. delbrueckii, a facultative fermentative species characterized by its exceptional resistance to osmotic stress (33,41). The aim of this work was to investigate the functional role of the HOG pathway in the stress response of T. delbrueckii and to determine whether this signaling route has evolved to enable this yeast to proliferate in highly osmotic environments. Surprisingly, we found that glycerol accumulation, a key feature of osmotic tolerance, is controlled mainly by regulatory mechanisms other than the HOG pathway. By contrast, the T. delbrueckii HOG pathway has undergone a functional specialization, being used as the central module of the stress crossprotection mechanism in this yeast.
Stress sensitivity tests. For stress experiments, cells were grown to mid-exponential phase at 30°C, collected, and transferred to fresh medium containing NaCl or sorbitol at the indicated concentrations. Plate phenotype experiments were done by diluting the cultures to an optical density at 600 nm (OD 600 ) of 0.3 and streaking the cells onto SD or YPD agar solid medium containing the stressor to be tested. More-detailed sensitivity assays were performed by spotting 10-fold serial dilutions (3 l) of the cell culture. Unless otherwise indicated, colony growth was inspected after 2 to 4 days of incubation at 30°C.
Strain and plasmid construction. Plasmid pMJH28, carrying a DNA fragment containing the HOG1 gene from T. delbrueckii (TdHOG1) and the flanking regions around this gene, was isolated from a genomic library (32) by complementation of the osmosensitivity phenotype of a hog1⌬ mutant strain in S. cerevisiae (see Results). Multicopy and centromeric plasmids containing the isolated TdHOG1 gene were constructed by cloning a 1,541-bp PCR fragment, which includes the complete open reading frame plus 143 bp upstream from the ATG and 140 bp of the 3Ј untranslated region. Amplification was carried out under standard conditions using synthetic oligonucleotides (see Table S1 in the supplemental material) and plasmid pMJH28 as a template. The amplified fragment was digested with XbaI/HindIII and ligated into the vectors YEplac195 and YCplac33 (26), which were previously digested with the same set of enzymes, resulting in plasmids YEpTdHOG1 and YCpTdHOG1, respectively.
The TdHOG1 disruption cassette containing the nourseothricin resistance natMX4 module (27) was constructed by restriction. First, the PCR-amplified fragment of TdHOG1, obtained as described above, was cloned into the pGEM-T Easy vector (Promega), resulting in plasmid pGEM-TdHOG1. This was digested with EcoRV and BamHI, and the released fragment was replaced by the natMX4 module obtained from plasmid pAG25 (27) by digestion with the same enzymes. Finally, the resulting plasmid, pGEM-TdHOG1-natMX4, was treated with XbaI and HindIII to release the disruption cassette.
The correct gene disruption of TdHOG1 was detected by diagnostic PCR using whole yeast cells (35) from isolated colonies and oligonucleotides designed to bind outside or inside the replaced TdHOG1 sequences and within the marker modules (see Table S1 in the supplemental material). Yeasts were transformed by the lithium acetate method (36). E. coli was transformed by electroporation using an electroporator according to the manufacturer's instructions (Eppendorf).
Northern blot. The preparation of total RNA and Northern blot hybridization were performed as described previously (58). PCR-amplified fragments of the S. cerevisiae GPD1 and ACT1 (loading control) genes were prepared and radiolabeled as previously described (1). Spot intensities were quantified with Image Gauge software, version 3.12 (Fuji, Kyoto, Japan). Values of spot intensities were corrected with respect to the ACT1 mRNA level and are represented as the relative mRNA levels. The highest relative GPD1 mRNA level for each sample analyzed was set at 100.
Western blot assay. Whole-cell extracts were prepared as previously described (50). Ten microliters of each sample was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Phospho-TdHog1p and TdHo1p were detected with anti-phospho-p38 MAPK (catalog no. 9215; Cell Signaling, Beverly, MA) and anti-Hog1p polyclonal antibody yC-20 (catalog no. sc-9079; Santa Cruz Biotechnology, Santa Cruz, CA), respectively, according to the manufacturers' instructions. The phosphorylated forms of Slt2p and Fus3p-Kss1p MAPKs were detected with anti-phospho-p44/42 antibody (catalog no. 9101; Cell Signaling) at a 1:1,000 dilution. As a secondary antibody, we used horseradish peroxidase-conjugated goat anti-rabbit antibody(1:2,000, catalog no. 7074; Cell Signaling). Blots were developed using the ECL Western blotting detection kit from Amersham Biosciences.
Glycerol determination. Total glycerol content was determined colorimetrically as previously described (50). Values are expressed as micrograms of glycerol per milligram (dry weight) of yeast cells. Growth throughout the experiment was estimated by measuring the OD 600 (an OD 600 of 1 equals 0.21 and 0.30 mg [dry weight] of cells/ml for T. delbrueckii and S. cerevisiae, respectively).
Sequencing and sequence analysis. DNA sequencing was performed in both strands by the dideoxy-chain termination procedure (56). Similarity searches were performed using the BLAST server at the Munich Information Center for Protein Sequences. We searched for TdHog1p domains by scanning the sequence against the protein profile databases PROSITE (available at http://www .expasy.org) and Pfam. Multiple sequence alignment was done using the ClustalW program (63).
Nucleotide sequence accession number. The nucleotide sequence for TdHOG1 has been deposited in the GenBank database (available at http://www.ncbi.nlm .nih.gov/GenBank/index.html) under accession number DQ020519.

RESULTS
Cloning and molecular characterization of the T. delbrueckii HOG1 gene. Functional complementation for growth of an S. cerevisiae hog1⌬ mutant by a T. delbrueckii genomic library (32) on 0.5 M NaCl was used to isolate DNA fragments encoding Hog1p. From more than 1,000 transformants, only one plasmid, pMJH28, which hybridized under heterologous conditions with a probe of the S. cerevisiae HOG1 gene (data not shown), was able to restore growth of the osmosensitive strain on selective medium. DNA sequencing of the fragment in pMJH28 revealed the presence of a 1,284-bp uninterrupted open reading frame encoding a putative 427-amino-acid protein, TdHog1p. This peptide showed 94 and 88% overall identity with Z. rouxii ZrHog2 and ZrHog1 proteins, respectively, 88% identity with S. cerevisiae Hog1p, and 85% identity with D. hansenii DHog1p. High percentages of identity between TdHog1p and other members of the Hog1/Spc/p38 MAPK subfamily, such as N. crassa Os-2 (83%), C. albicans CaHog1p (78%), A. nidulans HogA (76%), C. neoformans Hog1p (76%), and mammalian p38 (47%), were also recorded.
Protein domain analysis showed that TdHog1p contains a typical catalytic protein kinase domain (positions 23 to 302), which is also present in other Hog1p and p38 MAPK homologues. Within this region, we identified a protein kinase ATPbinding region (positions 29 to 53) and a consensus Ser/Thr protein kinase active site (positions 140 to 152). Moreover, the T. delbrueckii Hog1 protein contains a TGY dual phosphory-lation signature (amino acids 174 to 176) characteristic of MAPKs activated by hyperosmolarity (15), a common docking domain (60), and an alanine-rich region at the C terminus at positions 302 to 316 and 364 to 383, respectively, which is also found in S. cerevisiae Hog1p.
To further confirm the identity of the TdHOG1 gene product, we cloned the entire open reading frame into the yeast expression vectors YEplac195 and YCplac33, and the resulting constructs, YEpTdHOG1 and YCpTdHOG1, were transformed into the S. cerevisiae W303-1A hog1⌬ strain and examined for salt tolerance. As shown in Fig. 1A, expression of TdHOG1 at a high copy number complemented the osmosensitive phenotype of the S. cerevisiae hog1⌬ mutant strain on 1 M NaCl. However, the replacement of the Hog1p MAPK by its counterpart TdHog1p in a single copy did not improve the intrinsic salt sensitivity of S. cerevisiae. Indeed, YCpTdHOG1 and YCpHOG1 transformants, with the latter carrying the gene from S. cerevisiae, showed similar growth on SD liquid medium supplemented with 1.5 M NaCl (Fig. 1B).
TdHog1p is phosphorylated under osmotic stress. We examined the effect of a shift to NaCl-containing medium on the phosphorylation state of TdHog1p in cells of the wild-type strain. As shown in Fig. 2A, TdHog1p was clearly activated when cells grown in YPD medium (OD 600 of 1.0) were subjected to mild NaCl stress conditions (0.5 M). The kinetics of phosphorylation were similar to those previously reported for S. cerevisiae cells exposed to similar NaCl concentrations (65). Indeed, the phosphorylation level reached a maximum within 1 to 5 min of exposure to 0.5 M NaCl and decreased markedly after 10 min ( Fig. 2A). As expected, TdHog1p was also sensitive to the presence of external sorbitol (Fig. 2B). Thus, both NaCl and sorbitol trigger the activation of the HOG pathway.
Effect of TdHOG1 on growth under NaCl and sorbitol stress. In order to confirm the functional role of the T. delbrueckii HOG pathway, we constructed a Tdhog1⌬ mutant. Detection of the correct disruption of TdHOG1 was done by PCR and Western blotting. From a total of 10 transformants verified, all showed the pattern of PCR-amplified fragments expected for the correct gene disruption of TdHOG1. In addition, they lacked a specific band of hybridization with antiserum against total Hog1p (data not shown). Thus, it seems that a single TdHOG1 wild-type allele is responsible for the MAPK activity of the HOG pathway in the haploid T. delbrueckii strain PYCC5321. Figure 2C shows the results of the phenotypic characterization of Tdhog1⌬ mutant cells on YPD plates supplemented with 0.5 M NaCl or 1 M sorbitol, which both give approximately the same water activity, around 0.98 (53). In contrast to the dramatic osmosensitivity of the S. cerevisiae hog1⌬ mutant, the absence of TdHog1p had scarcely any effect on the growth of T. delbrueckii cells under conditions of moderate osmotic stress. Similar results were obtained when the phenotype was tested using 1 M NaCl (Fig. 2C). However, Tdhog1⌬ mutant cells were unable to proliferate at 1.5 M NaCl, while the wildtype strain showed noticeable growth (Fig. 2C).
Osmostress-induced glycerol overaccumulation and GPD1 expression. In S. cerevisiae, hyperosmotic shock triggers the Hog1p-dependent transcriptional induction of GPD1, the gene for glycerol production (2) and the main osmoregulator in this yeast (11). Likewise, T. delbrueckii overproduces glycerol in response to osmotic stress (33). Consequently, we analyzed the kinetics of glycerol production in YPD cultures of wild-type and Tdhog1⌬ mutant strains subjected to osmotic stress. As can be seen in Fig. 3A, the absence of MAPK slightly delayed the accumulation of total glycerol in response to 1 M NaCl. Similar results were obtained at different salt concentrations or in the presence of sorbitol (data not shown). However, after 3 h of incubation with 1 M NaCl, the glycerol content in the culture of the Tdhog1⌬ mutant increased to about 80% of the wild-type levels (Fig. 3A). Under identical conditions, the total glycerol content of S. cerevisiae hog1⌬ cultures treated with NaCl was found to be lower, around 30% of the wild-type levels (Fig. 3A, right panel).
We then used Northern blot analysis to examine the expression profile of GPD1 in NaCl-stressed Torulaspora cells. Since this gene has not been cloned, total RNA samples were hybridized with a probe of its S. cerevisiae counterpart under heterologous conditions. Previous work in our laboratory has Moreover, Southern blot experiments appear to indicate that a single GPD1 allele is present in the genome of T. delbrueckii (data not shown). As expected, expression of GPD1 was induced in wild-type cells exposed to 1 M NaCl, with an approximately 10-fold induction within 60 min (Fig. 3B). The absence of TdHog1p altered the osmostress-induced expression of GPD1. However, the effects were not as important as those reported previously for the S. cerevisiae hog1⌬ strain (53). Although maximum induction was shifted to later time points, the mutant was able to accumulate a GPD1 mRNA level of about 60% of the wild-type level (Fig. 3B). In addition, the absence of MAPK did not affect GPD1 expression in nonstressed control cells. Similar results were observed for cells exposed to 1 M sorbitol (data not shown).
Osmostress-induced morphology changes in Tdhog1⌬ mutants. Exposure of wild-type S. cerevisiae cells to osmotic stress has no effect on their morphology. By contrast, under the same conditions, a hog1⌬ mutant displays morphological alterations (shmoo-like cells) that are characteristic of pheromone-treated cells (12). Figure 4 shows the budded morphology of wild-type cells of S. cerevisiae strain W303-1A exposed to 1 M sorbitol or NaCl, in contrast to the shmoos formed in the S. cerevisiae hog1⌬ mutant. However, wild-type and Tdhog1⌬ mutant strains of T. delbrueckii displayed normal morphologies in both the presence and absence of 1 M sorbitol. Only exposure to 1 M NaCl (Fig. 4) or higher concentrations (data not shown) led to a clear morphological response, although shmoo-like cells were not observed in any case. Instead, cells showed a clear inability to separate normally. Thus, the response appeared to depend on the chemical used. However, blocking in cell separation was also evident after exposure to 2 M sorbitol (data not shown).
Cross talk between the T. delbrueckii HOG pathway and parallel MAPK pathways under hyperosmotic conditions. The formation of shmoo-like cells in S. cerevisiae Hog1p-deficient strains occurs by the inappropriate activation of two MAPKs (17,30,48), Kss1p, in the MAPK of the filamentation/invasion pathway (23), and Fus3p in the pheromone response pathway (19). Therefore, we investigated the phosphorylation states of these MAPKs in wild-type and Tdhog1⌬ cells exposed to high osmolarity. Protein extracts were examined with Western analysis by using a monoclonal anti-phospho-p44/42 MAPK antibody, which recognizes the TEY activation sequence found in mammalian p42 and p44 MAPKs (Erk1/Erk2) and yeast MAPKs Kss1p and Fus3p. As shown in Fig. 5, a single band of the expected mobility for either T. delbrueckii Kss1p or Fus3p MAPK (ϳ41 kDa) appeared in protein extracts from Tdhog1⌬ cells 30 min after osmotic shock. In contrast, there was no evidence of this effect in control cells (Fig. 5).
Interestingly, inspection of the Western blot revealed the presence of an additional ϳ61-kDa strong immunoreactive band in both wild-type and Tdhog1⌬ mutant cells (Fig. 5). This band might correspond to the phosphorylated form of Slt2p, the MAPK in the protein kinase C (PKC) pathway (28), which also contains the same TEY activation loop as Fus3p and Ssk1p and is therefore recognized by the antibody. Notably, we also observed that Slt2p exhibited sustained phosphorylation upon exposure to osmotic stress (Fig. 5). Indeed, only the 30- on January 9, 2021 by guest http://ec.asm.org/ to 60-min samples showed a significant reduction of the phospho-Slt2p signal. In addition, a loss of Slt2p phosphorylation was not observed in the Tdhog1⌬ strain (Fig. 5). This is in sharp contrast to the situation reported previously for S. cerevisiae, where Slt2p is rapidly dephosphorylated (within 1 min), in a Hog1p-independent way, upon osmotic stress (68). Hence, it seems that a functional T. delbrueckii HOG pathway is essential for the proper deactivation of the PKC signal in response to hyperosmolarity. The functional role of T. delbrueckii Hog1p in response to several types of stress. Functions other than osmostress protection have been identified for HOG homologue pathways in some yeast species. Based on this, we analyzed the need for TdHog1p in the response of T. delbrueckii to diverse stimuli (Fig. 6A). Clearly, the MAPK was essential for tolerance to methylglyoxal and citric acid. However, it appeared to be dispensable for growth at a high temperature, 34°C (Fig. 6A). We also noted that the T. delbrueckii MAPK was not essential for the response to H 2 O 2 . To further confirm this point, we ana-lyzed the kinetics of TdHog1p phosphorylation in T. delbrueckii wild-type cells following oxidative stress. As shown in and T. delbrueckii (PYCC5321) and the corresponding hog1⌬ mutants hog1⌬ and Tdhog1⌬ were pregrown in YPD medium, collected, and then transferred (initial OD 600 of 0.05) to the same medium (control) or YPD medium plus 1 M NaCl or sorbitol. Microphotographs were taken using an optical microscope after 16 h of culture. A representative cluster of cells is shown. observed for cells shifted to 34°C (Fig. 6B). Hence, the T. delbrueckii HOG pathway appears to be necessary under specific environmental conditions.
Stress cross-protection in T. delbrueckii. S. cerevisiae cells exposed to nonlethal doses of one type of stress develop tolerance to higher doses of the same stress as well as to unrelated stresses; these phenomena are described as induced tolerance and cross-protection, respectively (20,43). It is commonly accepted that yeasts other than Saccharomyces display induced tolerance. However, the existence of cross-protection mechanisms is not always evident and has frequently been the subject of controversy (18). Therefore, we were interested in investigating this phenomenon in the osmotolerant yeast T. delbrueckii.
As shown in Fig. 7A, cells of the T. delbrueckii wild-type strain were unable to grow at 37°C on YPD plates lacking (control) or containing 0.5 M sorbitol. However, this phenotype was suppressed when cells were challenged with 1 M (Fig.  7A) or 1.5 M (data not shown) sorbitol. Nevertheless, we reasoned that this phenomenon does not necessarily imply previous adaptation of the cells by osmotic stress. Reduction of water activity by sorbitol or salts is indeed an effective way to reduce thermal death, as demonstrated for osmotolerant yeasts like D. hansenii (3). Consequently, we looked into how TdHog1p is involved in this phenomenon. As can be seen in Fig. 7A, the thermal protection afforded by 1 M sorbitol was retained in the Tdhog1⌬ strain. Nevertheless, growth of mutant cells was clearly impaired at 37°C compared to the wild type, whereas at 30°C, the differences were less pronounced. Hence, adaptation to thermal stress appears to be dependent upon TdHog1p. Presumably, growth at high temperatures is also improved by water activity reduction mechanisms, being more pronounced at high osmolyte concentrations.
To investigate the role of TdHog1p in the above-described findings in greater depth, we first tested the levels of phospho-TdHog1p in cells exposed to increased temperatures plus high osmolarity (Fig. 7B). Exposure of yeast cells at a temperature of 37°C induced a modest increase in the level of phospho-TdHog1p, around 1.5-to 2-fold over basal levels, compared with that observed after exposure to 1 M sorbitol, about ninefold (Fig. 2B). Moreover, overphosphorylation of TdHog1p took place late and was only evident within 15 to 30 min after the thermal transfer, a temporary pattern often linked to an indirect mechanism of activation (34). In contrast, cells subjected to highly osmotic conditions at 37°C showed the same kinetics of TdHog1p phosphorylation that are characteristic for hyperosmolarity ( Fig. 2A and 7B).
Next, we investigated the thermal protection conferred by pretreatment of T. delbrueckii cells with sorbitol. Cells grown in YPD medium were exposed to 1 M sorbitol at 30°C for 3 h and then transferred to fresh prewarmed (37°C) YPD medium lacking sorbitol (Fig. 7C). As can be seen in Fig. 7C, pretreatment of wild-type cells with osmotic stress afforded higher growth ability at 37°C. However, this effect was lost in Tdhog1⌬ FIG. 6. TdHog1p is essential for the response to different stresses. (A) Exponentially growing cells of T. delbrueckii wild-type strain PYCC5321 (wt) and the corresponding Tdhog1⌬ mutant were spotted onto malt extract (ME), SD, or YPD plates containing 200 mM citric acid, 20 mM methylglyoxal (MG), and 4 mM H 2 O 2 , respectively. Control plates lacking the above-cited compounds were tested in parallel. Growth at high temperatures was inspected on YPD plates incubated at 34°C. For the rest, plates were incubated at 30°C for 2 to 4 days. (B) Cells of wild-type T. delbrueckii strain PYCC5321 were grown in YPD medium at 30°C until early exponential phase (OD 600 of 0.4 to 0.6) and then shifted to 34°C or transferred to medium containing 4 mM H 2 O 2 . At the indicated times, cells were harvested and processed, and the crude protein extracts were assayed for TdHog1p phosphorylation (P-TdHog1) levels as described in the legend of Fig. 2A

DISCUSSION
Phylogenetically, T. delbrueckii and S. cerevisiae are closely related, as previously demonstrated by sequence comparisons (10,32). Despite this genealogical relationship, T. delbrueckii is able to survive and proliferate at high osmolyte concentrations, a property not shared by S. cerevisiae (33). As a result, this nonconventional yeast is frequently found in different habitats compared to those of S. cerevisiae, including cane molasses, syrups, honey, and foods with a high sugar content (41). Hence, they would be expected to differ in the regulatory mechanisms and molecular targets that enable adaptability to high-osmolarity environments.
These differences were first evident in our analysis of the functional response of the T. delbrueckii HOG pathway to osmotic stress. Although the MAPK TdHog1p was rapidly phosphorylated after transfer to NaCl-or sorbitol-containing medium, this was not essential for the growth of T. delbrueckii cells under moderate stress conditions, 0.5 M NaCl or 1 M sorbitol. Only when severe stress conditions were tested, for example, 1.5 M NaCl, was the need for a functional HOG pathway clearly evident. This result fully agrees with previous observations of Z. rouxii hog1⌬ mutant cells, which show NaCl sensitivity only at concentrations above 2 M (37). Therefore, we predicted that this fact could reflect a distinct role of the HOG pathway in the osmoadaptation mechanisms employed by T. delbrueckii and S. cerevisiae. Consistent with this, neither osmostress-induced glycerol accumulation nor the expression of GPD1 was strongly affected in Tdhog1⌬ mutant cells exposed to moderate hyperosmolarity. This is in sharp contrast with the situation in S. cerevisiae, where Hog1p is the main regulator of these responses (2,53). Nevertheless, we noted that glycerol production and GPD1 mRNA levels were upregulated upon osmotic shock. Therefore, a sudden change in the water activity of the environment is perceived by T. delbrueckii as a stressful condition. However, T. delbrueckii, like S. cerevisiae, must be frequently exposed to significant osmolarity fluctuations in natural habitats. Therefore, adaptation to these FIG. 7. The HOG pathway mediates stress cross-protection in T. delbrueckii. (A) YPD-grown cells of T. delbrueckii PYCC5321 wild-type (wt) and Tdhog1⌬ mutant strains were spotted onto YPD plates (control) and YPD plates containing 0.5 or 1 M sorbitol. Plates were incubated at 30 or 37°C for 2 to 4 days. (B) Cells of the T. delbrueckii wild-type strain were grown in YPD medium at 30°C and then subjected to heat stress alone (37°C) or in combination with high osmolarity, 1 M sorbitol (37°C ϩ 1 M sorbitol). At the indicated times, cells were harvested and processed, and the crude protein extracts were assayed for TdHog1p phosphorylation (P-TdHog1) levels as described in the legend of Fig. 2A. (C) Exponentially growing cells of the wild-type (squares) and Tdhog1⌬ mutant (circles) strains in YPD medium were either left unstressed (open symbols) or exposed to 1 M sorbitol (closed symbols) at 30°C. After 3 h, cells were washed and shifted to prewarmed (37°C) YPD medium lacking sorbitol, and the OD 600 was measured at regular intervals. In all cases, representative experiments are shown.

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HERNANDEZ-LOPEZ ET AL. EUKARYOT. CELL changes requires a strict regulatory system that allows adaptability, thus avoiding the continuous stimulation of this response. Glycerol production is one of the processes requiring most energy by far (64). Hence, both yeasts appear to follow a similar strategy in response to osmotic stress. However, they have diverged in the regulatory mechanisms that control glycerol accumulation, at least under moderate osmotic stress conditions.
The differential specialization of Hog1p functions in S. cerevisiae and T. delbrueckii was further evidenced by analyzing the osmostress-induced morphological changes in the Tdhog1⌬ strain. High osmolarity induces an aberrant morphology in S. cerevisiae hog1⌬ mutants, resembling that of cells exposed to pheromones, shmoos, or pear-shaped cells (13). As we demonstrated, osmotic stress induced the overphosphorylation of Fus3p and Kss1p, the MAPKs in the pheromone response and filamentation/invasion pathways, respectively, in the TdHog1pdeficient strain. Thus, the MAPK TdHog1p prevents osmolarity-induced cross talk between the HOG and parallel MAPK pathways in T. delbrueckii. However, shmoos were not formed by Tdhog1⌬ mutant cells exposed to a high level of sorbitol or NaCl. Therefore, S. cerevisiae and T. delbrueckii appear to control a different set of genes by the Fus3p-Kss1p MAPKs. It is also possible that T. delbrueckii has evolved additional mechanisms to ensure the specificity of the osmostress signal. Nevertheless, further work is required to identify upstream elements of the T. delbrueckii HOG pathway and clarify the activation mechanisms operating in this yeast.
Regarding the morphological effects of osmotic stress, we also noted that Tdhog1⌬ mutant cells remain attached after budding at high osmolyte concentrations, a phenotype that was missing in the wild-type strain. This apparent cell division defect led to large branched aggregates, similar to those observed for chitinase-negative S. cerevisiae strains (40). This phenotype has also been reported for hog1⌬ mutant cells of C. albicans subjected to 1 M NaCl, suggesting a link between cell wall metabolism and the activity of CaHog1p in this pathogenic yeast (5). Such a relationship has also been suggested for S. cerevisiae, since components of the HOG pathway appear to be involved in cell wall maintenance (6).
A connection between cell wall metabolism and the T. delbrueckii HOG pathway was evidenced by the repressing effects that this pathway exerts on the activity of the PKC pathway following osmotic stress. The PKC signaling pathway is induced during budding and mating (69) as well as in response to environmental conditions such as high temperature, hypoosmotic stress, and cell wall-damaging conditions (38,44). As we have shown, overphosphorylation of Slt2p, the MAPK of the PKC pathway, was maintained in Tdhog1⌬ mutant cells after a shift to conditions of high osmolarity. This lack of repression might thus account for the cellular aggregation displayed by the Tdhog1⌬ mutant strain upon osmotic stress. On the other hand, the level of phospho-Slt2p was notably high in cells grown at 30°C. This observation indicates that T. delbrueckii perceives stressful temperature fluctuations that do not stimulate a stress response in S. cerevisiae. Moreover, the kinetics of Slt2p dephosphorylation after osmotic stress were unusually slow in T. delbrueckii compared to that exhibited by S. cerevisiae (68). Thus, this result suggests that there has been a di-vergence in the mechanisms that down-regulate the PKC pathway in T. delbrueckii and S. cerevisiae.
Another aspect of relevance addressed in our work is the functional role of TdHog1p under stress conditions other than high osmolarity. Interestingly, TdHog1p has no apparent function at supraoptimal temperatures or at high levels of H 2 O 2 . The lack of TdHog1p activity at high temperatures can account for the previous observation that S. cerevisiae Hog1p is not essential for growth at 37°C (67). Recently, Smith et al. (59) also showed that C. albicans Hog1p is not activated by temperature upshifts. However, the finding that TdHog1p is not essential for the oxidative stress response was quite unexpected. Previous reports demonstrated that the HOG pathway provides protection against this stressful condition in S. cerevisiae (29) as well as in other yeasts and fungi species such as C. albicans (7), Schizosaccharomyces pombe (14), and C. neoformans (8). Although we have no obvious explanation for this result, it could reflect a wider ability of this yeast to cope with high levels of reactive oxygen species. Consistent with this, we found that T. delbrueckii was able to grow at high relative levels of H 2 O 2 (4 mM) compared with other yeasts and in particular with S. cerevisiae. Thus, the T. delbrueckii HOG pathway appears to have evolved not to respond to oxidative stress.
In the present work, we have also observed stress crossprotection in T. delbrueckii, suggesting that a general stress response (20) exists in this yeast. In S. cerevisiae, the general stress response is mediated by a common pathway, the cyclic AMP-protein kinase A pathway (61), which controls the activity and nuclear localization of the transcriptional factors MSN2/MSN4 (45). In addition, S. cerevisiae uses strategies of coinduction of defense mechanisms. Instead of a common pathway, different stresses control a common set of genes via different signaling pathways and transcription factors (16,24). Far from the classical concept of a general stress response, we observed that growth of T. delbrueckii at 37°C, a temperature that inhibits the proliferation of this yeast, was dependent on the activity of TdHog1p coupled with the thermal protection provided by sorbitol. Furthermore, we find that pretreatment with sorbitol provides a growth advantage at 37°C for wild-type cells but not for Tdhog1⌬ mutant cells. Therefore, stress crossprotection exists in T. delbrueckii, but this is mediated by TdHog1p. Similarly, Smith et al. (59) previously reported Hog1p-dependent stress cross-protection in C. albicans, a result that fits well with the lack of a functional role of homologues of S. cerevisiae MSN2/MSN4 in this yeast (47).
Overall, the results presented in this study emphasize the divergence of a classical stress signaling pathway between different yeasts. Our current knowledge indicates that the HOG pathway has evolved in different yeasts in a niche-specific way (8,59). We have also demonstrated that changes have taken place in the Hog1p-GPD1 relationship during the evolutionary divergence of S. cerevisiae and T. delbrueckii. While it is easy to imagine the different mechanisms through which gene expression regulation evolves (25), it is much more difficult to understand how these events determine the adaptability of yeasts to changing environments.