The mitogen-activated protein kinase (MAPK) Hog1p plays an essential role in the yeast hyperosmotic response. A homolog of the HOG1 gene was isolated from the halophilic black yeast Hortaea werneckii encoding a putative 359 amino acid protein, HwHog1p, with high homology to Saccharomyces cerevisiae Hog1p and to other eukaryotic Hog1p homologs. HwHog1p contains a TGY motif within a protein kinase catalytic domain and a C-terminal common docking (CD) motif. Its activation by increased salinity is regulated at the posttranscriptional level. HwHog1p is located on the plasma membrane under nonstress conditions. Upon increased external salinity it is translocated from the membrane, presumably to the nucleus.
A group of halophilic and halotolerant melanized yeast-like fungi, also named black yeasts, was isolated from solar salterns as their natural habitat. The predominant species among them was Hortaea werneckii (Dothideales, Ascomycota), previously known as a causative agent of human tinea nigra, a superficial mycotic infection of the skin occurring in tropical countries and mainly affecting the palms . It has also been isolated from sea water, salted dried fish and salt marsh but its natural ecology was poorly understood . H. werneckii from salterns can grow at salinities ranging from 0% to a saturated solution of NaCl (32% NaCl (w/v)) . When compared to non-halophilic Saccharomyces cerevisiae and halotolerant yeasts, different responses to increased salinity were observed in halophilic H. werneckii at the level of membrane composition and fluidity (Turk, M., unpublished results) as well as in metabolic responses .
Exposure to high environmental osmolarity leads to dehydration and causes a decrease in cell viability. To adapt to salt stress, microorganisms balance high external osmotic pressure by accumulation of low molecular mass compounds which are compatible with cellular function and do not inhibit the enzymes . Increased synthesis and/or accumulation of glycerol and other compatible solutes, mainly polyols, have been observed to be the predominant way of yeast osmoregulation . Glycerol accumulates in H. werneckii when the cells are exposed to high salinities .
Of the five MAPK cascades in S. cerevisiae signal transduction pathways that enable yeast cells to regulate various aspects of cellular functions, the high-osmolarity glycerol response (HOG) signaling pathway senses and responds to hyperosmotic stress . Activation of the HOG pathway increases the transcription of some proteins, among others also enzymes involved in glycerol synthesis [8,9]. The resulting cytosolic glycerol accumulation leads to increased internal osmolarity and restores the osmotic gradient between the cells and their environment .
The key MAP kinase of the HOG pathway in S. cerevisiae is Hog1p. Upon osmotic stress the upstream kinase Pbs2p, which also acts as a scaffold protein, phosphorylates Hog1p . Activation of Hog1p leads to its transfer from the cytoplasm to the nucleus  where it phosphorylates transcription factors [12,13]. This transfer and retention in the nucleus are transient .
The presence of HOG1 homologous genes have been reported in animals , plants , and fungal species [17–23], indicating that this pathway is conserved among eukaryotes. No information however is available on the hyperosmotic signal pathway in halophilic organisms adapted to extremely high salinity. In this study we present a H. werneckii gene with homology to S. cerevisiae HOG1 from which a putative protein sequence was determined. Activation of the H. werneckii Hog1p homolog HwHog1p is discussed.
Materials and methods
Strains and growth condition
Cultures of H. werneckii (MZKI B-763) (Ascomycota, Dothideales) from the culture collection of the Slovenian National Institute of Chemistry (MZKI) and S. cerevisiae MZKI K-86 (wild-type) were used in this study. Cells were cultured as described previously  and harvested in the mid-exponential phase (OD600= 0.8–1.0) by centrifugation at 4000×g for 10 min.
Plasma membrane preparation
H. werneckii cells were grown in YNB media with 17% NaCl (w/v) or without NaCl until the mid-exponential phase; the cells were then harvested, frozen in liquid nitrogen and mechanically disintegrated. Plasma membranes were isolated on sucrose gradient . The purity of the plasma membranes was checked by measuring the plasma, mitochondrial, and vacuolar ATPase activity .
In vitro kinase assay and Western blot analysis
The in vitro kinase assay and Western blot analysis were performed as described previously . H. werneckii Hog1p and Pbs2p homologs (HwHog1p and HwPbs2p) were immunodetected by anti-Hog1p (yN-19) and anti-Pbs2p polyclonal antibodies (Santa Cruz Biotechnology) according to the manufacturer's recommendations.
DNA and RNA isolation
Highly purified genomic DNA was isolated according to the phenol/chlorophorm/isoamyl alcohol method from mid-exponential phase cells grown in YNB media without salt .
Total RNA was isolated by the acid guanidinium thiocyanate/phenol/chlorophorm method  from mid-exponential phase cells grown in YNB media with 5, 17 or 25% NaCl (w/v) or without salt that were later exposed to different concentrations of NaCl for 20–120 min.
H. werneckii genomic library construction
Genomic DNA fragments were generated by partial digestion with Sau 3AI (Roche). The digestion products were separated on a 1% agarose gel and fragments of 2 kb were recovered (Qiaquick PCR Purification Kits, Qiagen). The genomic H. werneckii library was constructed in a pBK-CMV phagemid vector (Zap Express® Predigested Vector Kit), packaged using Zap Express® Predigested Gigapack® Cloning Kit, titered and amplified in the Escherichia coli XL1-blue MRF′ bacterial strain (Stratagene) according to the manufacturer's recommendations.
Cloning the H. werneckii gene homologous to the S. cerevisiae HOG1 gene
A HOG1-harboring DNA fragment (approximately 580 bp) was obtained by polymerase chain reaction (PCR) amplification with oligonucleotides 5′-ACGGAATTACAAGGAACAGATTTAC-3′ and 5′-GCAGTGATTCTCTTCTTAGGATC-3′ as primers and S. cerevisiae genomic DNA as the template DNA. The PCR was performed as described elsewhere , with an annealing temperature of 56°C. After purification by agarose electrophoresis and Qiaquick PCR Purification Kits (Qiagen), the DNA fragment was labeled with [32P]dCTP using the Prime-It® RmT Random Primer Labeling Kit (Stratagene) according to the manual.
To clone the HwHOG1 gene, we used an H. werneckii genomic library constructed in pBK-CMV phagemid vector. 6×105 plaque-forming units of recombinant phage were screened with a radioactively labeled HOG1 probe. Plaque lifts were made on the positively charged nitrocellulose membranes which were used in Southern blot analysis. Hybridization using 32P-labeled HOG1 probe was carried out at 55°C in hybridization solution (6×SSC, 0.5% sodium dodecyl sulfate (SDS), 5×Denhardt reagent, 150 µg ml−1 salmon sperm DNA) overnight. The blots were then washed three times for 15 min with 2×SSC, 0.1% SDS at 55°C. Positive clones were visualized by autoradiography and plaque purified .
The H. werneckii DNA inserts in positive phagemid vectors pBK-CMV were PCR amplified with oligonucleotides T3 and T7. Sequence analysis of obtained PCR products was performed by Sequiserve (Dr. W. Metzger, Vatterstetten, Germany). Searches for homologs in DNA and protein sequence databases were performed with the Blast programs , and phylogenetic analysis with Clustal X  and Tree View (Win32) 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html).
Northern and Southern blot analysis
For the probe a HwHOG1-harboring DNA fragment (approximately 470 bp) was obtained by PCR amplification with oligonucleotides 5′-CGCAAATGACCGGTTACGTCTCGA-3′ and 5′-CAGATCCGCATCGTTGAAGGACCA-3′ as primers, H. werneckii genomic DNA as the template DNA, and 32P-labeled as described above.
For Northern blot analysis, total RNA (20 µg per lane) isolated from cells grown at different NaCl concentrations for different times, were separated on a 1.2% agarose formaldehyde-containing gel. For genomic Southern blot analysis, 25 µg of high purity genomic DNA was digested with restriction enzymes Eco RI, Hin dIII and Bam HI (Roche) followed by electrophoresis on a 1% agarose gel. Transfer to a nitrocellulose membrane (Amersham) was performed by capillary blotting with 20×SSC. Nucleic acids were cross-linked to the membranes by baking for 2 h at 80°C. Hybridization using a 32P-labeled HwHOG1 probe was carried out at 65°C as described above. The blots were then washed with 2×SSC, 0.1% SDS two times for 15 min at 65°C and two times for 10 min at 75°C, followed by autoradiography .
Chromosomal localization of the HwHOG1 gene through PFGE
The isolation of H. werneckii chromosomal DNA and pulsed-field gel electrophoresis (PFGE) were performed according to Raspor et al. , the Southern blot from PFGE gel a chromoblot, was performed according to Bignell and Evans . The analysis of the chromoblot was performed as described above.
Results and discussion
Since glycerol as a compatible solute accumulates in halophilic H. werneckii under increased salinity , we assumed that a pathway similar to the HOG signaling pathway in S. cerevisiae is present in H. werneckii and plays an important role in its adaptation to an extremely high salinity. To obtain an insight into the hyperosmotic signal pathway in H. werneckii, we wanted to identify and characterize a key MAP kinase, a S. cerevisiae Hog1p homolog involved in the transmission of the hyperosmolarity signal. First, we isolated and characterized the HOG1 homolog from H. werneckii.
Isolation and characterization of the HwHOG1 gene from H. werneckii
A HOG1-harboring fragment was prepared from S. cerevisiae DNA by PCR amplification and used as a probe for screening the genomic library of H. werneckii.
Two positive clones were isolated from the library with approximately 1.9 kb long DNA inserts. From the nucleotide sequence of the inserts, a coding region with seven exons (data not shown) was identified that represented an open reading frame corresponding to a putative protein with 359 amino acid residues. Fig. 1 shows the nucleotide and the amino acid sequence of the isolated gene. As the deduced primary protein structure was highly similar to other MAP kinases, this open reading frame was named HwHOG1. Similarly to other MAP kinases a catalytic protein kinase domain was found from Tyr-20 to Leu-299. Inside the catalytic domain an active site with Asp-141 and a TGY motif at amino acids 171–173, characteristic for MAP kinases activated by hyperosmolarity , were found. On the C-terminal a common docking (CD) motif outside the catalytic domain  was also identified from Asp-296 to Glu-331. The CD domain contains acidic and hydrophobic residues. Asp-304 and Asp-307 of HwHog1p could serve to establish critical electrostatic interactions with the positively charged residues of docking domains of upstream and downstream effectors together with the hydrophobic residues Tyr-312 and His-313.
To estimate the number of HwHOG1 copies in the H. werneckii, Southern blot analysis of the genomic DNA and chromoblot analysis (Fig. 2A and B) were performed. The bands obtained for genomic DNA cut with Eco RI, Hin dIII and Bam HI suggested the existence of only one copy of the HwHOG1 gene in the genome (Fig. 2A). The hybridization of the HwHOG1 probe to the Eco RI digested DNA gave a signal corresponding to one 7 kb fragment. After the hybridization to the Hin dIII and Eco RI+Hin dIII digested DNA a signal was detected, corresponding to one 1.4 kb fragment. A signal was also detected after the hybridization to the Bam HI digested DNA (5 kb band size). The results of the Southern blots were confirmed by chromoblot analysis (Fig. 2B). The karyotype of H. werneckii showed 13 bands, ranging in size from 510 to 1850 kb (Gorjan, A., personal communication). The chromoblot analysis revealed that the HwHOG1 was localized only on one chromosome of approximately 705 kb.
Activation of HwHog1p
Preliminary studies on the activation of the Hog1p homolog in H. werneckii (HwHog1p), using antibodies against S. cerevisiae Hog1p confirmed the presence of a pathway similar to the HOG pathway . In vitro kinase assay, used to measure the activity of HwHog1p, showed the highest activity of HwHog1p on 17 and 25% NaCl (w/v) while at lower NaCl concentrations activity was scarcely noticeable (Fig. 3A). Activation of the HOG pathway is regulated in different organisms at different levels. In S. cerevisiae the activation of Hog1p is regulated at the posttranscriptional level , however, in Aspergillus nidulans the regulation is also at the level of transcription and HOG genes are upregulated when exposed to high concentrations of salt . To establish whether the different activation of HwHog1p is due to a different transcription of HwHOG1 under different NaCl concentrations, Northern blot analysis on HwHOG1 was carried out. After NaCl shocks of different intensities the level of the transcription of HwHOG1 was the same (Fig. 3B), indicating that the regulation of HwHOG1 is posttranscriptional, most probably at the level of the phosphorylation of protein.
Activation of HwHog1p correlated well with the localization of HwHog1p. Under nonstress conditions HwHog1p is primarily located on the plasma membrane and is translocated from the membrane upon increased salinity (Fig. 4A). HwHog1p was also detected in the cytosol where no noticeable differences were observed in its presence before and after the osmotic stress (data not shown).
In S. cerevisiae studies with a functional Hog1p–green fluorescent protein (GFP) fusion reveal that under nonstress conditions Hog1p cycles between the cytoplasmic and nuclear compartments. Upon hyperosmotic stress, the Hog1p–GFP fusion protein rapidly transfers and accumulates in the nucleus by a mechanism that is not yet known. For Hog1p phosphorylation by an upstream MAP kinase, Pbs2p is essential. After return to an iso-osmotic environment or after adaptation to high osmolarity, Hog1p returns to the cytoplasm .
Western blot analysis, using anti-S. cerevisiae Pbs2p antibodies, revealed the presence of the Pbs2p homolog HwPbs2p. Under nonstress conditions no HwPbs2p was detected on the plasma membrane (Fig. 4B, left lane) in H. werneckii. Upon increased external salinity, HwPbs2p was recruited to the plasma membrane (Fig. 4B, central lane) where it presumably phosphorylates HwHog1p (Fig. 4A, left lane). This phosphorylation and activation of HwHog1p lead to its translocation from the plasma membrane (Fig. 4A, central lane) probably to the nucleus where it could stimulate expression of genes involved in glycerol synthesis, while HwPbs2p remained on the membrane (Fig. 4B, central lane). After prolonged hypersaline stress, HwPbs2p could be still detected on the plasma membrane (Fig. 4B, right lane), while HwHog1p was barely detectable (Fig. 4A, right lane). We assume that under constant hypersaline stress HwHog1p cycles between the plasma membrane, cytoplasm, and nucleus, and is constantly phosphorylated and dephosphorylated. Different localization of the Hog1p homolog under nonstress conditions in H. werneckii (plasma membrane) in comparison to S. cerevisiae (cytoplasm) could represent another adaptation to life in environments with extremely high salinity.
In conclusion, in the present paper we present the novel Hog1p homolog from the black yeast H. werneckii adapted to extremely high salinities. Although its protein sequence is quite similar to the Hog1p homologs from non-halophilic organisms, we observed important differences in its activation and localization. HwHog1p is totally activated only at extremely high NaCl concentrations. Under nonstress conditions HwHog1p is present also at the membrane while in non-halophilic S. cerevisiae it is localized in the cytoplasm. Since the HOG signaling pathway senses and responds to hyperosmotic stress, it is crucial in the response to the hypersaline stress, differences in this pathway between H. werneckii and non-halophilic organisms might represent an important molecular mechanism in adaptation to high salt concentrations.
We would like to thank Alenka Gorjan for communicating results prior to publication. We would also like to thank all our colleagues for their useful suggestions and technical assistance. M.T. is supported by a fellowship from the Slovene Ministry of Education, Science and Sport.