1 Ecology of Hortaea werneckii

Bacteria and Archaea dominate natural environments characterized by extreme physico-chemical properties. Nevertheless, eukaryotic microoorganisms such as the “black yeasts” (de Hoog and Hermanides-Nijhof 1977), microcolonial fungi (Staley et al. 1982) and meristematic ascomycetes (Sterflinger et al. 1999) are also remarkably successful in adapting to certain extreme environments (Staley et al. 1982; Gorbushina et al. 1993; Nienow and Friedmann 1993; Wollenzien et al. 1995; Gunde-Cimerman et al. 2000). Although they have been known since the end of the 19th century (de Hoog et al. 1999), difficulties in their morphological identification together with their slow growth and low competitive ability frequently hindered the isolation and identification of these fungi. The representatives of black yeasts belong to the ascomycetous orders Chaetothyriales, Dothideales and Pleosporales (Sterflinger et al. 1999). Several genera and species of black yeasts from the order Dothideales represent a group of rare halophilic eukaryotic microorganisms, highly appropriate for studying the mechanisms of salt tolerance in eukaryotes (Petrovič et al. 1999); (Petrovič et al. 2002); (Turk and Plemenitaš 2002). So far little is known about eukaryotic halophilic microorganisms, let alone the mechanisms of their adaptation to such conditions (Petrovič et al. 2002).

Microscopy and histological studies of black yeasts have revealed that their morphological ecotype is important for their survival in various extreme environments. Black yeasts are characterized by melanized slowly expanding colonies, with reproduction by isodiametric enlargement of subdividing cells (Sterflinger et al. 1999; Wollenzien et al. 1995). Their distinctive features are polymorphism, meristematic growth, endoconidiation or sarcinic conidiogenesis, frequently muriform cells which develop by conversion from undifferentiated hyphae, and thick, melanized cell walls (de Hoog 1993; Zalar et al. 1999; Sterflinger et al. 1999; Wollenzien et al. 1995).

Hortaea werneckii, the best described eukaryotic halophilic model organism to date, is a characteristic black yeast. In the past, its identification was based solely on morphological characteristics and has thus received many designations: Cladosporium werneckii, Exophiala werneckii, Pullularia werneckii, Aureobasidium werneckii, A. mansonii, Sarcinomyces crustaceus and Phaeoannellomyces werneckii. Nowadays, the identification is additionally based on nutritional physiology and molecular methods. The physiological tests include metabolism of different C and N sources with or without NaCl in high concentrations, as well as temperature-tolerance tests. Molecular differentiation is based on the sequencing of the ITS rDNA region and RFLP markers from SSU rDNA and ITS rDNA regions (de Hoog et al. 1999).

Until recently, the genus Hortaea contained only a single species, H. werneckii (Horta) Nishimura and Miyaji, with no known sexual stage (Zalar et al. 1999). In 2004, another species was described and named H. acidophila (Holker et al. 2004).

Hortaea werneckii was long known primarily as the etiological agent of human tinea nigra, a superficial infection of the human hand, particularly frequent in warmer areas of the world. Investigations have revealed that the fungus is strictly limited to the dead surface of the skin (stratum corneum), in particular to the grease on the skin. Since H. werneckii does not show any keratin-degrading activity, it does not invade the living tissue below and so the infection is only a cosmetic problem (Göttlich et al. 1995). Besides its involvement in tinea nigra, H. werneckii was also known as one of the few species of fungi capable of contaminating food preserved with high concentrations of NaCl (Mok and Barreto da Silva 1981), without showing any obligate requirement for NaCl (Andrews and Pitt 1987). In addition to human skin and salty food, the fungus has been isolated from seawater (Iwatsu and Udagawa 1988), marine fish (Todaro et al. 1983), beach soil (de Hoog and Guého 1998) and arid inorganic and organic surfaces (Krumbein et al. 1996). On the basis of H. werneckii random isolations from different low water activity substrates and in vitro ecophysiological studies it was suggested that salt might be the decisive factor in its ecology and therefore in the etiology of tinea nigra. Furthermore, it was thus speculated that H. werneckii might grow in drying salty ponds at the seaside (de Hoog and Gerrits van den Ende 1992). However, the primary environmental ecological niche of H. werneckii remained unknown until we investigated salterns along the Slovenian Adriatic coast for the potential presence of halophilic fungi. Their distribution was followed throughout two successive years in five different evaporitic ponds, covering the entire salinity range (3–32% NaCl) (Gunde-Cimerman et al. 2000; Butinar et al. 2005). This study revealed that hypersaline waters of the salterns harbour different species of melanized fungi from the order Dothideales. They appeared in three peaks, at the water salinities 5–8%, 10–20% and 18–25% NaCl, which correlated primarily with high environmental nitrogen values. At the highest environmental salinities, melanized fungi represented 85–100% of the total isolated mycobiota, but they were partially replaced by non-melanized fungi with lowering salinities and they were detected only occasionally at the end of the season, when NaCl concentrations were below 5%. H. werneckii was the dominant black-yeast species in the Adriatic salterns during the season of salt production (Gunde-Cimerman et al. 2000). Initially it was isolated from the crystallization ponds of the Adriatic salterns, but it was later also identified in hypersaline waters of six salterns on three continents (Butinar et al. 2005).

Besides being the dominant black yeast species in hypersaline water at salinities above 20%, H. werneckii was also isolated from various microniches within the salterns: the surface and interior of wood submerged in brine, from biofilms on the surface of hypersaline water, and from dry ponds and microbial mats (Butinar et al. 2005; Zalar et al. 2005). However, it appears that H. werneckii survives in eutrophic thalassohaline waters of salterns in temperate climatic zones, since it was only occasionally retrieved from salterns in Puerto Rico, but never from the oligotrophic salterns in Eilat at the Red Sea in Israel, or the athalassohaline waters of the Dead Sea, or those of Salt Lake, Utah (Gunde-Cimerman et al. 2005).

In contrast to most prokaryotic halophiles, which display better growth in the presence of NaCl, the growth salinity range for H. werneckii, defined in vitro, is from 0% to saturation (32%) NaCl, with a broad optimum from 6% to 14% NaCl. Its complex polymorphic life cycle enables H. werneckii to show versatile ecotype adaptations in response to changing environmental concentrations of NaCl, UV intensity, nutrients and water availability. If sufficient nutrients are available, the hydrophilic yeast phase rapidly colonizes hypersaline water. At salinites above 15% NaCl, yeast cells begin to differentiate into meristematic budding cells, and at the highest salinities they form dormant meristematic sclerotial bodies with endogenous conidiation. Clustered growth allows sheltering of interior cells and minimizes the number of cells directly in contact with the hostile environment. Under conditions of drought, with no water in the ponds, the fungus changes into an aerophilic, hydrophobic hyphal stage, producing conidia which can be dispersed by air currents. These conidia can germinate in saline water, giving rise to actively propagating yeast cells (Butinar et al. 2005).

2 Molecular adaptations of Hortaea werneckii

2.1 Membranes and cell-wall pigmentation

As the H. werneckii cell is in contact with its environment via the plasma membrane, the adaptability and flexibility of this membrane is of vital importance to the survival of the cell. The cell should be able to modify the lipid composition and consequently properties of its membranes if the conditions in the environment change. The fluidity of biological membranes is largely influenced by the length, branching and degree of saturation of the fatty acids, the amount of sterols and the nature of the phospholipids (Russell 1989a, b).

The influence of salt stress on lipid composition and membrane properties has been studied in bacteria (Russell et al. 1995) and in yeasts, including the salt-sensitive Saccharomyces cerevisiae (Sharma et al. 1996; Tunblad-Johansson and Adler 1987) and several halotolerant yeasts. These organisms showed different responses to salt stress. While Zygosaccharomyces rouxii showed increased amounts of free ergosterol, decreased amounts of unsaturated fatty acids and decreased membrane fluidity when grown with NaCl (Hosono 1992; Yoshikawa et al. 1995), high salinity did not induce significant changes in the unsaturation of fatty acids in Yarrowia lipolitica (Andreishcheva et al. 1999; Yoshikawa et al. 1995) but caused a decrease in phospholipid and sterol contents. In contrast, Candida membranefaciens grown at high NaCl concentrations exhibited increased unsaturation in fatty acids and an increase in the contents of phosphatidylinositol (PI) and phosphatidylethanolamine (PE), resulting in slightly higher membrane fluidity (Khaware et al. 1995). The plasma membrane of the marine yeast Debaryomyces hansenii adapts to stress conditions by decreasing fluidity and increasing the sterol-to-phospholipid ratio in the presence of salt (Turk et al., submitted). Our studies have shown that salt stress does not significantly influence the total sterol content in halophilic H. werneckii, but does cause an increase in the phospholipid content. The most abundant fatty acids in phospholipids contained C16 and C18 chain lengths with a high percentage of C18:2Δ9,12. Salt stress also caused an increase in the fatty acid unsaturation. Halophilic fungi maintained their sterol-to-phospholipid ratio significantly lower than the salt-sensitive S. cerevisiae. Additionally, EPR measurements showed that the membranes of H. werneckii were significantly more fluid in comparison with the membranes of the above mentioned fungi, of the halotolerant black yeast A. pullulans and also the salt-sensitive S. cerevisiae (Turk et al. 2004). Additionally, it was demonstrated that halophilic H. werneckii maintained this very high fluidity over a wide range of NaCl concentrations, indicating high intrinsic salt-stress tolerance. Results were in good agreement with eco-physiological data and the dominance of H. werneckii in hypersaline waters of salterns. As the membrane fluidity of the related halotolerant black yeast A. pullulans was different from that of halophilic H. werneckii and resembled more that of salt-sensitive S. cerevisiae (Turk et al. 2004), membrane fluidity appears to be a good indicator of the degree of salt tolerance.

It has been reported that hyperosmotic shock induces changes in the organization of cell wall, most probably as a result of displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane (Slaninova et al. 2000). No significant invaginations occurred in H. werneckii at low salinities, but such changes in its cell-wall structure were apparent at high salinities (Kogej 2006). However, we also observed salt-dependent changes of the cell wall structure previously unobserved in fungi, which was caused by the redistribution of the melanin which is responsible for the dark colour of H. werneckii (Kogej 2006). We demonstrated that H. werneckii synthesizes 1,8-dihydroxynaphthalene (DHN) melanin under saline as well as non-saline growth conditions (Kogej et al. 2004). While the biosynthesis was not salt-dependent, the ultrastructural studies of the cell wall of H. werneckii showed for the first time that the melanin granules in the outer part of the cell walls are loosely organized in the medium without salt, and are more densely packed as the salt concentration in the medium increases (Kogej 2006; Plemenitaš and Gunde-Cimerman 2005). Since melanins are known to confer protection to UV-irradiation, temperature extremes (Bell and Wheeler 1986), desiccation (Zhdanova et al. 1990; Zhdanova and Pokhodenko 1973) and have an osmotic role (Elliot and Henson 2001), it is reasonable to suggest a potential osmoprotectant role of melanin in the cell wall of halophilic H. werneckii.

2.2 Sodium and potassium in the cells of Hortaea werneckii

Cells living in natural saline systems, where high salt concentrations cause high osmotic pressure, must maintain lower water potential than their surroundings in order to survive and proliferate. At the same time they have to keep the intracellular concentrations of sodium ions bellow the toxic level for the cells. Halophilic microorganisms have developed different strategies for counterbalancing osmotic pressure. Extremely halophilic Archaea accumulate KCl up to molar concentrations when exposed to high external salinity (Oren 1999). In contrast, eukaryotic microorganisms cannot tolerate high intracellular ion concentrations. Mechanisms of salt tolerance have been studied on salt-sensitive S. cerevisiae (Blomberg 2000) and a few halotolerant fungi such as the filamentous yeasts Debaryomyces hansenii, Candida versatilis, Rhodotorula mucilaginosa and Pichia guillermondii (Andre et al. 1988; Ramos 1999, 2005; Almagro et al. 2000; Silva-Graça and Lucas 2003; Prista et al. 2005). Data on these fungi show that the maintenance of positive turgor pressure at high salinity is mainly due to an increased production and accumulation of glycerol, trehalose and other organic compatible solutes. However, it is also known that in D. hansenii osmotic adjustments of the major intracellular cations also occurs in response to osmotic stress (Blomberg and Adler 1992; Ramos 2005). It was shown that D. hansenii keeps relatively high amounts of internal sodium when grown under salt stress and so this yeast has been defined as a Na+-includer organism (Ramos 2005).

In contrast we demonstrated that H. werneckii keeps very low intracellular amounts of potassium and sodium even when grown in the presence of 25% NaCl, which prevents the growth of other investigated fungi, thus indicating the efficient Na-excluding character of this organism. Interestingly, in H. werneckii the amounts of K+ and Na+ were the lowest in the cells grown at 17% NaCl. At this salinity of the medium H. werneckii still grows well, but most probably this salinity represents a turning point, shown in restricted colony size, slower growth rate and characteristic changes of physiological behaviour (Plemenitaš and Gunde-Cimerman 2005). When cells of H. werneckii were exposed to hyperosmotic shock, both non-adapted cells grown without NaCl and salt-adapted cells grown at 10% NaCl reacted by an immediate drop in the amount of K+. On the other hand, the amount of Na+ in non-adapted cells remained almost unchanged after hyperosmotic shock, while the salt-adapted cells showed increased values and fluctuating values of sodium. The observed pattern of ion fluctuations after hyperosmotic shock is in accordance with the growth characteristics of H. werneckii. Twenty percent NaCl slowed the growth rate of H. werneckii, indicating the sensitivity to increased intracellular sodium. Our results nevertheless indicate that due to their low concentrations, cations probably do not contribute significantly to osmoadaptation in H. werneckii and that H. werneckii possesses a very efficient export system for Na+ and K+ (Kogej et al. 2005).

2.3 Sodium and potassium efflux through the membrane of Hortaea werneckii

When exposed to a hypersaline environment, many organisms exclude Na+ ions from the cytoplasm, due to the potentially toxic effects of these ions. Many fungi use ENA P-type ATPases as one of the mechanisms for Na+ and/or K+ export. However, most of the data on ENA ATPases are derived from studies on salt-sensitive and moderately halotolerant fungi. In S. cerevisiae, several ENA ATPases mediate sodium efflux processes (Garciadeblas et al. 1993), while most other fungi have only one ENA ATPase, like in Schizosaccharomyces pombe (Benito et al. 2002), Zygosaccharomyces rouxi (Watanabe et al. 1999) and Neurospora crassa (Benito et al. 2002) or two ENA ATPases, as found in Schwanniomyces occidentalis (Banuelos and Rodriguez-Navarro 1998) and D. hansenii (Almagro et al. 2000, 2001). All of these ENA ATPases are plasma membrane proteins. Most of them are equally effective in suppressing the sensitivity of cells to K+ or Na+, while NcENA1 from N. crassa was reported to be more effective for Na+.

We have recently isolated and characterized two HwENA genes coding for Ena ATPases from H. werneckii (Gorjan and Plemenitaš, submitted). Their protein sequences, deduced from the cDNA sequences of the respective genes, revealed that both of the amino acid sequences contain conserved domains significant for cation-transporting ATPases. Since that both HwENA genes were seen to be located on the same chromosome, we speculate that they are arranged in tandem, similarly to those in S. cerevisiae, where ENA genes constitute a tandem array of 4–5 genes on one chromosome.

We also found that the expressions of both HwENA genes, HwENA1 and HwENA2, were highly salt responsive. In adapted cells, the expression levels of both HwENA genes were relatively low below 17% NaCl, but became strongly induced in a hyper saline environment (25% NaCl), and this was particularly marked with the expression of the HwENA2 gene. In contrast, the expression profile of HwENA genes from the cells which were exposed to salt stress by sudden increase in NaCl concentration in the medium, revealed that the level of HwENA2 mRNA was lower then HwENA1 mRNA. We also found that mRNA expression of both genes was induced only after 90 min. These results suggest that HwENA genes are involved in the late response of the cells to salt stress, with HwENA1 being more responsive to sudden changes in the salt concentrations, and HwENA2 playing a more important role in the mechanism of maintaining low cation content in adapted cells. It was found that adapted cells of H. werneckii have low sodium and potassium intracellular contents, which does not vary much with increasing extracellular salt concentrations (Kogej et al. 2005). We assume that the adapted cells employ a variety of mechanisms to maintain low intracellular cation contents over a wide range of salinity, and we suggest that HwENA ATPases contribute to these mechanisms only at extremely high NaCl concentrations. A high concentration of Na+ ions in the environment probably causes the entry of more Na+ ions into the cells, so more Na+ exporters are needed. The higher demand for those exporters presumably triggers the additional transcription of HwENA genes.

The high similarity of ENA ATPases between H. werneckii and other fungal ENA ATPases involved in Na+/K+ transport, together with salt-responsive gene expressions described above, indicate the potential importance of this system in ion homeostasis in the halophilic black yeast H. werneckii. Phylogenetically, HwENA ATPases belong to a separate group of fungal alkali cation P-ATPases, evolving alongside other fungal P-type ATPases from a common ancestor (Gorjan and Plemenitaš, submitted). While the function of most of these P-type ATPases has not yet been determined, it was speculated that NcENA2 from N. crassa is involved in K+ metabolism (Benito et al. 2002). It is believed that genes encoding fungal K+- or Na+-ATPases (ENA P-type ATPases) have most probably evolved from an ancestral K+-ATPase through the processes of gene duplication and that the capacity of ENA ATPases to pump Na+ has evolved as an adaptation mechanism to increased salinity (Benito et al. 2002).

2.4 Compatible solutes

Halophilic microorganisms living in hypersaline conditions adapt to high osmotic pressure and avoid associated water loss by accumulation of osmolytes—either cations or various organic solutes in the cytoplasm. In eukaryotic species, exemplified by the salt-sensitive yeast S. cerevisiae and the green alga Dunaliella salina, osmoadaptation is predominantly achieved through accumulation of organic solutes, also termed compatible solutes. Glycerol is the predominant solute used for this purpose, increasing the intracellular osmotic potential and simultaneously protecting cellular structures from adverse conditions (Blomberg and Adler 1992; Oren 1999).

Our first measurements (Petrovič et al. 2002) showed that the intracellular glycerol concentration in halophilic H. werneckii cells grown at different salinities steadily increases from 0% to 10% NaCl. At higher salinities, the intracellular glycerol concentration remains virtually unchanged. On the other hand, the extracellular glycerol concentrations are seen to be low and independent of salt concentration between 0% and 17% NaCl and start to increase above this salinity. We hypothesized that synthesized glycerol is efficiently kept inside the cells; although we could not exclude the possibility that glycerol crosses the plasma membrane and is then actively taken up into the cells (Petrovič et al. 2002). This assumption is in accordance with previous reports from other microorganisms (Nevoigt and Stahl 1997; Blomberg 2000). The increase in glycerol synthesis in H. werneckii was supported with the identification of two genes coding for putative glycerol-3-phosphate dehydrogenase; HwGPD1 and HwGPD2. According to the expression profile obtained by RT-PCR, only HwGPD2 is expressed differentially at increased salinity (Petrovič et al. 2002). These results suggest that glycerol biosynthesis in H. werneckii is regulated at the level of transcription of glycerol-3-phosphate-dehydrogenase, like that of S. cerevisiae.

Although our results show that glycerol is the most important compatible solute, we investigated the presence of other compatible solute(s) in this halophilic black yeast. Indeed, H. werneckii accumulates other higher polyols besides glycerol in a salt-dependent manner (Kogej 2006). This is in accordance with the findings in other fungi, where polyols such as erythritol, inositol, arabinitol, xylitol and mannitol, were also found to be used for osmoadaptation (Pfyffer et al. 1986; Blomberg and Adler 1992). Furthermore, other compatible solutes besides polyols are also produced in response to salt stress in microorganisms, such as nitrogen-containing compounds like glycine betaine and free amino acids (Galinski 1995), while mycosporine-like amino acids (MAAs) were suggested as osmoprotectants in halophilic cyanobacteria (Oren 1997). MAAs preferentially contain an aminocyclohexenimine unit bound to an amino acid or amino alcohol group and absorb maximally in the range 310–360 nm (Badaranayake 1998; Libkind et al. 2004). Mycosporines are similar substances with an aminocyclohexenone unit bound to an amino acid or amino alcohol group (Bandaranayake 1998), which have been described in fungi as UV sunscreens (absorbing 310–320 nm) and as being involved in morphogenesis and sporulation (Leach 1965; Trione et al. 1966). We determined the mycosporine and MAA content in fungi grown in hypersaline conditions (Kogej et al. 2006). Mycosporine-glutaminol-glucoside and mycosporine-glutamicol-glucoside were two mycosporines detected in the extracts of H. werneckii. These mycosporines were previously identified as metabolites of microcolonial fungi (MCF) inhabiting UV-exposed rocks in arid and semi-arid regions. The amount of mycosporine-glutaminol-glucoside was clearly salt-dependent, whereas the amount of mycosporine-glutamicol-glucoside decreased as salt increased. It seems that mycosporine-glutaminol-glucoside might act as supplementary compatible solute in H. werneckii exposed to variations in water activity or to hypersaline conditions. Further studies are needed to confirm its role in osmoadaptation (Kogej et al. 2006).

In conclusion, H. werneckii uses a unique set of compatible solutes for osmoregulation. Whereas D. hansenii uses K+ (Blomberg and Adler 1992; Ramos 2005) and glycerol (Almagro et al. 2000) to achieve this purpose, H. werneckii uses no cations for osmoadaptation (Kogej et al. 2005), but rather accumulates compatible solutes including glycerol (Petrovič et al. 2002), higher polyols and mycosporine-glutaminol-glucoside (Kogej 2006).

3 Hortaea werneckii—a eukaryotic model organism for halophilic adaptations

The halophilic black yeast H. werneckii is one of the most salt tolerant eukaryotic organisms so far described. Our studies on its adaptations have revealed some new mechanisms that enable H. werneckii to thrive at extremely high NaCl concentrations and also to quickly adapt to a wide range of NaCl concentrations. This adaptive halophilic behaviour and complex polymorphic life cycle ensure H. werneckii dominance amongst fungi inhabiting the hypersaline waters of eutrophic salterns. Differences have been observed on the level of cell-wall pigmentation and structure, membrane fluidity and compatible solutes. Preliminary global studies on the transcriptome of H.werneckii allow us to speculate on the existence of double sets of genes involved in the mechanism of adaptation to life in hypersaline environments, such as the genes HwENA and HwGDP, which are expressed differentially at different salinities (Petrovič et al. 2002; Gorjan and Plemenitaš, submitted). This seems a plausible explanation for the successful euryhaline strategy employed by H. werneckii.