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1 University of Ljubljana, Biotechnology Faculty, Department of Biology, Ve
na pot 111, SI-1000 Ljubljana, Slovenia
2 Institut für Mikrobiologie und Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany
3 Geomicrobiology, ICBM, Carl von Ossietzky Universität Oldenburg, POB 2503, D-26111 Oldenburg, Germany
Correspondence
Nina Gunde-Cimerman
nina.gunde-cimerman{at}bf.uni-lj.si
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Environmental Biochemistry, Carl von Ossietzky Universität Oldenburg, POB 2503, D-26111 Oldenburg, Germany.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Strategies for cellular osmotic adaptations are conserved from bacteria to man (Klipp et al., 2005). A well-studied model system for research on osmotic adaptation and regulation is the moderately halotolerant baker's yeast Saccharomyces cerevisiae (Blomberg, 2000; Hohmann, 2002; Klipp et al., 2005; Mager & Siderius, 2002). A lot of information is available on its osmotic shock-induced signal transduction, accumulation of osmolytes and control of gene expression (Hohmann, 2002). However, while 3 % NaCl represents a concentration that is toxic for S. cerevisiae, the same concentration of NaCl is close to the growth optimum for the halotolerant yeast Debaryomyces hansenii (Prista et al., 1997) and the halophilic black yeast Hortaea werneckii (Gunde-Cimerman et al., 2000). The black yeast H. werneckii is a ubiquitous inhabitant of eutrophic hypersaline waters of salterns (Butinar et al., 2005; Cantrell et al., 2006; Gunde-Cimerman et al., 2000) and is one of the most salt-tolerant eukaryotic organisms so far described. It can grow, albeit extremely slowly, in a nearly saturated salt solution (30 % or 5.1 M), as well as completely without salt (Gunde-Cimerman et al., 2000). As not many extremely salt-tolerant eukaryotic micro-organisms are known, H. werneckii represents a novel model organism for studying the mechanisms of osmotic adaptation and salt tolerance in eukaryotes (Petrovi et al., 2002). Since the first isolation of H. werneckii from hypersaline water in 1997, we have studied various aspects of its adaptation to saline environments (see Plemenita
& Gunde-Cimerman, 2005 for a review).
Halophilic micro-organisms have developed different strategies to counterbalance changes in osmotic pressure. The strategy of the accumulation of KCl up to molar concentrations in extremely halophilic Archaea requires complete adaptation of the intracellular components, which can function at high intracellular salt concentrations (Oren, 1999). A more widely used strategy, used by most halophilic and halotolerant Bacteria and eukaryotic halophiles (Oren, 1999), involves the cytoplasmic accumulation of ‘compatible solutes’, low-molecular-mass organic compounds, which provide the cells with osmotic balance and turgor without interference with cellular metabolism (Brown, 1976). At high salinity, halotolerant and halophilic fungi maintain positive turgor pressure mainly by the increased production and accumulation of glycerol. Other compatible solutes include other polyols such as erythritol, inositol, arabitol, xylitol and mannitol (Blomberg & Adler, 1992; Pfyffer et al., 1986), nitrogen-containing compounds such as glycine betaine and free amino acids (Galinski, 1995), sodium (Prista et al., 1997), mycosporines (Kogej et al., 2006b; Oren & Gunde-Cimerman, 2007), and mycosporine-like amino acids (MAAs; Oren, 1997).
Preliminary studies have shown that glycerol is the most important compatible solute in halophilic H. werneckii, but also indicated the probable presence of other compatible solute(s) or ions (Petrovi et al., 2002). As we have shown that H. werneckii maintains very low intracellular amounts of potassium and sodium even when grown in the presence of high NaCl concentrations (Kogej et al., 2005), osmotic adaptation by using ions may be ruled out.
Based on its constitutive melanization under saline as well as non-saline growth conditions (Kogej et al., 2004), we hypothesized that melanin has a role in the osmoadaptation of H. werneckii. In appresoria of the rice pathogenic fungus Magnaporthe grisea, melanization of the cell wall is involved in the retention of glycerol and hence is responsible for the generation of very high osmotic pressure, which is important for penetration of plant cells (de Jong et al., 1997).
The objective of this study was to reveal the mechanisms of osmoadaptation of the extremely salt-tolerant black yeast H. werneckii. We hypothesized that H. werneckii uses a combination of compatible solutes and cell-wall melanization for growth at a range of NaCl concentrations. The types and amounts of intracellular osmolytes were determined at various salinities and after exposure of the salt-adapted H. werneckii to hypoosmotic stress. The potential role of melanin in the process of osmotic adaptation of H. werneckii was also studied. The results revealed how this eukaryotic organism is able to tolerate a wide range of salinities using a combination of osmoadaptation strategies.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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ovlje, on the eastern coast of the Adriatic Sea (Gunde-Cimerman et al., 2000). It is preserved in the Culture Collection of the National Institute of Chemistry (MZKI), Ljubljana, Slovenia. The strain was kept on solid yeast nitrogen-base (YNB) medium at 4 °C for a few weeks or at –80 °C in 15 % (v/v) glycerol for long-term storage.
Media and growth conditions.
The cultures for biochemical analyses were grown in a defined liquid YNB medium with casamino acids [complete supplement mixture (CSM), both Qbiogene] as described previously (Kogej et al., 2005). Malt-extract medium (MEA) was used for growth of the fungal cultures that were prepared for transmission electron microscopy (TEM). MEA contained 2 % (w/v) Bacto malt extract (Difco), 0.1 % (w/v) Bacto peptone (Difco), 2 % (w/v) glucose, 2 % (w/v) agar and NaCl. The salinity of the media was adjusted with various concentrations of NaCl.
Determination of dry weight.
For dry-weight determination, three 10 ml samples of fungal cultures grown in liquid YNB media amended with various concentrations of NaCl were filtered through Millipore membrane filters (0.45 µm pore-size) and dried at 80 °C to constant weight. The weight was corrected for the salt content of the growth media.
Osmolytes in salt-adapted cells.
Fungal cultures were grown in triplicate in 100 ml YNB amended with various NaCl concentrations, to the mid-exponential, late-exponential and stationary growth phases, and prepared for HPLC analysis of intracellular osmolytes. Each experiment was performed at least twice. Cultures grown in YNB with 20 % (w/v) NaCl were also sampled during the early exponential and late-stationary phases to determine the dynamics of the intracellular osmolyte pool.
Osmolytes in cells after hypoosmotic shock.
Fungal cultures were grown in 150 ml YNB with either low 5 % (w/v) NaCl or high 20 % (w/v) NaCl concentrations to the mid-exponential and stationary growth phases, as described above. Each of the four cultures was then divided into three equal volumes and centrifuged at 2655 g (Centrifuge 5810 R, Eppendorf) and 28 °C for 5 min. The supernatant was removed, filtered (0.22 µm pore-size filter) and frozen until analysis. The cells were resuspended in three different dilutions of the growth media [20 , 50 and 80 % (v/v)], which were prepared from medium of the same salinity by the addition of distilled water. Then the cells were incubated at 180 r.p.m. at 28 °C for 1 h to allow the release of solutes. All experiments were performed in triplicate.
Osmolytes in melanin-inhibited cultures.
To compare the amount of osmolytes in non-melanized and melanized H. werneckii, the cultures were grown in 100 ml YNB with either low [5 % (w/v) NaCl] or high [20 % (w/v) NaCl] salt content, with and without a melanin-biosynthesis inhibitor. A dihydroxynaphthalene (DHN)-melanin inhibitor, tricyclazole (5-methyl-1,2,4-triazolo(3,4,b)-benzothiazole) (Eli Lilly), was added to the autoclaved YNB as an ethanol solution, so that the final tricyclazole and ethanol concentrations in YNB were 30 µg ml–1 and 1 % (v/v), respectively. As a control, the same amount of ethanol, without tricyclazole, was added to YNB. The cultures were grown to the exponential or stationary phase then samples for the analysis of osmolytes were prepared.
Preparation of samples for HPLC analysis.
Samples of cells grown to the appropriate growth phase [or in the case of hypoosmotic shock, cells after incubation in diluted growth media originally containing 5 or 20 % NaCl (for details of dilution see Methods, Osmolytes in cells after hypoosmotic shock)] were filtered through Millipore membrane filters (0.45 µm pore-size), and washed with an isoosmotic (the same NaCl concentration as in the growth medium) ice-cold solution of the same composition as the respective growth medium but without glucose. The cells collected on the filter were washed onto a new filter, and washed again in the same manner. They were scraped off the filter, frozen in liquid nitrogen and freeze-dried. The filtrates were also frozen at –20 °C until analysis.
Extraction of polyols.
Polyols were extracted from 30 mg freeze-dried cells by a modification of the method of Bligh & Dyer (1959) (Galinski & Herzog, 1990). The cells were suspended in 500 µl Bligh & Dyer solution (composed of methanol/chloroform/water, 10/5/4) and vigorously shaken for approximately 30 min (IKA-Vibrax VXR, Janke & Kunkel). Then, 130 µl chloroform and 130 µl demineralized water were added and the suspension was again incubated for 30 min with shaking. The samples were centrifuged at 5510 g (Biofuge A, Heraeus Instruments) for 10 min for phase separation. The upper aqueous phase containing the solutes was transferred to a 1.5 ml tube and frozen at –20 °C until analysis. The samples were diluted with an appropropriate volume of 80 % (v/v) acetonitrile before analysis.
Detection and characterization of polyols.
HPLC analysis was performed with the Spectra system (Thermo Separation Products) equipped with a P100 pump, UV 1000 UV detector and Shodex RI-71 (Showa Denko) RI detector. HPLC separations were performed with a Grom-Sil Amino-1PR column (3 µm, 125x4 mm, LiChrocart system, Grom) with a LiChrospher 100 NH2 guard column (Merck) at ambient temperature. Acetonitrile (Merck; 80 %, v/v) in water (HPLC quality) was used as a mobile phase at a flow rate of 1 ml min–1. Then, 20 µl sample was injected using a Rheodyne injector number 7125 (Rheodyne). Chromatogram analysis was performed with the chromatography software ChromQuest version 2.51 (Thermo Quest).
NMR measurements were performed on a Bruker Avance 300DPX unit using D2O as a lock signal and trimethylsilylpropionic acid sodium salt (TMSP) as the internal standard. Cell extracts from 
0.5 g dried cells were freeze-dried and resuspended in 1 ml D2O.
Extraction of mycosporines.
The freeze-dried biomass was pulverized under liquid nitrogen using a pestle and mortar. Solid–liquid extraction procedures as described by Volkmann & Gorbushina (2006) were used to extract the UV-absorbing compounds from the samples.
Detection and characterization of mycosporines.
Mycosporines and MAAs were characterized by a Waters Alliance 2795 HPLC System, equipped with a diode-array detector. A Nucleosil 100RP-18 column (5 µm, 4.6x250 mm, Macherey Nagel), equipped with a guard pre-column containing the same material, was used for separation. Then, 20 µl of the sample was injected onto the column using an autosampler system. The solution primarily used for the extraction [0.2 % aqueous acetic acid+0.5 % methanol (v/v)] was used as a mobile phase at a flow rate of 0.7 ml min–1. The diode-array detector was continuously scanning from 
=250 to =400 nm. The UV-absorbing mycosporines were identified by their UV-absorption maxima and retention times (Volkmann & Gorbushina, 2006). The fraction containing mycosporine-glutaminol-glucoside was collected and measured spectrophotometrically at 310 nm. The amount of mycosporine-glutaminol-glucoside was quantified as in Libkind et al. (2004) using the molar absorption coefficient (25 000 M–1 cm–1) from Bouillant et al. (1981).
Ultrastructure.
H. werneckii was grown and prepared for TEM, as described previously for Trimmatostroma salinum (Kogej et al., 2006a). H. werneckii B-736 was grown on solid MEA amended with either no NaCl or 5, 10 or 20 % (w/v) NaCl. Tricyclazole was added to autoclaved MEA as an ethanol solution, so that the final concentrations of tricyclazole and ethanol were 30 µg ml–1 and 1 % (v/v), respectively. Preculturing on the same MEA media as used in the experiment was used to adapt the fungi to the media and growth conditions. The inocula from pre-cultures were prepared as cell suspensions in sterile water of the same salinity and drop-inoculated onto a piece of cellophane overlaid on the surface of solid MEA media. The plates were incubated at 22 °C for 10 days in the dark (Kogej et al., 2006a). Ten-day-old colonies were fixed in 2.5 % glutaraldehyde and 4 % formaldehyde in 0.1 M phosphate buffer, pH 7.2, for 2 h at room temperature, and post-fixed in 1 % OsO4 for 1 h at 4 °C. Embedding in 2 % agarose was used after fixation to allow the manipulation of cells. Dehydration was done in a series of graded ethanol solutions: 30 % (v/v) (15 min), 50 % (2x15 min), 70 % (2x15 min), 90 % (2x15 min), and absolute ethanol (2x15 min, 1x30 min). Spurr's resin was used for embedding. Sections were cut with an ultramicrotome (Ultracut, Reichert) and contrasted with uranyl acetate and lead citrate. The sections were examined with a Philips CM 100 transmission electron microscope (80 kV; digital camera Gatan Bioscan Camera 792) (Kogej et al., 2006a).
Statistical analyses.
The differences among the mean amounts of intracellular osmolytes after hypoosmotic shock determined by HPLC analysis were compared by one-way ANOVA (n=3). The total polyol amounts were checked for correlation with dilution of the media. The differences between the mean amounts of intracellular osmolytes in non-melanized and melanized H. werneckii were first compared by one-way ANOVA (n=3), and then by Tukey's HSD test using Excel and XLSTAT (Addinsoft). Tukey's HSD test was used to test whether the mean amounts of solutes of non-melanized cells differed significantly from the mean amounts of solutes of both melanized controls (melanized unamended cells and melanized ethanol-amended cells).
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RESULTS |
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). In all growth phases, there was a significant correlation between the intracellular amounts of glycerol and the salinity of the growth medium (exponential and late-exponential phases, P=0.01; stationary phase, P=0.05). The amount of erythritol was not correlated with salinity. It increased between 0 and 10 % (w/v) NaCl and remained at a similar value at higher salinities. Additionally, low amounts of arabitol and mannitol were detected in H. werneckii grown at up to 10 % NaCl, but their amounts decreased as the salinity increased. At salinities higher than 10 % NaCl, only trace amounts of both arabitol and mannitol were detected. The total amount of polyols was correlated with increasing salinity mostly due to increases in glycerol in all growth phases. The combined amounts of erythritol, arabitol and mannitol correlated with salinity only in the exponential growth phase (data not shown; sum of values in Fig. 1b–d).
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). Its amount increased steeply from 0 to 5 % (w/v) NaCl, and decreased at higher NaCl concentrations, whereas mycosporine-glutamicol-glucoside appeared only in very low amounts [up to 0.2 µmol (g dry weight) –1] in the stationary-phase cells at all salinities (data not shown). Interestingly, the amount of mycosporine-glutaminol-glucoside in the stationary-phase cells was strongly correlated (P=0.01) with H. werneckii growth rates at the same salinities (Kogej et al., 2005).
We followed the growth-phase dependence of polyol and mycosporine accumulation in H. werneckii grown at 20 % (w/v) NaCl (Fig. 2). Glycerol accumulated predominantly in the exponential growth phase and diminished steeply in the stationary phase. On the other hand, the amount of erythritol increased gradually during the exponential growth phase and reached its highest level in the stationary phase. The amounts of mycosporine-glutaminol-glucoside and mycosporine-glutamicol-glucoside also increased steeply as the culture entered the stationary phase.
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In cells grown at optimal salinity (5 % NaCl) to the mid-exponential growth phase, moderate hypoosmotic shock caused a decrease in the intracellular amount of glycerol and a slight decrease of arabitol that was correlated with dilution. However, the amounts of erythritol and mannitol did not change significantly (Fig. 3a). On the other hand, drastic hypoosmotic shock caused a significant decrease in the intracellular amount of glycerol and a slight decrease in erythritol in cells grown at high salinity (Fig. 3c). The amounts of mycosporine-glutaminol-glucoside and mycosporine-glutamicol-glucoside in mid-exponential cells of H. werneckii were unaffected by hypoosmotic shock (data not shown).
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). Similarly, there was no change in the amount of mycosporine-glutaminol-glucoside or mycosporine-glutamicol-glucoside (data not shown). On the other hand, the strong decrease in both glycerol and erythritol in the drastically hypoosmotically shocked stationary-phase cells of H. werneckii grown at 20 % NaCl strongly correlated (P=0.05) with the dilution of the medium (Fig. 3d). The amount of mycosporine-glutaminol-glucoside increased significantly compared to the non-shocked cells, but the total amounts were low [up to 0.008 µmol (g dry weight) –1]. In conclusion, moderate hypoosmotic shock in mid-exponential phase cells of H. werneckii was most efficiently prevented by lowering the amount of the main compatible solute, glycerol, whereas in stationary-phase cells there was no significant change in polyols. On the other hand, drastic hypoosmotic shock in either mid-exponential or stationary-phase cells grown at 20 % NaCl caused loss of glycerol and, to a lesser extent, of erythritol also (mainly in stationary-phase cells).
Salinity affects cell-wall melanization in H. werneckii
The melanization of H. werneckii was studied at various salinities by TEM. The ultrastructure of melanized cells was compared to that of cells grown in the presence of the melanization inhibitor tricyclazole, a specific inhibitor of trihydroxynaphthalene reductase (Andersson et al., 1996). In melanized cells, melanin was observed as electron-dense granules (dark) in or on the electron-translucent (light-coloured) cell walls, whereas the cells with blocked melanin biosynthesis either had no electron-dense granules or these were smaller and lighter in colour (compare left and right columns in Fig. 4).
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). At 10 % NaCl, the electron-dense granules were deposited mostly on the cell wall, they were larger and scarce, and they did not form a continuous layer. On the other hand, in cells grown at 20 % NaCl, melanin granules were smaller and deposited in the outer layer of the cell wall (Fig. 4e, g). On tricyclazole-amended media, no melanin granules were visible in the cell wall, but separate smaller and lighter electron-dense granules were present on the outside of the cells (Fig. 4, right column). In conclusion, H. werneckii is most melanized at low salinities close to the growth optimum, whereas melanization is reduced at higher salinities.
Non-melanized H. werneckii cells retain less glycerol than melanized cells
We assessed the effect of blocked cell-wall melanization on polyol and mycosporine amounts in mid-exponential and stationary-phase H. werneckii cells grown with 5 and 20 % NaCl.
In mid-exponential phase H. werneckii cells grown at 5 % NaCl, non-melanized cells contained a significantly lower amount of glycerol (P=0.01) than melanized control cells, while the amounts of erythritol, arabitol and mannitol were higher than in melanized cells. The increased levels of erythritol and arabitol apparently replaced the diminished glycerol content (Fig. 5a). As a result, the total amount of polyols in melanin-inhibited and melanized cells did not change. The comparison of mean amounts of polyols by Tukey's HSD test showed that the amounts of glycerol, erythritol and arabitol in melanin-inhibited cells differed significantly (P=0.01) from those in melanized cells. On the other hand, the test showed no significant differences between the amounts of mannitol in melanized and non-melanized cells (P=0.05). Interestingly, in mid-exponential H. werneckii cells grown at 20 % NaCl, tricyclazole inhibition resulted in a significant increase in erythritol content in non-melanized cells compared to the melanized controls, while there was no statistically significant change in glycerol content (Fig. 5c). The amounts of mycosporines were low and were unaffected by tricyclazole inhibition (data not shown).
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), and also mycosporine-glutaminol-glucoside (Fig. 6a). Stationary-phase cells with blocked melanin synthesis grown at optimal salinity (5 % NaCl) contained significantly less erythritol (P=0.01) and significantly more arabitol (P=0.01) than melanized cells, while the amount of mannitol was significantly higher (P=0.01) in both types of control cells (melanized ethanol-amended and non-melanized tricyclazole-amended) than in melanized cells grown in unamended medium. The amount of mycosporine-glutaminol-glucoside was also significantly lower in non-melanized cells than in melanized cells (Fig. 6a). Melanin inhibition of stationary-phase cells grown at 20 % NaCl resulted in significantly lower amounts of glycerol (P=0.05), while the amounts of erythritol were increased compared to both controls (Fig. 5d). The tricyclazole-inhibited cells contained no mycosporines, while the control cells contained some mycosporine-glutaminol-glucoside (Fig. 6b).
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DISCUSSION |
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et al., 2002) and ion measurements (Kogej et al., 2005). It has been determined that the amounts of ions remain low at all salinities (Kogej et al., 2005), while the amount of glycerol correlates with increasing salinity in the medium (Petrovi et al., 2002). The main goal of this study was to unravel the complete osmoadaptation strategy of H. werneckii. Our results confirm that glycerol is its main compatible solute, since its intracellular amount was the highest of all solutes and it increased with salinity. It accumulated maximally up to 2.94 mmol (g dry weight) –1 during exponential growth at 25 % NaCl. However, H. werneckii also compensated for the external osmotic pressure caused by increased NaCl concentrations by accumulating various additional substances (erythritol, arabitol, mannitol and mycosporines). The composition of this mixture depended not only on the external salinity, but also on the age of the cells. Similar observations have been made for some halotolerant/halophilic eubacteria (Galinski & Trüper, 1994), which can change the composition of the solute pool according to growth phase and growth medium. In some halotolerant yeast species, the amount and type of polyol accumulation are also related to the growth phase (Nobre & da Costa, 1985) and the carbon sources used for growth (Van Eck et al., 1989).
Glycerol is known to be the primary osmolyte in many eukaryotic cells, such as in the halophilic green algae Dunaliella sp. (Ben-Amotz & Avron, 1973) and Asteromonas gracilis (Oren, 2002), and in fungi and yeasts, such as Aspergillus niger (Witteveen & Visser, 1995), S. cerevisiae (Blomberg & Adler, 1992), Debaryomyces hansenii (Adler et al., 1985), Zygosaccharomyces rouxii (Kayingo et al., 2001) and Yarrowia lipolytica (Andreishcheva et al., 1999). Besides glycerol, other polyols such as erythritol, arabitol and mannitol are also recognized to be compatible solutes in fungi. The strategy of accumulating a mixture of polyols is also common in many fungi (Davis et al., 2000) and is not unique to H. werneckii. For example, Candida sake accumulates equimolar amounts of glycerol and arabitol (Abadias et al., 2000). Erythritol is produced by osmophilic yeasts such as Pichia, Candida, Torulopsis, Trigonopsis, Moniliella and Aureobasidium sp. (Kim et al., 1997). All four polyols have been detected in some Aspergillus species (Beever & Laracy, 1986; Nesci et al., 2004) and in C. sake (Abadias et al., 2000). In addition to their use as compatible solutes, polyols can also serve as carbon storage compounds, they may help in balancing the cellular redox potential (Diano et al., 2006), and they can act as scavengers of reactive oxygen species (Voegele et al., 2005).
At optimal growth salinity, H. werneckii accumulated a mix of polyols with different lengths of carbon backbone (from three to six atoms) in various ratios. Experimental osmometric data of Davis et al. (2000) show that complex patterns of osmolyte accumulation cannot be explained by their effects upon the cytoplasmic osmotic pressure, because millimolar concentrations of the individual polyols, accumulated by H. werneckii, generate similar osmotic pressures (Davis et al., 2000). Such mixtures may reduce the toxicity associated with high concentrations of a single osmolyte, and obviate feedback mechanisms that down-regulate metabolic pathways in the presence of a high concentration of the product (Davis et al., 2000). As salinity increased over the optimal range for H. werneckii, the production and accumulation of glycerol rose, while the amounts of other polyols remained low. Interestingly, the osmometric data of Davis et al. (2000) also show that at high salt concentrations high-molecular-mass polyols generate higher osmotic pressures than lower-molecular-mass polyols at the same concentration. On the other hand, glycerol synthesis is energetically the least expensive option for cells growing at the highest salinities, since glycerol is energetically the cheapest polyol to produce (Oren, 1999).
Accordingly, glycerol is the main compatible solute in actively growing fungal cells in most cases. However, in H. werneckii, the amount of glycerol was drastically reduced in the stationary phase. Since glycerol was only partially replaced by higher levels of erythritol, the total amount of polyols decreased in the stationary phase. A similar shift in polyol pools has been observed in halotolerant D. hansenii, which accumulates glycerol in the exponential phase (André et al., 1988) and arabitol in stationary-phase cells (Adler & Gustafsson, 1980), whereas A. niger replaces glycerol with mannitol and erythritol in older mycelia (Witteveen & Visser, 1995). Some investigated fungi use high-affinity glycerol transporters to maintain high levels of glycerol, despite continuous efflux (Blomberg & Adler, 1992). Assuming that such transporters exist in H. werneckii, a possible explanation for glycerol loss and its partial replacement with erythritol in the stationary phase is a lack of energy for the functioning of glycerol-uptake systems due to a gradual depletion of the substrate. However, to our knowledge, no such transporters have been identified in H. werneckii to date.
The response of H. werneckii to increased salinities is in some way similar to that of other halotolerant yeast species. Both glycerol and erythritol have been documented as compatible osmolytes in yeast, but not mycosporines, which might act as additional compatible solutes in H. werneckii. Mycosporines, substances with an aminocyclohexenone unit bound to an amino acid or amino alcohol group, were initially known as morphogenetic factors in fungal sporulation and as UV-protecting compounds (Bandaranayake, 1998). Recently, it has been hypothesized that in certain micro-organisms, mycosporines or MAAs play a role as complementary compatible solutes (Kogej et al., 2006b; Oren, 1997; Oren & Gunde-Cimerman, 2007). In an earlier study, a higher amount of mycosporine-glutaminol-glucoside was measured in H. werneckii and in the cells of other black yeasts from a hypersaline environment (Phaeotheca triangularis and Aureobasidium pullulans) grown at 10 % NaCl than on non-saline growth medium (Kogej et al., 2006b), with a marked increase of mycosporines in the stationary-phase cells of H. werneckii. Interestingly, the amounts of mycosporines correlated with growth rates of H. werneckii. The highest amount of mycosporine-glutaminol-glucoside [158 µmol (g dry weight) –1] was detected in H. werneckii cells grown at optimal salinity (5 % NaCl) in the stationary phase. Here, mycosporine-glutaminol-glucoside represented 18.9 mol% of the total osmolytes, while there were 12 mol% glycerol and 50.3 mol% erythritol, in comparison. These results indicate that mycosporine-glutaminol-glucoside can be considered to be a complementary osmolyte in H. werneckii in the stationary phase.
Cells slowly adjusting to various salt concentrations, as described above, differed considerably from cells of H. werneckii exposed to sudden hypoosmotic shock. The shocked cells responded by lowering glycerol and erythritol, while the amounts of higher polyols and mycosporines remained unchanged. Thus, the hypoosmotic shock confirmed the role of glycerol as the main compatible solute, which was lowered to balance the sudden change in osmotic pressure. It seems that erythritol acts in the same way in the stationary-phase cells. At present, it is not, to our knowledge, known how H. werneckii is able to lower the amount of both polyols. In S. cerevisiae, an aquaglyceroporin channel Fps1p opens during steady-state growth. The combination of a closed Fps1p channel and upregulated glycerol production has been found to cause a marked increase in steady-state intracellular glycerol (Klipp et al., 2005). Fps1p is also open when S. cerevisiae cells are exposed to a sudden lowering of salinity in their environment, and allows for quick glycerol expulsion during a hypoosmotic shock (Luyten et al., 1995).
Glycerol presents a paradox for cells that hyperaccumulate compatible osmolytes. Due to its small molecular mass, it has an unusually high permeability coefficient for passage through the lipid bilayers relative to other uncharged, polar molecules. Thus, passive efflux from the cytoplasm would seem likely to limit glycerol accumulation (Blomberg & Adler, 1992). In the halophilic alga Dunaliella sp., the membrane permeability for glycerol is considerably lowered (Brown et al., 1982; Gimmler & Hartung, 1988), which is correlated with its high sterol content (Oren, 1999; Sheffer et al., 1986). In H. werneckii and other halophilic and halotolerant melanized fungi, ergosterol as the principal sterol and 23 other types of sterols (Turk et al., 2004) constitute the most distinctive lipid fraction of the cell membranes (Méjanelle et al., 2000). However, in H. werneckii the total sterol content remained largely unchanged with increased salinity, and the cells maintained high membrane fluidity over a broad range of salinities. Membrane fluidity was lower than in S. cerevisiae and in the halotolerant A. pullulans (Turk et al., 2004). Seemingly, there is a contradiction in the facts that (i) H. werneckii can grow at very high salinities, which require a high intracellular amount of glycerol, and (ii) at the same time it maintains a very fluid membrane by keeping a low sterol-to-phospholipid ratio, which does not prevent glycerol leakage. However, adaptation at the cell-wall level can minimize glycerol loss from the cells. For example, the melanized appresorial cell wall in the plant-pathogenic fungus M. grisea limits the loss of intracellular glycerol by creating a permeability barrier. This adaptation is crucial for generating a very high turgor by glycerol accumulation (Davis et al., 2000; de Jong et al., 1997).
H. werneckii also has melanized cell walls. The ultrastructural studies showed that the salinity of the growth medium affects the melanization patterns of H. werneckii. At 0–5 % NaCl, the melanin granules form a thin continuous layer in the outer part of the cell wall (Fig. 4a, c) and could thus create a mechanical permeability barrier for glycerol. Melanin granules probably limit cell-wall permeability by reducing the size of pores in the cell wall, as has been shown for Cryptococcus neoformans, in which cell-wall melanization reduces the size of pores from 10.6 nm in non-melanized to 4 nm in melanized cell walls (Jacobson & Ikeda, 2005). Indeed, when cell-wall melanization in H. werneckii was blocked by tricyclazole, the non-melanized cells accumulated significantly lower amounts of glycerol and significantly higher amounts of higher-molecular-mass polyols (see Fig. 6). This indicates that melanization limits the permeability of the cell wall to glycerol, the smallest of the detected polyols and the major compatible solute in H. werneckii. However, in H. werneckii grown at 10 and 20 % NaCl, the melanin granules are separate and do not form a continuous layer. Here, melanin probably has a less pronounced effect on the permeability, as is shown by the smaller difference in the glycerol content between melanized and non-melanized cells (Fig. 5c, d). Higher permeability to glycerol would also explain the higher extracellular levels of glycerol detected at high salinities in a previous study of H. werneckii (Petrovi et al., 2002).
Cell-wall melanization is an important mechanism that helps to maintain a high intracellular concentration of glycerol in H. werneckii despite its highly fluid membrane. A melanized cell wall might decrease the energetic needs of the cell, as glycerol accumulation from the medium as well as de novo synthesis can be reduced due to more effective glycerol retention. At optimal salinities, H. werneckii probably maintains a balance between two options: (i) an energetically cheap production of glycerol, which partially leaks out of the cells and therefore needs to be recovered by energy-dependent processes; and (ii) an energetically more costly synthesis of other compatible solutes, which escape less easily from the cells and are therefore retained more efficiently. At higher salinities, the cost of maintaining osmotic balance in the cells is probably much higher because of the higher concentration of glycerol needed for osmoadaptation, but also due to increased leakage of glycerol as H. werneckii cell walls are less melanized. Despite the choice of the shortest polyol as osmolyte at high salinities, which is energetically the cheapest to produce, the higher energetic demands of H. werneckii are reflected in reduced growth rates and biomass yield at salinities above 17 % NaCl (T. Kogej & N. Gunde-Cimerman, unpublished data). Perhaps the higher proportion of polymorphic cells that were observed with increasing salinity (T. Kogej & N. Gunde-Cimerman, unpublished data) is another mechanism for reducing glycerol leakage when melanization is diminished. Similar observations on the salinity-affected cell-wall melanization patterns of the other halophilic black yeast species T. salinum (Kogej et al., 2006a) and P. triangularis (T. Kogej & N. Gunde-Cimerman, unpublished data), which grow at similar salinities as H. werneckii, confirm our findings. This might indicate that melanization also has a role in the osmoadaptation of these fungi, but further studies are needed to confirm this.
In conclusion, the main compatible solute of H. werneckii is glycerol, which is replaced by erythritol and partially by mycosporine-glutaminol-glucoside in stationary-phase cells. At low salinities, H. werneckii accumulates a mixture of glycerol, erythritol, arabitol and mannitol, whereas glycerol and erythritol prevail at high salinities. The role of glycerol and erythritol as the most important osmolytes was confirmed by hypoosmotic shock. At optimal growth salinities, the melanized cell walls help in retaining high concentrations of glycerol in the cells of H. werneckii, despite the highly fluid membrane. The novelty of osmoadaptation of the halophilic fungus H. werneckii, probably contributing to its growth in a wide salinity range, is an effective combination of the accumulation of known compatible solutes (polyols) and of melanized cell walls for improved osmolyte retention. In combination, an array of adaptations at the levels of osmolyte production, membrane structure and cell-wall melanization interact to make H. werneckii a very versatile halophile, able to grow in a broader salinity range than most known micro-organisms.
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ACKNOWLEDGEMENTS |
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Edited by: N. L. Glass
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REFERENCES |
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Received 18 June 2007;
revised 28 August 2007;
accepted 7 September 2007.
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), erythritol (
) and mycosporines (
) in the cells of H. werneckii grown at 20 % (w/v) NaCl. The cultures were grown in liquid YNB medium with 20 % (w/v) NaCl at 27±1 °C on a rotary shaker and harvested at various growth phases. Growth was monitored by OD600 (
). The cell samples were extracted and measured by HPLC, as described in Methods. Data are taken from a single experiment.
