Tina Kogej

The Halophilic Fungus Hortaea werneckii and the Halotolerant Fungus Aureobasidium pullulans Maintain Low Intracellular Cation Concentrations in Hypersaline Environments

ABSTRACT Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na+ even when grown at high NaCl concentrations and can be thus considered Na+ excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K+ or Na+ for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H.werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment.

Mycosporines in Extremophilic Fungi—Novel Complementary Osmolytes?

Fungi isolated from hypersaline waters and polar glacial ice were screened for the presence of mycosporines and mycosporine-like amino acids under non-saline and saline growth conditions. Two different mycosporines and three unidentified UV-absorbing compounds were detected by high performance liquid chro- matography in fungal isolates from hypersaline waters and polar glacial ice. It was shown for the first time that the mycosporine-glutaminol-glucoside in halophilic and halotolerant black yeasts from salterns was higher on saline growth medium. This substance might act as a supplementary compatible solute in some extremophilic black yeasts exposed to saline growth conditions.

Morphological Response of the Halophilic Fungal Genus Wallemia to High Salinity

ABSTRACT The basidiomycetous genus Wallemia is an active inhabitant of hypersaline environments, and it has recently been described as comprising three halophilic and xerophilic species: Wallemia ichthyophaga, Wallemia muriae, and Wallemia sebi. Considering the important protective role the fungal cell wall has under fluctuating physicochemical environments, this study was focused on cell morphology changes, with particular emphasis on the structure of the cell wall, when these fungi were grown in media with low and high salinities. We compared the influence of salinity on the morphological characteristics of Wallemia spp. by light, transmission, and focused-ion-beam/scanning electron microscopy. W. ichthyophaga was the only species of this genus that was metabolically active at saturated NaCl concentrations. W. ichthyophaga grew in multicellular clumps and adapted to the high salinity with a significant increase in cell wall thickness. The other two species, W. muriae and W. sebi, also demonstrated adaptive responses to the high NaCl concentration, showing in particular an increased size of mycelial pellets at the high salinities, with an increase in cell wall thickness that was less pronounced. The comparison of all three of the Wallemia spp. supports previous findings relating to the extremely halophilic character of the phylogenetically distant W. ichthyophaga and demonstrates that, through morphological adaptations, the eukaryotic Wallemia spp. are representative of eukaryotic organisms that have successfully adapted to life in extremely saline environments.

Adaptation of extremely halotolerant black yeast Hortaea werneckii to increased osmolarity: a molecular perspective at a glance.

Halophilic adaptations have been studied almost exclusively on prokaryotic microorganisms. Discovery of the black yeast Hortaea werneckii as the dominant fungal species in hypersaline waters enabled the introduction of a new model organism to study the mechanisms of salt tolerance in eukaryotes. Its strategies of cellular osmotic adaptations on the physiological and molecular level revealed novel, intricate mechanisms to combat fluctuating salinity. H. werneckii is an extremely halotolerant eukaryotic microorganism and thus a promising source of transgenes for osmotolerance improvement of industrially important yeasts, as well as in crops.

Evidence for 1,8‐dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non‐saline conditions

The ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum are halophilic fungi that inhabit hypersaline water of solar salterns. They are characterized by slow, meristematic growth and very thick, darkly pigmented cell walls. The dark pigment, generally thought to be melanin, is consistently present in their cell walls when they grow under saline and non-saline conditions. We used the inhibitor tricyclazole to test the fungi in this study for the presence of 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, since fungal melanins reportedly are derived either from DHN, tyrosine via 3,4-dihydroxyphenylalanine, gamma-glutaminyl-3,4-dihydroxybenzene, or catechol. Tricyclazole-treated cultures of the fungi were reddish-brown in color and contained typical intermediates of the DHN-melanin pathway, as demonstrated by high-performance liquid chromatography. This investigation showed that the three fungi synthesized DHN-melanin under saline and non-saline growth conditions.

Osmoadaptation Strategy of the Most Halophilic Fungus, Wallemia ichthyophaga, Growing Optimally at Salinities above 15% NaCl

ABSTRACT Wallemia ichthyophaga is a fungus from the ancient basidiomycetous genus Wallemia (Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilic W. ichthyophaga and to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates of W. ichthyophaga were higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth of W. ichthyophaga was between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition, W. ichthyophaga maintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence, W. ichthyophaga should become well established as a novel model organism for studies of halophily in eukaryotes.

Fungi in Salterns

Salterns provide special living conditions for microorganisms. They are extreme environments because of high concentrations of NaCl and other salts, occasional rapid changes in water activity, low oxygen concentration, and high UV radiation (Brock 1979). It is generally assumed that microbial life in concentrated seawater at the highest salinities is mainly composed of Archaea and Bacteria and one eukaryotic species, the alga Dunaliella salina. Other eukaryotic microorganisms usually appear at lower salinities and are represented by different species of algae and protozoa (Ramos-Cormenzana 1991; Pedros-Alio et al. 2000). Surprisingly, until recently, fungi have not been isolated from natural hypersaline environments (Buchalo et al.1998; GundeCimerman et al. 2000), although xerophilic fungi able to grow on media with low water activities are frequently isolated from food preserved with high concentrations of salt or sugar (Filtenborg et al. 2000). It seems that growth of the few known xerophilic species of food-borne fungi in the presence of high concentrations of the solute is determined primarily by the water activities of the medium and not by the chemical nature of the solute. This explains why only as late as 1975 the term halophilic fungi was introduced for those few xerophilic food-borne species that exhibit superior growth on media with NaCl as controlling solute (Pitt and Hocking 1985). Only a few reports describe the isolation of fungi from natural moderately saline environments such as salt marshes (Newell 1996), saline soil (Guiraud et al.1995) and seawater (Kohlmeyer and Volkmann-Kohlmeyer 1991). Recently, however, we made a novel observation that fungi, representing the only kingdom so far not known to sustain extremely saline conditions, populate salterns nearly saturated with NaCl (Gunde-Cimerman et al. 2000).

Inhibition of DHN-Melanin Biosynthesis by Tricyclazole in Hortaea Werneckii

To demonstrate the type of melanin biosynthetic pathway in the black yeasts Trimmatostroma salinum, Phaeotheca triangularis and Hortaea werneckii by use of tricyclazole, a specific inhibitor for polyketide melanin biosynthesis.