Janja Zajc

Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective

Molecular studies of salt tolerance of eukaryotic microorganisms have until recently been limited to the bakers yeast Saccharomyces cerevisiae and a few other moderately halotolerant yeast. Discovery of the extremely halotolerant and adaptable fungus Hortaea werneckii and the obligate halophile Wallemia ichthyophaga introduced two new model organisms into studies on the mechanisms of salt tolerance in eukaryotes. H. werneckii is unique in its adaptability to fluctuations in salt concentrations, as it can grow without NaCl as well as in the presence of up to 5 M NaCl. On the other hand, W. ichthyophaga requires at least 1.5 M NaCl for growth, but also grows in up to 5 M NaCl. Our studies have revealed the novel and intricate molecular mechanisms used by these fungi to combat high salt concentrations, which differ in many aspects between the extremely halotolerant H. werneckii and the halophilic W. ichthyophaga. Specifically, the high osmolarity glycerol signaling pathway that is important for sensing and responding to increased salt concentrations is here compared between H. werneckii and W. ichthyophaga. In both of these fungi, the key signaling components are conserved, but there are structural and regulation differences between these pathways in H. werneckii and W. ichthyophaga. We also address differences that have been revealed from analysis of their newly sequenced genomes. The most striking characteristics associated with H. werneckii are the large genetic redundancy, the expansion of genes encoding metal cation transporters, and a relatively recent whole genome duplication. In contrast, the genome of W. ichthyophaga is very compact, as only 4884 protein-coding genes are predicted, which cover almost three quarters of the sequence. Importantly, there has been a significant increase in their hydrophobins, cell-wall proteins that have multiple cellular functions.

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.

Chaophilic or chaotolerant fungi: a new category of extremophiles?

It is well known that few halophilic bacteria and archaea as well as certain fungi can grow at the highest concentrations of NaCl. However, data about possible life at extremely high concentrations of various others kosmotropic (stabilizing; like NaCl, KCl, and MgSO4) and chaotropic (destabilizing) salts (NaBr, MgCl2, and CaCl2) are scarce for prokaryotes and almost absent for the eukaryotic domain including fungi. Fungi from diverse (extreme) environments were tested for their ability to grow at the highest concentrations of kosmotropic and chaotropic salts ever recorded to support life. The majority of fungi showed preference for relatively high concentrations of kosmotropes. However, our study revealed the outstanding tolerance of several fungi to high concentrations of MgCl2 (up to 2.1 M) or CaCl2 (up to 2.0 M) without compensating kosmotropic salts. Few species, for instance Hortaea werneckii, Eurotium amstelodami, Eurotium chevalieri and Wallemia ichthyophaga, are able to thrive in media with the highest salinities of all salts (except for CaCl2 in the case of W. ichthyophaga). The upper concentration of MgCl2 to support fungal life in the absence of kosmotropes (2.1 M) is much higher than previously determined to be the upper limit for microbial growth (1.26 M). No fungal representatives showed exclusive preference for only chaotropic salts (being obligate chaophiles). Nevertheless, our study expands the knowledge of possible active life by a diverse set of fungi in biologically detrimental chaotropic environments.

The Mycobiota of the Salterns

Solar salterns are constructed as shallow multi-pond systems for the production of halite through evaporation of seawater. The main feature of salterns is the discontinuous salinity gradient that provides a range of well-defined habitats with increasing salinities, from moderate to hypersaline. These present one of the most extreme environments, because of the low levels of biologically available water and the toxic concentrations of ions. Up to the year 2000, hypersaline environments were considered to be populated almost exclusively by prokaryotic microorganisms till fungi were reported to be active inhabitants of solar salterns. Since then, numerous fungal species have been described in hypersaline waters around the world. The mycobiota of salterns is represented by different species of the genus Cladosporium and the related meristematic melanized black yeasts, of non-melanized yeasts, of the filamentous genera Penicillium and Aspergillus and their teleomorphic forms (Eurotium and Emericella), and of the basidiomycetous genus Wallemia. Among these, two species became new model organisms for studying the mechanisms of extreme salt tolerance: the extremely halotolerant ascomycetous black yeast Hortaea werneckii and the obligate halophilic basidiomycete Wallemia ichthyophaga.

Xerophilic fungal genus Wallemia - bioactive inhabitants of marine solar salterns and salty food.

Wallemia is a genus of cosmopolitan xerophilic fungi, frequently involved in food spoilage of particularly sweet, salty, and dried food. Until recently, only a single species, Wallemia sebi, was recognized in the genus. When a large group of strains globally collected in salterns and other different ecological niches was analyzed on the level of physiological, morphological and molecular characteristics, a new basidiomycetous class, Wallemiomycetes, covering an order of Wallemiales was proposed and three Wallemia species were recognized: W. ichthyophaga, W. sebi and W. muriae. Wallemia ichthyophaga was recognized as the most halophilic eukaryote known, thus representing an appropriate eukaryotic model for in depth studies of adaptation to hypersaline conditions. Our preliminary studies indicated that all three Wallemia species synthesized a yet undescribed haemolytic compound under, surprisingly, low water activity conditions. Due to the taxonomic status w hich was unrevealed only recently, there were so far no reports on the production of any bioactive compounds by the three newly described species. The article aims to present the taxonomy, ecology, physiology and so far described molecular mechanisms of adaptations to life at low water activity, as well as bioactive potential of the genus Wallemia, a phylogenetically ancient taxon and a taxonomic maverick within Basidiomycota.

Yeasts in Hypersaline Habitats

For a long time, halotolerant yeasts were known exclusively as contaminants of food preserved with high concentrations of salt or sugar. Their presence in natural thalassohaline hypersaline environments was unknown until 2000, when they were first reported to be active inhabitants of man-made solar salterns in Slovenia. Since then, they have been described on the surface of halophytic plants, in salt mines, in cold and temperate saline lakes, in brine and bittern of different solar salterns on three continents and in MgCl2-dominated waters of the Dead Sea. Yeasts in these environments can be described as halotolerant, extremely halotolerant and even extremely chaotolerant. The dominant representatives are different ascomycetous black yeast species, mainly of the genera Hortaea and Phaeotheca; non-melanised ascomycetous yeasts from the genera Candida, Debaryomyces, Meyerozyma, Metschnikowia, Pichia and Yarrowia; and basidiomycetous yeasts from the genera Bulleromyces, Cryptococcus, Cutaneotrichosporon, Papiliotrema, Rhodosporidium, Rhodotorula, Solicoccozyma, Sterigmatomyces and Vishniacozyma. Until the discovery and description of indigenous saltern mycobiota, the physiological and molecular mechanisms relating to salt tolerance in eukaryotic microorganisms were studied using salt-sensitive Saccharomyces cerevisiae as the model organism. Nowadays, most studies focus on halotolerant yeast species like Debaryomyces hansenii, Aureobasidium pullulans and Hortaea werneckii, which have been isolated globally from natural hypersaline environments and can tolerate up to 10%, 15% and 30% NaCl, respectively. Studies of halotolerant yeasts at the molecular level continue to unravel the complexity of the adaptations needed for yeasts to cope with the problems of ion toxicity and low water activity that are characteristic of hypersaline environments.

The Genus Wallemia—From Contamination of Food to Health Threat

The fungal genus Wallemia of the order Wallemiales (Wallemiomycotina, Basidiomycota) comprises the most xerotolerant, xerophilic and also halophilic species worldwide. Wallemia spp. are found in various osmotically challenged environments, such as dry, salted, or highly sugared foods, dry feed, hypersaline waters of solar salterns, salt crystals, indoor and outdoor air, and agriculture aerosols. Recently, eight species were recognized for the genus Wallemia, among which four are commonly associated with foods: W. sebi, W. mellicola, W. muriae and W. ichthyophaga. To date, only strains of W. sebi, W. mellicola and W. muriae have been reported to be related to human health problems, as either allergological conditions (e.g., farmer’s lung disease) or rare subcutaneous/cutaneous infections. Therefore, this allergological and infective potential, together with the toxins that the majority of Wallemia spp. produce even under saline conditions, defines these fungi as filamentous food-borne pathogenic fungi.

Occultifur mephitis f.a., sp. nov. and other yeast species from hypoxic and elevated CO 2 mofette environments

An inventory of culturable yeasts from the soil and water of natural CO2 springs (mofettes) in northeast Slovenia is presented. In mofettes, CO2 of geological origin reaches the soil surface causing temporarily and spatially stable hypoxic environments in soil and water. In total, 142 yeast strains were isolated and identified from high CO2 and control meadow soil, meadow ground-water, forest pond and stream water. All water locations showed below-ground CO2 release. They were assigned to six basidiomycetous yeast genera (six species) and 11 ascomycetous genera (18 species). All ascomycetous yeasts, with the exception of Debaryomyces hansenii, were able to grow under elevated CO2 and fermented glucose. Candida sophiae-reginae, Pichia fermentans and Candida vartiovaarae were the dominating species in meadow and forest high CO2 exposed water. Meyerozyma guilliermondii and Wickerhamomyces anomalus predominated in high CO2 exposed soils. Using high dilution plating of a mofette soil sample, four strains of an unknown basidiomycetous species were isolated and are here newly described as Occultifur mephitis based on molecular phylogenetic and phenotypic criteria. The type strain of Occultifur mephitis is EXF-6436T[CBS 14611=PYCC 7049, LT594852 (D1/D2), KX929055 (ITS)]. An additional three isolated strains are EXF-6437 (LT594853, KX929056), EXF-6473 (LT594863, KX929057) and EXF-6482 (LT594867, KX929054), as well as a strain reported from previous studies isolated from a leaf of Cistus albidus in Portugal (CBS 10223=PYCC 6067), EU002842 (D1/D2), KY308183 (ITS).