BackgroundAureobasidium pullulans is a black-yeast-like fungus used for production of the polysaccharide pullulan and the antimycotic aureobasidin A, and as a biocontrol agent in agriculture. It can cause opportunistic human infections, and it inhabits various extreme environments. To promote the understanding of these traits, we performed de-novo genome sequencing of the four varieties of A. pullulans.ResultsThe 25.43-29.62 Mb genomes of these four varieties of A. pullulans encode between 10266 and 11866 predicted proteins. Their genomes encode most of the enzyme families involved in degradation of plant material and many sugar transporters, and they have genes possibly associated with degradation of plastic and aromatic compounds. Proteins believed to be involved in the synthesis of pullulan and siderophores, but not of aureobasidin A, are predicted. Putative stress-tolerance genes include several aquaporins and aquaglyceroporins, large numbers of alkali-metal cation transporters, genes for the synthesis of compatible solutes and melanin, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins. All of these genomes contain a homothallic mating-type locus.ConclusionsThe differences between these four varieties of A. pullulans are large enough to justify their redefinition as separate species: A. pullulans, A. melanogenum, A. subglaciale and A. namibiae. The redundancy observed in several gene families can be linked to the nutritional versatility of these species and their particular stress tolerance. The availability of the genome sequences of the four Aureobasidium species should improve their biotechnological exploitation and promote our understanding of their stress-tolerance mechanisms, diverse lifestyles, and pathogenic potential.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-549) contains supplementary material, which is available to authorized users.

This study was intended to determine the osmoadaptation strategy of Hortaea werneckii, an extremely salt-tolerant melanized ascomycetous fungus that can grow at 0-5.1 M NaCl. It has been shown previously that glycerol is the major compatible solute in actively growing H. werneckii. This study showed that the exponentially growing cells also contained erythritol, arabitol and mannitol at optimal growth salinities, but only glycerol and erythritol at maximal salinities. The latter two were both demonstrated to be major compatible solutes in H. werneckii, as their decrease correlated with the severity of hypoosmotic shock. Besides higher amounts of erythritol and lower amounts of glycerol, stationary-phase cells also contained mycosporineglutaminol-glucoside, which might act as a complementary compatible solute. H. werneckii is constitutively melanized under various salinity conditions. Ultrastructural study showed localization of melanin in the outer parts of the cell wall as a distinct layer at optimal salinity (0.86 M NaCl), whereas cell-wall melanization diminished at higher salinities. The role of melanized cell wall in the effective retention of glycerol is already known, and was also demonstrated in H. werneckii by lower retention of glycerol in cells with blocked melanization compared to melanized cells. However, these non-melanized cells compensated for the lower amounts of glycerol with higher amounts of erythritol and arabitol. We hypothesize that H. werneckii melanization is effective in reducing the permeability of its cell wall to its major compatible solute glycerol, which might be one of the features that helps it tolerate a wider range of salt concentrations than most organisms.

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.

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.Sodium is a very abundant cation in nature; nevertheless, it is toxic for most living cells, even in small concentrations. Cells living in natural saline systems, where high salt amounts cause high osmotic pressure, must maintain lower water potential than their surroundings in order to survive and proliferate and at the same time adjust to increased concentrations of sodium ions in the cells. Halophilic microorganisms have developed different strategies for counterbalancing osmotic pressure. Extremely halophilic archaea accumulate potassium up to molar levels when exposed to high external salinity (14). In contrast, eukaryotic microorganisms cannot tolerate such high intracellular ion concentrations.In the absence of appropriate eukaryotic model organisms, mechanisms of salt tolerance have been studied mostly in saltsensitive Saccharomyces cerevisiae (3, 4, 10, 11) and in some halotolerant fungi, such as filamentous Aspergillus nidulans, and yeasts such as Debaryomyces hansenii (1,2,13,17,19), Candida versatilis (22), and Rhodotorula mucilaginosa and Pichia guillermondii (12). The 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 certain f...

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.