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.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...

Growth at High pH and Sodium and Potassium Tolerance in Media above the Cytoplasmic pH Depend on ENA ATPases in Ustilago maydis

“…In S. cerevisiae, an Na ϩ /K ϩ ratio of 1 is completely nontoxic (39), and in D. hansenii, the Na ϩ /K ϩ ratio can be as high as 4 without detrimental effects (39). Similarly, Hortaea werneckii and Aureobasidium pullulans show comparable K ϩ and Na ϩ contents when actively growing at 0.8 M NaCl (37). In contrast, in Neurospora crassa (52), Candida albicans (16), and Candida tropicalis (24), low Na ϩ contents are toxic, which suggests high cytoplasmic Na ϩ toxicity.…”

Properties of the halophyte microbiome and their implications for plant salt tolerance

“…Exclusion of ions have been postulated for fungi, if they are exposed to high salt concentrations and this strategy seems to be more effective for the halophilic H. werneckii compared with the only halotolerant Aureobasidium pullulans (Kogej et al 2005). Corresponding proteins or genes have not been yet identified, but in Saccharomyces cerevisiae gene duplication was detected mostly for genes encoding stressresponsive and transport proteins (Kondrashov et al 2002).…”

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

“…The cation contents of cells were determined in the mid-exponential growth phase by a modified procedure previously described (14). In short, Millipore membrane filters (0.8-m pore size) were prewashed with 2% HCl and three times with MilliQ water.…”

“…The cultures of W. ichthyophaga for the analysis of intracellular compatible solutes after a hypo-osmotic shock were grown in triplicate in liquid YNB medium with 20% (wt/vol) NaCl (14,17). Hypo-osmotic shocks were performed as follows; the cells were grown in YNB with 20% NaCl to the mid-exponential growth phase.…”

“…The mechanisms of salt tolerance in fungi have been studied mostly in salt-sensitive Saccharomyces cerevisiae (reviewed in reference 13), which cannot grow in hypersaline environments. Therefore, more suitable model organisms for halotolerance studies in eukaryotes were suggested, such as the extremely halotolerant black yeast Hortaea werneckii, the moderately halotolerant yeast Debaryomyces hansenii, and the halophilic W. ichthyophaga (all reviewed in reference 9) and the polyextremotolerant Aureobasidium pullulans (14,15). H. werneckii can grow in vitro at a wide salinity range, from 0% to 32% (saturation) NaCl, and has a broad growth optimum, from 6 to 10% NaCl (16,17), whereas the halotolerant A. pullulans grows best without NaCl but can tolerate up to 17% NaCl and the marine yeast D. hansenii grows better at low sodium and tolerates up to 24% NaCl (18)(19)(20).…”