Osmoadaptation-dependent activity of microsomal HMG-CoA reductase in the extremely halotolerant black yeast Hortaea werneckii is regulated by ubiquitination

We have investigated regulation of HMG-CoA reductase (HMGR) in one of the most salt-tolerant fungi, Hortaea werneckii, under different salinities and at the level of protein degradation. Two different HwHMGR isoenzymes were identified, specific to mitochondria and endoplasmic reticulum: HwHmg1 and HwHmg2, respectively. The activity of microsomal HwHmg2 is highest under hypo-saline and extremely hyper-saline conditions, and down-regulated under optimal growth conditions. We show that this is due to intense ubiquitination and proteasomal degradation of HwHmg2. The activity of the truncated mitochondrial HwHmg1 is constant under different growth conditions, suggesting an osmoadaptation-directed fate for mevalonate utilization in H. werneckii.     

Occurrence of the black yeast Hortaea werneckii in the Mediterranean Sea

Extremophiles - The occurrence of cultivable fungi was investigated along the water column (25–2500 m depth) of four off-shore stations in the Mediterranean basin. An unexpected high...     

Role of oxidative stress in the extremely salt-tolerant yeast Hortaea werneckii

Eukaryotic halotolerant microorganisms are important as model organisms to understand the general mechanisms of resistance to environmental salinity. The ability of the extremely halotolerant black yeast Hortaea werneckii to combat oxidative stress was addressed, using hydrogen peroxide to generate the reactive oxygen species. Increasing environmental salinity was found to have no effect on its high ability to degrade hydrogen peroxide but resulted in a decrease in viability in response to externally added hydrogen peroxide, suggesting that the latter property determines the upper limit of the salt tolerance of H. werneckii. A refinement of the model of adaptation of H. werneckii to high-salinity environments is proposed.     

Identification and characterization of putative osmosensors, HwSho1A and HwSho1B, from the extremely halotolerant black yeast Hortaea werneckii

In Saccharomyces cerevisiae, the Sho1 protein is one of two potential osmosensors that can activate the kinase cascade of the HOG pathway in response to increased extracellular osmolarity. Two novel SHO1-like genes, HwSHO1A and HwSHO1B, have been cloned from the saltern-inhabiting, extremely halotolerant black yeast Hortaea werneckii. The HwSho1 protein isoforms are 93.8% identical in their amino-acid sequences, and have a conserved SH3 domain. When the HwSHO1 genes were transferred into S. cerevisae cells lacking the SHO1 gene, both of the HwSho1 isoforms fully complemented the function of the native S. cerevisiae Sho1 protein. Through microscopic and biochemical validation, we demonstrate that in S. cerevisiae, both of the HwSho1 proteins have characteristic subcellular localizations similar to the S. cerevisiae Sho1 protein, and they can both activate the HOG pathway under conditions of osmotic stress. To a lower extent, crosstalk to the mating pathway expressing HwSho1 proteins is conserved in the PBS2 deleted S. cerevisiae strain. These data show that the HwSho1 proteins from H. werneckii are true functional homologs of the Sho1 protein of S. cerevisiae.     

Salinomyces polonicus: A moderately halophilic kin of the most extremely halotolerant fungus Hortaea werneckii

A clade where the most halotolerant fungus in the world – Hortaea werneckii, belongs (hereafter referred to as Hortaea werneckii lineage) includes five species: Hortaea werneckii, H. thailandica, Stenella araguata, Eupenidiella venezuelensis, and Magnuscella marina, of which the first species attracts increasing attention of mycologists. The species diversity and phylogenetic relationships within this lineage are weakly known. In this study two moderately halophilic black yeast strains were isolated from brine of graduation tower in Poland. Molecular phylogenetic analyses based on the rDNA ITS1-5.8S-ITS2 (=ITS), rDNA 28S D1–D2 (=LSU), and RNA polymerase II (rpb2) sequences showed that the two strains belong to Hortaea werneckii lineage but cannot be assigned to any described taxa. Accordingly, a new genus and species, Salinomyces and Salinomyces polonicus, are described for this fungus. Furthermore, molecular phylogenetic analyses have revealed that Hortaea thailandica is more closely related to S. polonicus than to H. werneckii. A new combination Salinomyces thailandicus is proposed for this fungus.     

Ecology and molecular adaptations of the halophilic black yeast Hortaea werneckii

Molecular studies on halophilic adaptations have focused on prokaryotic microorganisms due to a lack of known appropriate eukaryotic halophilic microorganisms. However, the black yeast Hortaea werneckii has been identified as the dominant fungal species in hypersaline waters on three continents. It represents a new model organism for studying the mechanisms of salt tolerance in eukaryotes. Ultrastructural studies of the H. werneckii cell wall have shown that it synthesizes dihydroxynaphthalene (DHN) melanin under both saline and non-saline growth conditions. However, melanin granules in the cell walls are organized in a salt-dependent way, implying the potential osmoprotectant role of melanin. At the level of membrane structure, H. werneckii maintains a sterol-to-phospholipid ratio significantly lower than the salt-sensitive Saccharomyces cerevisiae. Accordingly, membranes of H. werneckii are more fluid over a wide range of NaCl concentrations, indicating high intrinsic salt stress tolerance. Even H. werneckii grown in high NaCl concentrations maintains very low intracellular amounts of potassium and sodium, demonstrating the sodium-excluder character of this organism. The salt-dependent expressions of two HwENA genes suggest roles for them in the adaptation to changing salt concentrations. The high similarity of these ENA ATPases to other fungal ENA ATPases involved in Na⁺/K⁺ transport indicates their potential importance in H. werneckii ion homeostasis. Glycerol is the main compatible solute which accumulates in the cytoplasm of H. werneckii at high salinity, although it seems that mycosporines may also act as supplementary compatible solutes. Salt dependent increase in glycerol synthesis is supported by the identification of two copies of a gene putatively coding for glycerol-3-phosphate-dehydrogenase. Expression of only one of these genes is salt dependent.     

Salt stress affects sterol biosynthesis in the halophilic black yeast Hortaea werneckii

Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia. Some of the adaptations of halophilic microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties. By modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes. We studied the regulation of sterol biosynthesis in H. werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols. We found some differences in the characteristics of HMG R and in its regulation by different environmental salinities in H. werneckii when compared to the mesophilic baker's yeast, Saccharomyces cerevisiae. Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts. From this perspective, H. werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on sterol biosynthesis in eukaryotes.     

Novel 3'-phosphoadenosine-5'-phosphatases from extremely halotolerant Hortaea werneckii reveal insight into molecular determinants of salt tolerance of black yeasts

The 3'-phosphoadenosine-5'-phosphatase encoded by HAL2 gene, is a ubiquitous enzyme required for the removal of the cytotoxic 3'-phosphoadenosine-5'-phosphate produced during sulfur assimilation in eukaryotes. Salt toxicity in yeast and plants results from Hal2 inhibition by sodium or lithium ions. Two novel HAL2-like genes, HwHAL2A and HwHAL2B, have been cloned from saltern-inhabited extremely halotolerant black yeast Hortaea werneckii. Expression of both HwHAL2 isoforms was differentially inducible upon salt. When the HwHAL2 genes were transferred from such a halotolerant species into the salt sensitive Saccharomyces cerevisiae, the resulting organism can tolerate 1.8M NaCl or 0.8M LiCl, the highest reported salt concentrations at which S. cerevisiae can grow. With genetic and biochemical validation we demonstrated the critical HwHal2B sequence motif--the META sequence--common only to Dothideales fungi, with evident effect on the HwHal2B-dependent salt tolerance. These results may have significance for biosaline agriculture in coastal environments.     

Global Analysis of the Hortaea werneckii proteome: studying steroid response in yeast

The response of the halophilic black yeast Hortaea werneckii to the steroid hormone progesterone has been studied at the protein level using fluorescent two-dimensional differential gel electrophoresis (2D-DIGE) technology in combination with mass spectrometry. Data on protein identification from this study reveal molecular mechanisms of the response to progesterone. In particular, the overexpression of Pck2 and Pac2 in the stimulated cells indicates the interactions of progesterone with the cell growth and reproduction signaling pathways.     

The halophilic fungus Hortaea werneckii and the halotolerant fungus Aureobasidium pullulans maintain low intracellular cation concentrations in hypersaline environments

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.     

Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence

Two general strategies exist for the growth and survival of prokaryotes in environments of elevated osmolarity. The 'salt in cytoplasm' approach, which requires extensive structural modifications, is restricted mainly to members of the Halobacteriaceae. All other species have convergently evolved to cope with environments of elevated osmolarity by the accumulation of a restricted range of low molecular mass molecules, termed compatible solutes owing to their compatibility with cellular processes at high internal concentrations. Herein we review the molecular mechanisms governing the accumulation of these compounds, both in Gram-positive and Gram-negative bacteria, focusing specifically on the regulation of their transport/synthesis systems and the ability of these systems to sense and respond to changes in the osmolarity of the extracellular environment. Finally, we examine the current knowledge on the role of these osmostress responsive systems in contributing to the virulence potential of a number of pathogenic bacteria.     

Osmotic adaptation in halotolerant yeast, Debaryomyces nepalensis NCYC 3413: role of osmolytes and cation transport

Debaryomyces nepalensis NCYC 3413, a food spoiling yeast isolated from rotten apple, has been previously demonstrated as halotolerant yeast. In the present study, we assessed its growth, change in cell size, and measured the intracellular polyol and cations (Na⁺ or K⁺) accumulated during growth in the absence and presence of different concentrations of salts (NaCl and KCl). Cells could tolerate 2 M NaCl and KCl in defined medium. Scanning electron microscopic results showed linear decrease in mean cell diameter with increase in medium salinity. Cells accumulated high amounts of K⁺ during growth at high concentrations of KCl. However, it accumulated low amounts of Na⁺ and high amounts of K⁺ when grown in the presence of NaCl. Cells grown in the absence of salt showed rapid influx of Na⁺/K⁺ on incubation with high salt. On incubation with 2 M KCl, cells grown at 2 M NaCl showed an immediate efflux of Na⁺ and rapid uptake of K⁺ and vice versa. To withstand the salt stress, osmotic adjustment of intracellular cation was accompanied by intracellular accumulation of polyol (glycerol, arabitol, and sorbitol). Based on our result, we hypothesize that there exists a balanced efflux and synthesis of osmolytes when D. nepalensis was exposed to hypoosmotic and hyperosmotic stress conditions, respectively. Our findings suggest that D. nepalensis is an Na⁺ excluder yeast and it has an efficient transport system for sodium extrusion.     

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

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.