Martina Turk

Salt-induced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized fungi.

The halophilic melanized yeast-like fungi Hortaea werneckii, Phaeotheca triangularis, and the halotolerant Aureobasidium pullulans, isolated from salterns as their natural environment, were grown at different NaCl concentrations and their membrane lipid composition and fluidity were examined. Among sterols, besides ergosterol, which was the predominant one, 23 additional sterols were identified. Their total content did not change consistently or significantly in response to raised NaCl concentrations in studied melanized fungi. The major phospholipid classes were phosphatidylcholine and phosphatidylethanolamine, followed by anionic phospholipids. The most abundant fatty acids in phospholipids contained C16 and C18 chain lengths with a high percentage of C18:2Δ9,12. Salt stress caused an increase in the fatty acid unsaturation in the halophilic H. werneckii and halotolerant A. pullulans but a slight decrease in halophilic P. triangularis. All the halophilic fungi maintained their sterol-to-phospholipid ratio at a significantly lower level than did the salt-sensitive Saccharomyces cerevisiae and halotolerant A. pullulans. Electron paramagnetic resonance (EPR) spectroscopy measurements showed that the membranes of all halophilic fungi were more fluid than those of the halotolerant A. pullulans and salt-sensitive S. cerevisiae, which is in good agreement with the lipid composition observed in this study.

Whole Genome Duplication and Enrichment of Metal Cation Transporters Revealed by De Novo Genome Sequencing of Extremely Halotolerant Black Yeast Hortaea werneckii

Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern. Two of its most striking characteristics that may be associated with a halotolerant lifestyle are the large genetic redundancy and the expansion of genes encoding metal cation transporters. Although no sexual state of H. werneckii has yet been described, a mating locus with characteristics of heterothallic fungi was found. The total assembly size of the genome is 51.6 Mb, larger than most phylogenetically related fungi, coding for almost twice the usual number of predicted genes (23333). The genome appears to have experienced a relatively recent whole genome duplication, and contains two highly identical gene copies of almost every protein. This is consistent with some previous studies that reported increases in genomic DNA content triggered by exposure to salt stress. In hypersaline conditions transmembrane ion transport is of utmost importance. The analysis of predicted metal cation transporters showed that most types of transporters experienced several gene duplications at various points during their evolution. Consequently they are present in much higher numbers than expected. The resulting diversity of transporters presents interesting biotechnological opportunities for improvement of halotolerance of salt-sensitive species. The involvement of plasma P-type H+ ATPases in adaptation to different concentrations of salt was indicated by their salt dependent transcription. This was not the case with vacuolar H+ ATPases, which were transcribed constitutively. The availability of this genomic sequence is expected to promote the research of H. werneckii. Studying its extreme halotolerance will not only contribute to our understanding of life in hypersaline environments, but should also identify targets for improving the salt- and osmotolerance of economically important plants and microorganisms.

Coenzyme Q10 Supplementation Decreases Statin-Related Mild-to-Moderate Muscle Symptoms: A Randomized Clinical Study

Background Statin use is frequently associated with muscle-related symptoms. Coenzyme Q10 supplementation has yielded conflicting results in decreasing statin myopathy. Herein, we tested whether coenzyme Q10 supplementation could decrease statin-associated muscular pain in a specific group of patients with mild-to-moderate muscle symptoms. Material/Methods Fifty patients treated with statins and reporting muscle pain were recruited. The Q10 group (n=25) received coenzyme Q10 supplementation over a period of 30 days (50 mg twice daily), and the control group (n=25) received placebo. The Brief Pain Inventory (BPI) questionnaire was used and blood testing was performed at inclusion in the study and after 30 days of supplementation. Results The intensity of muscle pain, measured as the Pain Severity Score (PSS), in the Q10 group was reduced from 3.9±0.4 to 2.9±0.4 (P<0.001). The Pain Interference Score (PIS) after Q10 supplementation was reduced from 4.0±0.4 to 2.6±0.4 (P<0.001). In the placebo group, PSS and PIS did not change. Coenzyme Q10 supplementation decreased statin-related muscle symptoms in 75% of patients. The relative values of PSS and PIS significantly decreased (−33.1% and −40.3%, respectively) in the Q10 group compared to placebo group (both P<0.05). From baseline, no differences in liver and muscle enzymes or cholesterol values were found. Conclusions The present results show that coenzyme Q10 supplementation (50 mg twice daily) effectively reduced statin-related mild-to-moderate muscular symptoms, causing lower interference of statin-related muscular symptoms with daily activities.

The Evolution of Fatty Acid Desaturases and Cytochrome b5 in Eukaryotes

Desaturases that introduce double bonds into the fatty acids are involved in the adaptation of membrane fluidity to changes in the environment. Besides, polyunsaturated fatty acids (PUFAs) are increasingly recognized as important pharmaceutical and nutraceutical compounds. To successfully engineer organisms with increased stress tolerance or the ability to synthesize valuable PUFAs, detailed knowledge about the complexity of the desaturase family as well as understanding of the coevolution of desaturases and their cytochrome b5 electron donors is needed. We have constructed phylogenies of several hundred desaturase sequences from animals, plants, fungi and bacteria and of the cytochrome b5 domains that are fused to some of these enzymes. The analysis demonstrates the existence of three major desaturase acyl-CoA groups that share few similarities. Our results indicate that the fusion of Δ6-desaturase-like enzymes with their cytochrome b5 electron donor was a single event that took place in the common ancestor of all eukaryotes. We also propose the Δ6-desaturase-like enzymes as the most probable donor of the cytochrome b5 domain found in fungal Δ9-desaturases and argue that the recombination most likely happened soon after the separation of the animal and fungal ancestors. These findings answer some of the previously unresolved questions and contribute to the quickly expanding field of research on desaturases.

The expressions of Δ9-, Δ12-desaturases and an elongase by the extremely halotolerant black yeast Hortaea werneckii are salt dependent

The black yeast-like fungus Hortaea werneckii is the predominant fungal species in salterns, and it is extremely halotolerant. The restructuring of the H. werneckii membrane lipid composition is one of its adaptations to high concentrations of salt, which is mainly achieved by increasing the unsaturation of its phospholipid fatty acids. Genes encoding three fatty acid-modifying enzymes, Delta(9)-, Delta(12)-desaturases and an elongase, have been identified in the genome of H. werneckii, each in two copies. Their transcription profiles show responsiveness to different salinity conditions, with the lowest expression at optimal salinity. Transcriptional responses to hyperosmotic and hypo-osmotic shock show substantial differences between cells exposed to osmotic shock and cells adapted to an osmotically stressful environment.

Expression of fatty-acid-modifying enzymes in the halotolerant black yeast Aureobasidium pullulans (de Bary) G. Arnaud under salt stress

Multiple tolerance to stressful environmental conditions of the black, yeast-like fungus Aureobasidium pullulans is achieved through different adaptations, among which there is the restructuring of the lipid composition of their membranes. Here, we describe three novel genes encoding fatty-acid-modifying enzymes in A. pullulans, along with the levels of their mRNAs under different salinity conditions. High levels of Δ9-desaturase and Δ12-desaturase mRNAs were seen at high salinities, which were consistent with an increased desaturation of the fatty acids in the cell membranes. Elevated levels of elongase mRNA were also detected. Surprisingly, increases in the levels of these mRNAs were also seen following hypo-osmotic shock, while hyperosmotic shock had exactly the opposite effect, demonstrating that data that are obtained from up-shift and down-shift salinity studies should be interpreted with caution.

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).

Genetic resources of extremotolerant fungi: a method for identification of genes conferring stress tolerance.

Fungal species from extreme environments represent an underexploited source of stress-resistance genes. These genes have the potential to improve stress tolerance of economically important microorganisms and crops. An efficient high-throughput method for the identification of biotechnologically interesting genes of extremotolerant fungi was developed by constructing a cDNA expression library in Saccharomyces cerevisiae and screening for gain-of-function transformants under stress conditions. The advantages and possible modifications of this method are discussed, and its efficiency is demonstrated using the stress-tolerant basidiomycetous yeast Rhodotorula mucilaginosa. Twelve R. mucilaginosa genes are described that increase halotolerance in S. cerevisiae. These include genes encoding a phosphoglucomutase and a phosphomannomutase. All 12 investigated genes might be useful for the improvement of halotolerance in genetically modified crops or industrial microorganisms.

Salt and oxidative stress tolerance in Debaryomyces hansenii and Debaryomyces fabryi

We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K(+) and Na(+) ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na(+) content and its tolerance to NaCl or between its ROS content and its tolerance to H(2)O(2). Results show that, although in general, human origin D. fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D. hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied.

Extremotolerant fungi as genetic resources for biotechnology

Increased stress tolerance of economically important plants and microorganisms can improve yields in agriculture and industrial microbiology. The pool of resources used for the genetic modification of crops and industrial fungal strains in the past has been relatively limited, and has frequently included only stress-sensitive organisms. However, certain groups of fungi have evolved specialized mechanisms that enable them to thrive under even the most extreme of environmental conditions. These species can be considered as promising sources of biotechnologically interesting genes. Together with a powerful and convenient high-throughput functional screening method, extremotolerant fungi represent a new opportunity for the identification of stress-tolerance-conferring genes. The approaches described here should provide important contributions to the enhancing of the properties of economically important organisms in the future.