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.     

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.     

Salt stress and plasma-membrane fluidity in selected extremophilic yeasts and yeast-like fungi

We have investigated changes in plasma-membrane fluidity in relation to NaCl concentrations in yeasts and yeast-like fungi that were isolated from either subglacial ice or hypersaline waters. In both of these natural environments, these organisms are exposed to low water activity, due to either high NaCl concentrations or low temperatures. Our data indicate that the fluidity of the plasma membrane can be used as an indicator of fitness for survival in extreme environments. Fungi that can survive in such extreme environments, such as Hortaea werneckii in the hypersaline waters of salterns, and Cryptococcus liquefaciens in subglacial environments, showed similar profiles of plasma-membrane fluidity in response to raised salinity. The same was seen for ubiquitous fungi, which are generally adapted for different types of stress, such as Aureobasidium pullulans and Rhodotorula mucilaginosa. Representatives of both of these groups modulated their plasma-membrane fluidity differently. When salinity exceeded their optimal range, the ubiquitous stress-tolerant species (A. pullulans, Rh. mucilaginosa) showed increased plasma-membrane fluidity, whereas in the dominant extremophiles (H. werneckii, Cr. liquefaciens), it decreased. On the other hand, the plasma membranes of the fungi with a narrow ecological amplitude (Arctic A. pullulans and Rhodosporium diobovatum) showed different responses.     

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.     

Mitochondrial mediation of environmental osmolytes discrimination during osmoadaptation in the extremely halotolerant black yeast Hortaea werneckii

We have investigated the mitochondrial responses to hyperosmotic environments of ionic (4.5 M NaCl) and non-ionic (3.0 M sorbitol) osmolytes in the most halo/osmo-tolerant black yeast, Hortaea werneckii. Adaptation to both types of osmolytes resulted in differential expression of mitochondria-related genes. Live-cell imaging has revealed a condensation of mitochondria in hyperosmotic media that depends on osmolyte type. In the hypersaline medium, this was accompanied by increased ATP synthesis and oxidative damage protection, whereas adaptation to the non-ionic osmolyte resulted in a decrease in ATP synthesis and lipid peroxidation level in mitochondria. A proteomic study of the mitochondria revealed preferential accumulation of energy metabolism enzymes in the hypersaline medium, and accumulation of protein chaperones in the non-ionic osmolyte. The HwBmh1/14-3-3 protein, localized to mitochondria in hypersaline conditions, and not at optimal salinity, suggesting its role in differential perception of ionic and non-ionic osmolytes in H. werneckii.     

Modeling and simulation‐based design of electroenzymatic batch processes catalyzed by unspecific peroxygenase from A. aegerita

Unspecific peroxygenases have attracted interest due to their ability to catalyze the oxygenation of various types of C–H bonds using only hydrogen peroxide as a cosubstrate. Due to the instability of these enzymes at even low hydrogen peroxide concentrations, careful fed‐batch addition of the cosubstrate or ideally in situ production is required. While various approaches for hydrogen peroxide addition have been qualitatively assessed, only limited kinetic data concerning enzyme inactivation and peroxide accumulation has been reported so far. To obtain quantitative insights into the kinetics of such a process, a detailed data set for a peroxygenase‐catalyzed benzylic hydroxylation coupled with electrochemical hydrogen peroxide production is presented. Based on this data set, we set out to model such an electroenzymatic process. For this, initial velocity data for the benzylic hydroxylation is collected and an extended Ping‐Pong‐Bi‐Bi type rate equation is established, which sufficiently describes the enzyme kinetic. Moreover, we propose an empirical inactivation term based on the collected data set. Finally, we show that the full model does not only describe the process with sufficient accuracy, but can also be used predictively to control hydrogen peroxide feeding rates To limit the concentration of this critical cosubstrate in the system.     

分离自青海湖可培养耐盐真菌的多样性研究

青海湖位于青藏高原东北部,属于典型的高原盐湖沉积环境。作者以常规的分离培养方法对青海湖中的耐盐真菌多样性进行了研究。从黑马河、耳海和尕海共分离得到617株耐盐真菌,结合形态学和分子生物学的方法将菌株鉴定到18个属和45个种。子囊菌的菌株数比接合菌和担子菌多,枝孢菌和青霉菌为优势耐盐真菌。在这45种耐盐真菌中,轻度、中度和极端嗜盐真菌分别有19、25和1种。分离自黑马河地区的耐盐真菌,其物种丰富度指数Ma（3.12）、多样性指数H′（1.11）和均匀度指数J（0.267）均比耳海和尕海的高。从1.5%浓度下     

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.     

Oxidative stress in Synechococcus sp. strain PCC 7942: various mechanisms for H2O2 detoxification with different physiological roles

This study focuses on the mechanisms for hydrogen peroxide detoxification in Synechococcus sp. strain PCC 7942. To gain better understanding of the role of different routes of hydrogen peroxide detoxification, we inactivated TplA (thioredoxin-peroxidase-like), which we recently identified. In addition, we inactivated the gene encoding catalase-peroxidase and examined the ability to detoxify H(2)O(2) and to survive oxidative stress in both of the single mutants and in the double mutant. Surprisingly, we observed that the double mutant survived H(2)O(2) concentrations that the single catalase-peroxidase mutant could not tolerate. This phenotype correlated with an increased ability of the double mutant to detoxify externally added H(2)O(2) compared to the catalase-peroxidase mutant. Therefore, our studies suggested the existence of a hydrogen peroxide detoxification activity in addition to catalase-peroxidase and thioredoxin-peroxidase. The rate of detoxification of externally added H(2)O(2) was similar in the wild-type and the TplA mutant cells, suggesting that, under these conditions, catalase-peroxidase activity was essential for this process and TplA was dispensable. However, during excessive radiation, conditions under which the cell might experience oxidative stress, TplA appears to be essential for growth, and cells lacking it cannot compete with the wild-type strain. Overall, these studies suggested different physiological roles for various cellular hydrogen peroxide detoxification mechanisms in Synechococcus sp. strain PCC 7942.     

A systematic strain selection approach for halotolerant and halophilic bioprocess development: a review

Halotolerant and halophilic microorganisms have potential applications in a number of very relevant environmental and industrial bioprocesses, from wastewater treatment to production of value-added chemicals. While numerous microbial strains have been identified and studied in the literature, the number of those successfully used in industrial applications is comparatively small. Literature is abundant in terms of characterisation of specific strains under a microbiology perspective; however, there is a need for studies tackling the selection of strains for bioprocess applications. This review presents a database of over 200 halophilic and halotolerant prokaryote strains compiled from taxonomic microbiological resources and classified by trophic groups as well as by their salinity, pH and temperature tolerance and optimum ranges, all under a process development perspective. In addition to this database, complementary systematic approaches for the selection of suitable strains for a given trophic activity and environmental conditions are also presented. Both the database and the proposed selection approaches together constitute a general tool for process development that allows researchers to systematically search for strains capable of specific substrate degradations under specific conditions (pH, T, salinity). Many exiting established halotolerant and halophilic environmental and industrial bioprocesses appear to have been developed following strategies in line with the systematic approaches proposed here.     

微生物高盐渗透适应策略及其耐盐强化研究进展

高盐废水因具有硬度高、可生化性差、水质成分复杂等特点,是较难处理的工业废水之一。现有物化处理技术存在运行成本高、处理效率低、二次污染重等诸多瓶颈。耐盐/嗜盐微生物可在高盐环境下进行正常生理代谢,因此,开发经济、高效、可靠的高盐废水生物处理技术有望成为高盐废水处理的主流方向之一。本文系统综述了耐盐/嗜盐微生物盐溶、胞内小分子相容溶质积累、蛋白质稳定和细胞表面稳定等高渗透压适应策略。由于嗜盐微生物存在生长条件苛刻、功能微生物种类稀缺等问题,因此,耐盐微生物在高盐废水处理的未来应用空间更大。最新研究发现强化调控技术(电、光、磁)可提升微生物的高渗透压适应能力,其中电调控技术或是未来高盐废水生物处理的重点研究方向。     

Catalase induction protects Haemonchus contortus against hydrogen peroxide in vitro

The response of Haemonchus contortus to oxidative stress in vitro was examined by measuring catalase activities in adult and L4 stage worms exposed to hydrogen peroxide generated by a glucose/glucose oxidase system. Adult nematodes showed increases of up to 2.3-fold in catalase activity after 42 h exposure to the peroxide. L4 nematodes showed up to 4.6-fold induction. A two-stage dose-response was apparent, with catalase activities increasing as the peroxide levels increased, before a return to control levels at higher peroxide concentrations, most likely reflecting a balance between induction and toxicity of the inducing agent itself. Adult nematodes exposed to low levels of peroxide for 24h (hence, having enhanced catalase activities) showed an ability to tolerate subsequent exposure to toxic levels of the peroxide compared to worms with no pre-exposure. An increase of up to approximately threefold in the LC(50) of the hydrogen peroxide generating system was observed after hydrogen peroxide pre-exposure. This indicates that exposure to low oxidant levels lead to an increase in defensive enzyme activities, which allows the nematode to survive subsequent oxidant threats more effectively. The ability of H contortus to increase its catalase activity may be crucial in allowing it to respond to the production of reactive oxygen species by host phagocytes in vivo.     

Organic solute accumulation in osmotically stressed cyanobacteria

Abstract Three groups of cyanobacteria are recognized on the basis of their organic osmotica and upper salinity limit for growth. In general, the least halotolerant forms accumulate disaccharides, while cyanobacteria of intermediate halotolerance synthesize the heteroside glucosylglycerol and the most halotolerant isolates accumulate betaines in response to salt stress. However, certain strains also accumulate additional organic solutes, depending upon the growth temperature, the ambient salinity and the duration of salt stress.     

Identity and biodegradative abilities of yeasts isolated from plants growing in an arid climate

Plants harvested in the Canary Islands Lanzarote and Fuerteventura were analyzed for the yeasts inhabiting their surface. Half of the isolates (22 out of 44) were identified as Debaryomyces hansenii. Black ascomycetes, viz. Hortaea werneckii and two Hormonema species were represented by 7 strains. Basidiomycetous yeasts, viz. Cryptococcus sp. (8 strains), Rhodotorula sp. (5 strains), Cerinosterus cyanescens (1 strain) and Pseudozyma sp. (1 strain) constituted a minority of 33%. Thirty strains were screened for their ability to assimilate various plant constituents including lipids of the cuticle and the cell membrane, hemicelluloses, nitrogenous compounds (protein, nucleic acids, amino acids) and benzene compounds. All strains were able to assimilate or to hydrolyze lipids, lecithin included. Many strains of D. hansenii, H. dematioides, H. werneckii, C. cyanescens, Cr. laurentii, Pseudozyma sp. and Rh. glutinis were proteolytic. Hemicelluloses like xylan and pectin were assimilated by black ascomycetous yeasts, Cryptococcus sp., Pseudozyma sp. and Rh. glutinis. Ferulic and hydroxycinnamic acids, gallic and tannic acids were assimilated by some strains of H. dematioides, C. cyanescens, Pseudozyma sp. and Rhodotorula sp.     

Quantitative analysis of hydrogen peroxide with special emphasis on biosensors

Bioprocess and Biosystems Engineering - Determination of hydrogen peroxide (H2O2) has become essential in pharmaceutical, biological, clinical and environmental studies. The conventional detection...