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-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 C18:2^9,12. 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.Communicated by W.D. Grant     

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.     

Intracellular Changes in Ions and Organic Solutes in Halotolerant Brevibacterium sp. Strain JCM 6894 after Exposure to Hyperosmotic Shock

In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halotolerant organism Brevibacterium sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl. Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline. The intracellular free Na⁺ concentrations remained low (80 nmol mg of protein⁻¹) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium. In contrast, K⁺ ions immediately accumulated in the cells in fresh culture medium, whereas K⁺ ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not. Growth of the Brevibacterium strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture. In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein⁻¹ at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock. These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.     

Melanin is crucial for growth of the black yeast Hortaea werneckii in its natural hypersaline environment

Melanin has an important role in the ability of fungi to survive extreme conditions, like the high NaCl concentrations that are typical of hypersaline environments. The black fungus Hortaea werneckii that has been isolated from such environments has 1,8-dihydroxynaphthalene-melanin incorporated into the cell wall, which minimises the loss of glycerol at low NaCl concentrations. To further explore the role of melanin in the extremely halotolerant character of H. werneckii, we studied the effects of several melanin biosynthesis inhibitors on its growth, pigmentation and cell morphology. The most potent inhibitors were a 2,3-dihydrobenzofuran derivative and tricyclazole, which restricted the growth of H. werneckii on high-salinity media, as shown by growth curves and plate-drop assays. These inhibitors promoted release of the pigments from the H. werneckii cell surface and changed the medium colour. Inhibitor-treated H. werneckii cells exposed to high salinity showed both decreased and increased cell lengths. We speculate that this absence of melanin perturbs the integrity of the cell wall in H. werneckii, which affects its cell division and exposes it to the harmful effects of high NaCl concentrations. Surprisingly, melanin had no effect on H. werneckii survival under H₂O₂ oxidative stress.     

Osmoregulation in the Parasitic Protozoan Tritrichomonas foetus

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K⁺ concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K⁺ concentrations and was inhibited when K⁺ and/or Cl⁻ was absent from the medium. The results suggested that the well-documented Na⁺-K⁺-2Cl⁻ cotransport system was responsible for the K⁺ influx activated during the RVI. However, inhibitors of Na⁺-K⁺-2Cl⁻ cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K⁺ influx in T. foetus.     

Physiological Response of Lactobacillus plantarum to Salt and Nonelectrolyte Stress

In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K⁺ (or Na⁺) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.     

Glycine Betaine, Carnitine, and Choline Enhance Salinity Tolerance and Prevent the Accumulation of Sodium to a Level Inhibiting Growth of Tetragenococcus halophila

Natural-abundance ¹³C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na⁺ content and confer a much higher salt tolerance to T. halophila.     

Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H⁺-translocating ATPase (V-ATPase) and Na⁺/H⁺ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na⁺ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H⁺ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na⁺/H⁺ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na⁺/H⁺ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C₃ metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na⁺ sequestration, mediated by the V-ATPase and Na⁺/H⁺ antiport, is regulated through ABA-independent pathways.     

AtCCX3 is an Arabidopsis endomembrane H+ -dependent K+ transporter

The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na⁺, K⁺, and Mn²⁺ transport. We also report high-capacity uptake of ⁸⁶Rb⁺ in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of ⁸⁶Rb⁺ uptake in AtCCX3 cells by excess Na⁺, K⁺, and Mn²⁺. Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl₂. Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na⁺ accumulation and increased ⁸⁶Rb⁺ transport. Uptake of ⁸⁶Rb⁺ increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H⁺-dependent K⁺ transporter with apparent Na⁺ and Mn²⁺ transport properties distinct from those of previously characterized plant transporters.     

Resistance of the oil-oxidizing microorganism <Emphasis Type="Italic">Dietzia</Emphasis> sp. to hyperosmotic shock in reconstituted biofilms

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY medium) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo-and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10–12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5–20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypoor under hyperosmotic shock. Neither could halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without the addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.     

Bioenergetic Aspects of Halophilism

Examinination of microbial diversity in environments of increasing salt concentrations indicates that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis for H₂ + CO₂ or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. Occurrence of the different metabolic types is correlated with the free-energy change associated with the dissimilatory reactions. Life at high salt concentrations is energetically expensive. Most bacteria and also the methanogenic archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. All halophilic microorganisms expend large amounts of energy to maintain steep gradients of NA⁺ and K⁺ concentrations across their cytoplasmic membrane. The energetic cost of salt adaptation probably dictates what types of metabolism can support life at the highest salt concentrations. Use of KCl as an intracellular solute, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic-compatible solutes. This may explain why the anaerobic halophilic fermentative bacteria (order Haloanaerobiales) use this strategy and also why halophilic homoacetogenic bacteria that produce acetate from H₂ + CO₂ exist whereas methanogens that use the same substrates in a reaction with a similar free-energy yield do not.     

Halotolerant Cyanobacterium Aphanothece halophytica Contains NapA-Type Na+/H+ Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na⁺/H⁺ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na⁺/H⁺ and Li⁺/H⁺ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K⁺ uptake-deficient E. coli mutant and exhibited K⁺ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.     

A Putative Multisubunit Na+/H+ Antiporter from Staphylococcus aureus

We cloned several genes encoding an Na⁺/H⁺ antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na⁺/H⁺ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na⁺/H⁺ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na⁺/H⁺ antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na⁺/H⁺ antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na⁺/H⁺ antiporter. We observed a large Na⁺ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na⁺ extrusion was sensitive to an H⁺ conductor, supporting the idea that the system is not a respiratory Na⁺ pump but an Na⁺/H⁺ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.     

Alkali Metal Cation Transport and Homeostasis in Yeasts

Summary: The maintenance of appropriate intracellular concentrations of alkali metal cations, principally K⁺ and Na⁺, is of utmost importance for living cells, since they determine cell volume, intracellular pH, and potential across the plasma membrane, among other important cellular parameters. Yeasts have developed a number of strategies to adapt to large variations in the concentrations of these cations in the environment, basically by controlling transport processes. Plasma membrane high-affinity K⁺ transporters allow intracellular accumulation of this cation even when it is scarce in the environment. Exposure to high concentrations of Na⁺ can be tolerated due to the existence of an Na⁺, K⁺-ATPase and an Na⁺, K⁺/H⁺-antiporter, which contribute to the potassium balance as well. Cations can also be sequestered through various antiporters into intracellular organelles, such as the vacuole. Although some uncertainties still persist, the nature of the major structural components responsible for alkali metal cation fluxes across yeast membranes has been defined within the last 20 years. In contrast, the regulatory components and their interactions are, in many cases, still unclear. Conserved signaling pathways (e.g., calcineurin and HOG) are known to participate in the regulation of influx and efflux processes at the plasma membrane level, even though the molecular details are obscure. Similarly, very little is known about the regulation of organellar transport and homeostasis of alkali metal cations. The aim of this review is to provide a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants.     

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

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.     

Diversity in Expression Patterns and Functional Properties in the Rice HKT Transporter Family

Plant growth under low K⁺ availability or salt stress requires tight control of K⁺ and Na⁺ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K⁺ Transporters), permeable either to K⁺ and Na⁺ or to Na⁺ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na⁺ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na⁺ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na⁺-K⁺ symport, Na⁺ uniport, or inhibited states, depending on external Na⁺ and K⁺ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K⁺ and Na⁺ accumulation in monocots.Although it is not clear what levels of Na⁺ are toxic in the plant cell cytosol and actually unacceptable in vivo, the hypothesis that this cation must be excluded from the cytoplasm is widely accepted. The most abundant inorganic cation in the cytosol is K⁺, in plant as in animal cells. This cation has probably been selected during evolution because it is less chaotropic than Na⁺ (i.e. more compatible with protein structure even at high concentrations; Clarkson and Hanson, 1980). Its selection might also be due to the fact that in primitive cells, which originated in environmental conditions (seawater) where Na⁺ was more abundant than K⁺, a straightforward process to energize the cell membrane was to accumulate the less abundant cation and to exclude the most abundant one.In the cell, K⁺ plays a role in basic functions, such as regulation of cell membrane polarization, electrical neutralization of anionic groups, and osmoregulation. Concerning the latter function, K⁺ uptake or release is the usual way through which plant cells control their water potential and turgor. Although toxic at high concentrations, Na⁺ can be used as osmoticum and substituted for K⁺, mainly in the vacuole, when the plant is facing low K⁺ conditions and Na⁺ is available in the soil solution. This use of Na⁺, however, requires a tight regulation of K⁺ and Na⁺ transport and compartmentalization that becomes crucial in conditions of high Na⁺ concentrations in the soil solution. Control of Na⁺ and K⁺ uptake, long-distance transport in the xylem and phloem vasculatures, accumulation in aerial parts, and compartmentalization at the cellular and tissue levels have actually been shown to be essential in plant adaptation to salt stress (Greenway and Munns, 1980; Flowers, 1985; Hasegawa et al., 2000; Mühling and Läuchli, 2002). Thus, accumulation of Na⁺ as osmoticum during K⁺ shortage or plant adaptation to salt stress requires integration at the whole plant level of Na⁺ and K⁺ membrane transport system activities (Apse et al., 1999; Shi et al., 2002; Qi and Spalding, 2004; Ren et al., 2005; Maathuis, 2006; Pardo et al., 2006; Horie et al., 2007).This report concerns transport systems named HKT upon first identification (for High-Affinity K⁺ Transporters) that are active at the plasma membrane and permeable to either K⁺ and Na⁺ or to Na⁺ only (Schachtman and Schroeder, 1994; Rodríguez-Navarro and Rubio, 2006). Several members of the HKT family have already been shown, by genetic approaches, to play important roles in plant salt tolerance (Berthomieu et al., 2003; Ren et al., 2005; Huang et al., 2006; Byrt et al., 2007) or growth in conditions of K⁺ shortage (Horie et al., 2007). In Arabidopsis (Arabidopsis thaliana), the HKT family comprises a single member, AtHKT1;1, which is permeable to Na⁺ only (Uozumi et al., 2000) and contributes to Na⁺ removal from the ascending xylem sap and recirculation from the leaves to the roots via the phloem vasculature (Berthomieu et al., 2003; Sunarpi et al., 2005). Interestingly, the HKT family comprises a much larger number of members in rice (Oryza sativa), with seven to nine genes depending on the cultivar (Garciadeblás et al., 2003). In line with previous reports using rice as a model species to decipher the roles that HKT transporters can play in the plant, we have analyzed the expression patterns of three rice HKT genes, OsHKT2;1, OsHKT1;1, and OsHKT1;3, and investigated the functional properties of these transporters after heterologous expression, revealing new patterns of expression for HKT transporters and striking functional diversity.