Comparative study of physiological adaptation to salt stress in the genome shuffled Candida versatilis and a wild-type salt-tolerant yeast strain

Candida versatilis is a yeast with a complex salt-tolerant system. It can maintain normal physiological activities and metabolic fermentation under a high-salt environment. The cellular mechanisms of adaptation to salt stress in strains of a wild type of C. versatilis (WT) and S3–5, genome shuffling strains of C. versatilis with improved tolerance to salt, were investigated. The content of intra- and extra-cellular glycerol, intra-cellular Na⁺, as well as membrane fluidity and permeability, were determined under salt-stressed yeast growth conditions. The results showed that Na⁺/H⁺-antiporter played a primary role in Na⁺ extrusion and H⁺-ATPase has been associated with yeast survival under salt stress. Considerable amounts of glycerol were produced and secreted by the yeast to outside the cell under this salt stress. Changes in the portion of membrane saturated and unsaturated fatty acid composition of C. versatilis in response to osmotic stress lead to membrane permeability and fluidity decreases. They could restrict the influx of Na⁺, enhance H⁺-ATPase activity, and prevent leakage of glycerol across the cell membrane under osmotic stress. The salt tolerance of genome shuffled strain S3–5 was higher than WT. It could be correlated with a higher level of intra-cellular accumulation of glycerol and sodium ions in cells of S3–5 than WT as well as a higher portion of oleic fatty acid (C18: 1) and a lower level of linoleic acid (C18: 2) in cell membranes of the studied yeast mutant. It can be concluded that S3–5 improved physiological regulatory mechanisms of response to salt stress, such as decreased membrane fluidity and a permeability that rapidly adjusted to osmotic stress.     

Mechanisms of Salt Tolerance Conferred by Overexpression of the HAL1 Gene in Saccharomyces cerevisiae

Overexpression of the HAL1 gene improves the tolerance of Saccharomyces cerevisiae to NaCl by increasing intracellular K⁺ and decreasing intracellular Na⁺. The effect of HAL1 on intracellular Na⁺ was mediated by the PMR2/ENA1 gene, corresponding to a major Na⁺ efflux system. The expression level of ENA1 was dependent on the gene dosage of HAL1 and overexpression of HAL1 suppressed the salt sensitivity of null mutants in calcineurin and Hal3p, other known regulators of ENA1 expression. The effect of HAL1 on intracellular K⁺ was independent of the TRK1 and TOK1 genes, corresponding to a major K⁺ uptake system and to a K⁺ efflux system activated by depolarization, respectively. Overexpression of HAL1 reduces K⁺ loss from the cells upon salt stress, a phenomenon mediated by an unidentified K⁺ efflux system. Overexpression of HAL1 did not increase NaCl tolerance in galactose medium. NaCl poses two types of stress, osmotic and ionic, counteracted by glycerol synthesis and sodium extrusion, respectively. As compared to glucose, with galactose as carbon source glycerol synthesis is reduced and the expression of ENA1 is increased. As a consequence, osmotic adjustment through glycerolsynthesis, a process not affected by HAL1, is the limiting factor for growth on galactose under NaCl stress. © 1997 John Wiley & Sons, Ltd.     

The role of K+ and H+ transport systems during glucose‐ and H2O2‐induced cell death in Saccharomyces cerevisiae

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K⁺ and vacuolar collapse. K⁺ is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H⁺‐ATPase are more tolerant to glucose‐induced cell death and exhibit less ROS production. A triple mutant ena1‐4 tok1 nha1, devoid of K⁺ efflux systems, is more tolerant to both glucose‐ and H₂O₂‐induced cell death. We hypothesize that ROS production, activated by glucose and H⁺‐ATPase and inhibited by K⁺ uptake, triggers leakage of K⁺, a process favoured by K⁺ efflux systems. Loss of cytosolic K⁺ probably causes osmotic lysis of vacuoles. The nature of the ROS‐producing system sensitive to K⁺ and H⁺ transport is unknown. Copyright © 2010 John Wiley & Sons, Ltd.     

吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响

为了提高鲁氏接合酵母抗高盐胁迫能力,作者探索了添加吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响。通过测量甘油、海藻糖含量以及Na⁺/K⁺-ATP酶活性等一系列生理表征来验证鲁氏接合酵母高盐适应性的变化。实验结果表明,鲁氏接合酵母在高盐胁迫条件下外源添加吡哆醇,在延滞期时,菌株提前24 h积累细胞内甘油,并且加快了细胞内海藻糖分解代谢速率;在对数期时,细胞内钠钾比率（Na⁺/K⁺）降低82.3%,细胞膜上Na⁺/K⁺-ATP酶活性增加16.9%,2-苯乙醇产量（质量浓度）提高6.42倍;在稳定期时,生物量提高10.6%,乙醇产量（质量浓度）提高5%,2-苯乙醇产量（质量浓度）提高1.26倍。综上所述,吡哆醇的添加能有效提高鲁氏接合酵母的高盐胁迫耐受能力,进一步增强了鲁氏接合酵母在酱油等高盐发酵食品中的应用前景。     

Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K⁺ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K⁺ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K⁺ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb⁺. Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K⁺ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li⁺ ions.     

Change in transport activities of vacuoles of the yeast Yarrowia lipolytica during its growth on glucose

Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H⁺-ATPase (0·9–1·1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca²⁺- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.     

Salt-induced modulation in inorganic nutrients, antioxidant enzymes, proline content and seed oil composition in safflower (Carthamus tinctorius L.)

BACKGROUND: Safflower (Carthamus tinctorius L.) has gained considerable ground as a potential oil‐seed crop. However, its yield and oil production are adversely affected under saline conditions. The present study was conducted to appraise the influence of salt (NaCl) stress on yield, accumulation of different inorganic elements, free proline and activities of some key antioxidant enzymes in plant tissues as well as seed oil components in safflower. Two safflower accessions differing in salt tolerance (Safflower‐33 (salt sensitive) and Safflower‐39 (salt tolerant)) were grown under saline (150 mmol L^?1) conditions and salt‐induced changes in the earlier‐mentioned physiological attributes were determined. RESULTS: Salt stress enhanced leaf and root Na⁺, Cl^? and proline accumulation and activities of leaf superoxide dismutase, catalase and peroxidase, while it decreased K⁺, Ca²⁺ and K⁺/Ca²⁺ and Ca²⁺/Na⁺ ratios and seed yield, 100‐seed weight, number of seeds, as well as capitula, seed oil contents and oil palmitic acid. No significant effect of salt stress was observed on seed oil α‐tocopherols, stearic acid, oleic acid or linoleic acid contents. Of the two safflower lines, salt‐sensitive Safflower‐33 was higher in leaf and root Na⁺ and Cl^?, while Safflower‐39 was higher in leaf and root K⁺, K⁺/Ca²⁺ and Ca²⁺/Na⁺ and seed yield, 100‐seed weight, catalase activity, seed oil contents, seed oil α‐tocopherol and palmitic acid. Other attributes remained almost unaffected in both accessions. CONCLUSION: Overall, high salt tolerance of Safflower‐39 could be attributed to Na⁺ and Cl^? exclusion, high accumulation of K⁺ and free proline, enhanced CAT activity, seed oil α‐tocopherols and palmitic acid contents. Copyright © 2011 Society of Chemical Industry     

Effects of Cation Properties on Sol-gel Transition and Gel Properties of κ-carrageenan

The phase transition temperatures, rheological properties and gel‐network characteristics for gelation of κ‐carrageenan‐salt (NaCl, KCl and CaCl₂) solutions and their aged gels were investigated. The effectiveness of increasing gelling and gel‐melting temperatures at the salt concentrations examined followed the sequence of K⁺ > Ca2⁺ > Na⁺. This sequence was also true for the gel strength and the melting enthalpy (DH) of the most crosslinked junction zones of aged gels at low salt concentrations. Nonetheless, a different order (Ca⁺⁺ > K⁺ and Na⁺) was found for increasing storage modulus and gelation rate during early‐stage gelation, thermal hysteresis and the DH of aged gels in some salt‐carrageenan systems.     

The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H+-ATPase

Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1·2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25 286 Daltons, has two distinct membrane-spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H⁺-ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM -CaCl₂ or 4 mM -ZnCl₂ or buffered to pH 7·5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H⁺-ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).     

Monovalent cation transporters at the plasma membrane in yeasts

Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H⁺-ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na⁺, K⁺, and H⁺, both under normal growth conditions and in response to stress.     

Characterization of Na+/H+-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii

In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na⁺/H⁺-antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na⁺/H⁺-antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra-hydrophilic stretch in the C-terminal region. The expression of Z-SOD2 was constitutive and independent of NaCl-shock. Z-SOD2-disruptants of Z. rouxii did not grow in media supplemented with 3 M -NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt-tolerance of Z. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number: D43629.     

Factors influencing permeability of the cell membrane of the osmotolerant yeast Saccharomyces rouxii grown in the presence and absence of 18% NaCl. I. Na+/K+-activated Mg2+-dependent ATPase

It was found that cells of Saccharomyces rouxii contain an ouabain-inhibited ATPase, assumed to be an Na⁺/K⁺-activated Mg²⁺-dependent ATPase, which could serve as a sodium pump protecting the cells in a high salt environment. Twenty-two cell homogenates or supernatants (centrifuged at 3000 × g) grown without added salt in the medium contained sufficient total ATPase activity to liberate (on average) 0.225 μM P_i min^?1 mg^?1 protein. The percentage of total ATPase inhibited by the addition of ouabain (1 × 10^?4 M) varied from 7 to 100%. Cell homogenates or supernatants from cells grown in the presence of 18% NaCl in the media contained sufficient ATPase activity to liberate (on average) 0.114 μM P_i min^?1 mg^?1 protein, about 50% of the total ATPase activity found in the non-salt grown cells. The percentage of total ATPase activity inhibited by ouabain ranged from 16 to 100%. Although the non-salt-grown cells contained approximately double the total ATPase activity of the salt-grown cells. there was evidence that the percentage of total ATPase that is ouabain sensitive (Na⁺/K⁺-activated ATPase) is higher in the salt-grown cells. Also, cells of S. rouxii grown in media without added NaCl, recovered by centrifugation and transferred to media containing 18% NaCl for 16 h and again recovered by centrifugation, homogenized and centrifuged at 10 000 × g contained 61.2% ouabain-sensitive ATPase compared with 21.3% ouabain-sensitive ATPase in the cells before adaptation to the high salt environment.     

Stability and Activity of β-Galactosidase in Sonicated Cultures of Lactobacillus delbrueckii ssp. bulgaricus 11842 as Affected by Temperature and Ionic Environments

Stability of β‐galactosidase and related lactose hydrolyzing activity in sonicated cultures of Lactobacillus bulgaricus 11842 was investigated in the presence of Na⁺ or K⁺ ions. After sonication in Na⁺ or K⁺ buffers at various pH levels, cultures were held at various temperatures, before adding test lactose solutions. Hydrolysis was monitored by cryoscopy. Cultures sonicated in K⁺ buffer had higher activity and stability than those in Na⁺ buffer; both were highest at pH 6 and 7. Stability was unaffected at pH 6 and 7 below 56 °C. Holding at 61 °C for 60 min caused 70% activity loss with K⁺ ions at pH 6 and 7, while with Na⁺, activity loss was almost complete.     

KCI, CaCI2, Na+ Binding, and Salt Taste of Gum Systems

Aqueous nonionic (0.3% w/v) and ionic (0.1% and 0.3% w/v) gum systems containing NaCl, or equal weights of NaCl plus KCl, or NaCl plus CaCl, were examined. At equivalent molar concentrations of added ions, ²³Na NMR transverse relaxation rates (R₂, set^?1) showed an increase in average Na⁺ mobility with the addition of K⁺ or Ca²⁺ to ionic gum systems. Correspondingly, salt taste increased with addition of KCl as determined by Decision Boundary modeling of subject identification data. Viscosity did not affect saltiness. Na⁺ was free to induce salt taste when K⁺ was bound to the gum. Enhancement of salt taste by KCl is due, in part, to competitive binding of Na⁺ and K⁺ in a system.     

Mdm31 protein mediates sensitivity to potassium ionophores but does not regulate mitochondrial morphology or phospholipid trafficking in Schizosaccharomyces pombe

Mdm31p is an inner mitochondrial membrane (IMM) protein with unknown function in Saccharomyces cerevisiae. Mutants lacking Mdm31p contain only a few giant spherical mitochondria with disorganized internal structure, altered phospholipid composition and disturbed ion homeostasis, accompanied by increased resistance to the electroneutral K⁺/H⁺ ionophore nigericin. These phenotypes are interpreted as resulting from diverse roles of Mdm31p, presumably in linking mitochondrial DNA (mtDNA) to the machinery involved in segregation of mitochondria, in mediating cation transport across IMM and in phospholipid shuttling between mitochondrial membranes. To investigate which of the roles of Mdm31p are conserved in ascomycetous yeasts, we analysed the Mdm31p orthologue in Schizosaccharomyces pombe. Our results demonstrate that, similarly to its S. cerevisiae counterpart, SpMdm31 is a mitochondrial protein and its absence results in increased resistance to nigericin. However, in contrast to S. cerevisiae, Sz. pombe cells lacking SpMdm31 are also less sensitive to the electrogenic K⁺ ionophore valinomycin. Moreover, mitochondria of the fission yeast mdm31Δ mutant display no changes in morphology or phospholipid composition. Therefore, in terms of function, the two orthologous proteins appear to have considerably diverged between these two evolutionarily distant yeast species, possibly sharing only their participation in ion homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

An overview of membrane transport proteins in Saccharomyces cerevisiae

All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to ～60 transport proteins whose function was at least partially known, ～100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K⁺ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na⁺-solute symporters, a protein very similar to electroneural cation-chloride co-transporters, and a putative Na⁺-H⁺ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.     

Activity and Activity Coefficient of Counter Monovalent Ions in Aqueous Polyelectrolyte Solutions from Carboxyalkylated Starch

Activity and activity coefficient were determined and compared for counter H⁺, Li⁺, Na⁺, K⁺ and Ag⁺ ions in polyelectrolyte solutions prepared from the condensation products of starch and sodium salts of mono-, di-, and trichloroacetic acids. Na-glass electrode was used for measuring the activity of Na⁺ ions; Ag-electrode for the measurement of the activity of Ag⁺ ions, and ion-exchange permselective membrane were also used in concentration cells to determine the activities of H⁺, Na⁺, and K⁺ ions. For salt-free polyelectrolyte solutions, the activity coefficient of each ion, at equal concentrations, increases in the above mentioned order of the polymer used. In all cases the activity coefficient increases in the following order of the studied monovalent cations: Ag⁺ < K⁺ < Na⁺ < Li⁺ < H⁺.     

Characterization of the respiration‐induced yeast mitochondrial permeability transition pore

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration‐induced mannitol permeability in mitochondria from this strain. Potassium (K⁺) had varied effects on the respiration‐induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K⁺ inhibited respiration‐induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K⁺ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration‐induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration‐induced yPTP was also inhibited by NAD, Mg²⁺, NH₄⁺ or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration‐induced yPTP as those previously described for the ATP‐induced yPTP and reconcile previous strain‐dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.