Enhancing ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae by Mg2+ via reduction in plasma membrane permeability

Mg²⁺ at 3.5 mM increased the tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae to ethanol. After 9 h of exposure to 20% (v/v) ethanol at 30 °C, all cells died whereas over 50% remained viable for the cells grown with Mg²⁺. The effect of Mg²⁺ is closely related to its ability to decrease plasma membrane permeability of cells subjected to ethanol stress.     

The function of Alr1p of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in cadmium detoxification: Insights from phylogenetic studies and particle-induced X-ray emission

Two genes in Saccharomyces cerevisiae, ALR1 and ALR2, encode transmembrane proteins involved in Mg²⁺ uptake. The present study investigates the phylogenetic relationship of Alr1p/Alr2p with bacterial CorA proteins and some proteins related to Mg²⁺influx/efflux transport in mitochondrial and bacterial zinc transporters; including hydrophobic cluster analysis (HCA). The phylogenetic results indicate that the Alrp sequences of S. cerevisiae share a common carboxy-terminus with proteins related to zinc efflux transport. We also analyse the intracellular metal content by particle-induced X-ray emission (PIXE) after cell exposure to cadmium. The PIXE analysis of cadmium-exposed ALR mutants and wild-type yeast cells suggests that Alrp has a central role in cell survival in a cadmium-rich environment. Published online December 2004 Ana Lúcia Kern, Diego Bonatto: Both authors contributed equally to this work.     

Optimization of an ethanol production medium in very high gravity fermentation

Concentrations of Mg²⁺, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg²⁺ and peptone were identified as the critical factors: 50 mM Mg²⁺ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.     

Enhancement of yeast ethanol tolerance by calcium and magnesium

Ethanol-induced changes of CO₂ production were compared in three strains ofSaccharomyces cerevisiœ. CaCl₂ and MgCl₂ exerted protective effects against the action of ethanol. Optimal concentrations ensuring maximum of CO₂ production at 10% (V/V) of ethanol under non-growing conditions were 3 mmol/L Ca²⁺ and 2 mmol/L Mg²⁺. Yeast growth with and without ethanol addition was stimulated by Mg²⁺ more than by Ca²⁺ during fermentation, whereas ethanol production was more efificient when both Ca²⁺ and Mg²⁺ were added.     

Influence of silicon on cobalt, zinc, and magnesium in baker's yeast, Saccharomyces cerevisiae

Silicon (Si, as silicate) is involved in numerous important structure and function roles in a wide range of organisms, including man. Silicate availability influences metal concentrations within various cell and tissue types, but, as yet, clear mechanisms for such an influence have been discovered only within the diatoms and sponges. In this study, the influence of silicate on the intracellular accumulation of metals was investigated in baker's yeast (Saccharomyces cerevisiae). It was found that at concentrations up to 10 mM, silicate did not influence the growth rate of S. cerevisiae within a standard complete medium. However, an 11% growth inhibition was observed when silicate was present at 100 mM. Intracellular metal concentrations were investigated in yeast cultures grown without added silicate (−Si) or with the addition of 10 mM silicate (+Si). Decreased amounts of Co (52%), Mn (35%), and Fe (20%) were found within +Si-grown yeast cultures as compared to −Si-grown ones, whereas increased amounts of Mo (56%) and Mg (38%) were found. The amounts of Zn and K were apparently unaffected by the presence of silicon. +Si enhanced the yeast growth rate for low-Zn²⁺ medium, but it decreased the growth rate under conditions of a low Mg²⁺ medium and did not alter the growth rates in high Zn²⁺ and Co²⁺ media. +Si doubled the uptake rate of Co²⁺ but did not influence that of Zn²⁺. We propose that a possible explanation for these results is that polysilicate formation at the cell wall changes the cell wall binding capacity for metal ions. The toxicity of silicate was compared to germanium (Ge, as GeO₂), a member of the same group of elements as Si (group 14). Hence, Si and Ge are chemically similar, but silicate starts to polymerize to oligomers above 5 mM, whereas Ge salts remain as monomers at such concentrations. Ge proved to be far more toxic to yeast than Si and no influence of Si on Ge toxicity was found. We propose that these results relate to differences in cellular uptake.     

Nickel resistance in fission yeast associated with the magnesium transport system

We isolated and characterized a nickel (Ni²⁺)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni²⁺ and Zn²⁺, but not to Cd²⁺, Co²⁺, and Cu²⁺. The growth rate of GA1 increased proportionally with increasing Mg²⁺ concentrations until 50 mM Mg²⁺. The GA1 mutation phenotype suggests a defect in Mg²⁺ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg²⁺ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro 153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni²⁺ resistance in fission yeast. Specifically, that reducing Mg²⁺ influx through the CorA Mg²⁺ transport membrane protein confers Ni²⁺ resistance in S. pombe. We also report that Ni²⁺ ion detoxification of the fission yeast is related to histidine metabolism and pH.     

Ionophores and intact cells II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg²⁺ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.     

Factors affecting the susceptibility of sensitive yeast cells to killer toxin K1

Optimum conditions for action of the killer toxin K1 on sensitive strainS. cerevisiae S6 were established. Maximum killing was reached in a very narrow pH range of 4.5–4.6. Maximum susceptibility to toxin was displayed by highly energized fresh cells from the early exponential phase in the presence of an external energy source (at least 200 mmol/L glucose). Further, maintenance of maximum membrane potential was necessary for killer action, as documented by decreasing toxin activity in the presence of increasing concentrations of KCl. The killing was strongly stimulated in the presence of millimolar concentrations of Ca²⁺ and Mg²⁺.     

Manganese uptake and toxicity in magnesium-supplemented and unsupplemented Saccharomyces cerevisiae

The magnesium content of Saccharomyces cerevisiae was found to vary by up to fivefold at differing␣ stages of batch growth and during growth in the presence of differing magnesium concentrations. Excess Mg was primarily sequestered in vacuoles. Mn²⁺-uptake experiments revealed that Mg-enriched cells had a markedly reduced capacity for Mn²⁺ accumulation. For example, after 6 h incubation in the presence of 50 μM Mn²⁺, Mn levels were approximately twofold higher in cells previously grown in unsupplemented medium than in those from Mg-supplemented medium. These differences were further accentuated at higher Mn²⁺ concentrations and were not attributable to altered cell-surface charge or altered cell-surface Mn²⁺ binding. Cellular Mg status also influenced Mn toxicity towards S. cerevisiae. During exposure to 5 mM Mn²⁺, 50% reductions in the viability of cells with initial Mg contents of approximately 1400 and 2700 nmol (10⁹ cells)⁻¹ occurred after approximately 1.6 h and 3.6 h respectively. In cells containing 3300 nmol Mg (10⁹ cells)⁻¹, more than 75% viability was still maintained after 7 h incubation with 5 mM Mn²⁺. It is concluded that Mn²⁺ uptake and toxicity in S. cerevisiae are strongly influenced by intracellular Mg, possibly through Mg-dependent regulation of divalent-cation transport activity. Received: 15 May 1996 / Received revision: 13 September 1996 / Accepted: 22 September 1996     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.     

Calcium and Saccharomyces cerevisiae

The claim that Ca may be a dispensable element for yeast Saccharomyces cerevisiae has been reexamined. The cells of S. cerevisiae could grow in media which contained no added Ca and were deprived of contaminating Ca²⁺ by filtration through a Chelex 100 column. Also, the cells were able to grow in the presence of fairly high concentrations of EGTA. The apparent intracellular concentrations of Ca, assessed from the content of radioactive ⁴⁵Ca in cells preloaded with ⁴⁵CaCl₂, could vary within the range of approx. 2 nM to 2.8 mM, without adversively affecting growth or morphology of the cells. An extremely low affinity for Ca²⁺ of the system taking up Ca into the cells was corroborated. However, even the Chelex 100-treated media were found in contain 1–5 μM Ca when maintained in glass culture vessels. Also, the ability of the cells to take up Ca from a medium containing surplus of EGTA or EDTA was demonstrated. su14CEDTA, alone or in the presence of Ca, could also be transported into the cells. It has been inferred that Ca must be as essential for yeast as it is for other eucaryotic organisms. The omnipresence of contaminating Ca and peculiarities of the Ca transporting system, combined with an intricate intracellular compartmentation of Ca, would account for the impossibility to prove the importance of Ca for yeast by direct growth studies.     

Yeast mitochondrial calcium uptake: Regulation by polyamines and magnesium ions

Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca²⁺ transport system ofEndomyces magnusii mitochondria. Mg²⁺ at concentrations up to 5 mM partially inhibits Ca²⁺ transport with a half-maximal inhibiting concentration of 0.5 mM. In the presence of 2 mM MgCl₂, theS _0.5 value of the Ca²⁺ transport system increases from 220 to 490 µM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 µM, respectively. In the case of spermine, theS _0.5 value falls to 50–65 µM, which implies an increase in the transport system affinity for Ca²⁺. Both Mg²⁺ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca²⁺ from the media completely. In contrast, Mg²⁺ lowers the mitochondrial buffer capacity. When both Mg²⁺ and spermine are present in the medium, their effects on theS _0.5 value and the free extramitochondrial Ca²⁺ concentration are additive. The ability of spermine and Mg²⁺ to regulate yeast mitochondrial Ca²⁺ transport is discussed.     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress. Received: 17 April 1996 / Accepted: 21 October 1996     

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu²⁺ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd²⁺ and Mn²⁺. By adding Cd²⁺ and Mn²⁺ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO₄, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu²⁺ and 1 M-Cd²⁺. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd²⁺ environment. In contrast, the presence of Mn²⁺ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu²⁺. The recovery from growth inhibition by Mn²⁺ was due to decreased Cu²⁺ accumulation. Inhibitory concentrations of Co²⁺, Ni²⁺ and Zn²⁺ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd²⁺ and Mn²⁺. These results are discussed in relation to Cu²⁺ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.     

Ethanol induces calcium influx via the Cch1-Mid1 transporter in <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis>

Yeast suffers from a variety of environmental stresses, such as osmotic pressure and ethanol produced during fermentation. Since calcium ions are protective for high concentrations of ethanol, we investigated whether Ca²⁺ flux occurs in response to ethanol stress. We find that exposure of yeast to ethanol induces a rise in the cytoplasmic concentration of Ca²⁺. The response is enhanced in cells shifted to high-osmotic media containing proline, galactose, sorbitol, or mannitol. Suspension of cells in proline and galactose-containing media increases the Ca²⁺ levels in the cytoplasm independent of ethanol exposure. The enhanced ability for ethanol to induce Ca²⁺ flux after the hypertonic shift is transient, decreasing rapidly over a period of seconds to minutes. There is partial recovery of the response after zymolyase treatment, suggesting that cell wall integrity affects the ethanol-induced Ca²⁺ flux. Acetate inhibits the Ca²⁺ accumulation elicited by the ethanol/osmotic stress. The Ca²⁺ flux is primarily via the Cch1 Ca²⁺ influx channel because strains carrying deletions of the cch1 and mid1 genes show greater than 90% reduction in Ca²⁺ flux. Furthermore, a functional Cch1 channel reduced growth inhibition by ethanol.     

Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

Effects of cations on Methanosarcina thermophila TM-1 growing on moderate concentrations of acetate: production of single cells

Summary The effects of different concentrations of Mg²⁺, Ca²⁺, or Na⁺ on the morphology and growth of Methanosarcina thermophila TM-1 growing on acetate at concentrations comparable with those found in anaerobic digestors was studied. At 30 mm Mg²⁺ or less, M. thermophila grew as large aggregates that settled rapidly. At 100 mm Mg²⁺ or more, the bacteria grew as single cells or a mixture of single cells and small aggregates is suspended culture. Mg²⁺ was necessary for growth and could not be substituted by addition of either Ca²⁺ or Na⁺. The optimal Mg²⁺ concentration was 30 mm and no growth was observed at 400 mm Mg²⁺. Cultures could be adapted to 300 mm Mg²⁺ without a change in growth rate. Added Ca²⁺ was not required for growth and had no effect on cell morphology. Inhibition by Na⁺ was directly related to the Mg²⁺ concentration. When the Mg²⁺ was 0.05 mm or less, 0.35 m Na⁺ completely inhibited growth. However, more Na⁺ was required for inhibition at higher Mg²⁺ concentrations. The same inhibitory effect of Na⁺ was observed when the temperature was 52°C or 45°C. The potential for disaggregation of Methanosarcina aggregates in anaerobic digestor environments was discussed. Offprint requests to: B. K. Ahring     

Effect of <Emphasis Type="Italic">m</Emphasis>-carbonyl cyanide 3-chlorophenylhydrazone on inorganic polyphosphates synthesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under different growth conditions

The effects of different concentrations of the protonophore uncoupler m-carbonyl cyanide 3-hchlorophenylhydrazone (CCCP) on the synthesis of inorganic polyphosphates (polyP) during the first 0.5 h of hypercompensation in the yeast Saccharomyces cerevisiae VKM Y-1173 growing on media with 2% glucose under low (hypoxia) or high aeration or with 1% (vol/vol) ethanol under high aeration were studied. It was shown that the yeast growth on ethanol was completely inhibited by 5 μM CCCP, while growth on glucose was inhibited by 25 μM CCCP, independently of aeration of the medium. The maximum rate of H2 absorption was shown at 2, 5, and 25 μM CCCP for the cells grown on ethanol, on glucose under high aeration, and on glucose under hypoxia, respectively. Against the decrease of total ATP level and total polyP, CCCP had a nonuniform effect on the synthesis of individual polyP fractions. CCCP maximally inhibited synthesis of the most actively formed fractions: polyPI during growth on glucose under hypoxia, polyPIII during growth on glucose under aeration, and polyPIII and polyPV during growth on ethanol. CCCP had no substantial effect on the synthesis of polyPII and polyPIV fractions, the formation of which seems to be less related to the electrochemical potential gradient of H⁺ ions.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Ni²⁺, Zn²⁺, and Li⁺) were analyzed. AtCCX5 expression was found to affect the response to K⁺ and Na⁺ in yeast. The AtCCX5 transformant also showed a little better growth to Zn²⁺. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K⁺ (0.5 mM), and also suppressed its Na⁺ sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K⁺ uptake and was also involved in Na⁺ transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K⁺ uptake and Na⁺ transport in yeast.