Reversible loss of affinity induced by glucose in the maltose-H+ symport of Saccharomyces cerevisiae.

Glucose represses and inactivates maltose transport in Saccharomyces cerevisiae. The inactivation has been described as an irreversible process involving proteolysis. We have studied the inactivation of the maltose-H+ symport in this yeast and have observed that the mechanism of inactivation depends on the physiological conditions. In resting cells there was a decrease in transport capacity. The rate of decrease was enhanced nonspecifically by the presence of a sugar, glucose being more effective than maltose. In growing cells, glucose induced a decrease in affinity of the H+-symport which could be recovered by starvation, even in the presence of cycloheximide; there was no loss in capacity or, if present, this loss could be explained fully by the dilution due to repression during growth on glucose. We submit that in growing cells inactivation consists in a reversible modification of the permease not involving proteolysis.

A model of the specific growth rate inhibition by weak acids in yeasts based on energy requirements.

Zygosaccharomyces bailii, a spoilage yeast, capable of metabolic activity in food environments with low pH, low a(w) and in the presence of weak acid preservatives was chosen for a study on the effect of benzoic acid on growth parameters. In batch cultures, under controlled pH, this food preservative inhibited growth, decreasing the specific growth rate (mu) and the yield coefficient (Y(S)) on glucose. Data obtained at pH 3.5, 4.0 and 4.5 showed that this inhibition was exclusively promoted by the undissociated form of the acid since the effect was independent of pH when the concentration of the acid was expressed in this form. Moreover, the relationship between the values for mu and Y(S), provided evidence that the specific consumption rate of glucose (q(S)) was not affected by benzoic acid, indicating that the inhibition of growth should be completely explained by a decrease of Y(S). The outcome of parallel experiments performed in continuous culture was that the decrease of Y(S) was due to an increase of the maintenance coefficient (m), defined as the fraction of q(S) diverted from growth to cope with stress, represented in this case by the presence of the preservative. Based on these results a model was built, assuming that m increased hyperbolically with the concentration of benzoic acid, from zero in the absence of the acid up to q(S) when growth was completely inhibited. The concentration of the acid, for which m=q(S)/2, is a constant (K(W)), and represents a measure of the tolerance for a preservative, in this case benzoic acid. The simple equation mu/mu(0)=1+W/K(W) predicts the value of mu for a concentration (W) of the preservative, requiring the knowledge of two parameters: the specific growth rate in the absence of the preservative (mu(0)) and K(W). The equation fitted very well the data of the effect of benzoic acid on the specific growth rate of Z. bailii, having K(W)=0.96 mM benzoic acid. The model was also validated with other spoilage yeasts grown in the presence of benzoic acid in microtiter plates in an automated spectrophotometer. The values obtained for K(W) under these conditions confirm Z. bailii as the most tolerant (K(W)=2.1 mM) followed by Pichia sp. (K(W)=0.78 mM), Saccharomyces cerevisiae (K(W)=0.53 mM) and Debaryomyces hansenii (K(W)=0.11 mM).

Effects of 2-deoxyglucose on Saccharomyces cerevisiae as observed by in vivo 31P-NMR.

Saccharomyces cerevisiae cells were treated with 2-deoxyglucose (1 mM) and the effects induced in the levels of phosphorus compounds and in the internal pH were monitored using 31P-NMR. Upon incubation with 2-deoxyglucose a strong decrease in the polyphosphate level was observed and the cytoplasmic pH decreased by about 0.4 units. This shows that 2-deoxyglucose strongly interferes with the cell conditions and consequently, the results of experiments in which 2-deoxyglucose was used to obtain deenergized cells should be carefully reanalysed.

Energetics of the effect of acetic acid on growth of Saccharomyces cerevisiae.

In batch cultures of a respiratory deficient mutant of Saccharomyces cerevisiae the maximum specific growth rate and the yield coefficient decreased, but the specific glucose consumption rate increased, in the presence of acetic acid. The ATP yield decreased from approximately 14 to 4 g biomass (mol ATP)(-1) when the concentration of acetic acid increased from 0 to 170 mM. Intracellular acidification was much weaker than previously reported for non-adapted cells. A linear relation was obtained between the ATP specific production rate and the uptake rate of acetic acid, suggesting that about 1 mol ATP is consumed per mol of acetic acid diffusing into the cells.

Cyanide-resistant respiration is frequent, but confined to yeasts incapable of aerobic fermentation.

In Pichia membranifaciens, cyanide-resistant respiration (CRR) sensitive to salicylhydroxamic acid emerged after forced aeration of starved cells for 4 h. Surveying a large number of species by this simple methodology, we found that CRR is very frequent among yeasts. Remarkably, considering our results together with previous data in the literature, CRR was present in 24 out of 28 non-fermentative or Crabtree-negative yeasts and absent in 10 out of 12 Crabtree-positive yeasts. We submit that, as alternatives to cytochromic respiration, yeasts developed two strategies: either aerobic fermentation in Crabtree-positive yeasts or CRR in non-fermentative or Crabtree-negative yeasts.

Weak acid inhibition of fermentation by Zygosaccharomyces bailii and Saccharomyces cerevisiae.

The inhibition kinetics of fermentation by Zygosaccharomyces bailii and Saccharomyces cerevisiae were evaluated for weak carboxylic acids. Several regression equations were tried to fit the experimental data, most cases being best fitted to exponential curves. The following parameters were determined: i) acid concentration responsible for 50% inhibition of fermentation (C50%); ii) area under the regression curve up to that concentration (A50%) and iii) exponential inhibition constant (k(i)). These parameters were compared according to their ability to express the inhibitory effect of each acid. In broad terms, the values of k(i) in association with minimum inhibitory concentrations (x(min)), were found best to express the inhibitory effect of the weak acids. However, C50% values were satisfactorily correlated with k(i). The value of A50% more precisely reflected the occasional stimulatory effect of low concentrations of weak acids. Comparison of inhibition parameters for Z. bailii and for S. cerevisiae revealed a higher resistance of the former to acetic, propionic, butyric and benzoic acids and similar resistance to caproic, caprylic and sorbic acids. Previous cultivation in the presence of acetic, propionic and benzoic acids showed a distinct influence on the resistance of both yeasts, although it did not always induce cellular adaptation. Fermentation inhibition showed a good correlation with the lipid solubility of weak acids suggesting that the acids interact with the hydrophobic regions of cell membranes.

Effects of salts on Debaryomyces hansenii and Saccharomyces cerevisiae under stress conditions.

The effect of Na+ and K+ on growth and thermal death of Debaryomyces hansenii and Saccharomyces cerevisiae were compared under stress conditions as those commonly found in food environments. At the supraoptimal temperature of 34 degrees C both cations at concentrations of 0.5 M stimulated growth of D. hansenii, while K+ had no effect and Na+ inhibited growth of S. cerevisiae. At 8 degrees C, close to the minimum temperature for growth in both species, both cations inhibited both yeasts, this effect being more pronounced with Na+ in S. cerevisiae. At extreme pH values (7.8 and 3.5) both cations at concentrations of 0.25 M stimulated D. hansenii while Na+ inhibited S. cerevisiae. K+ inhibited this yeast at pH 3.5. Thermal inactivation rates, measured at 38 degrees C in D. hansenii and at 48 degrees C in S. cerevisiae, decreased in the presence of both cations. This protective effect could be observed in a wider range of concentrations in D. hansenii. These results call the attention to the fact that not all yeasts have the same behaviour on what concerns synergy or antagonism of salt together with other stress factors and should be taken into consideration in the establishment of food preservation procedures.

Movements of protons coupled to glucose transport in yeasts. A comparative study among 248 yeast strains.

In 248 strains representing 205 yeast species, changes in pH coupled to glucose addition were followed in unbuffered cell suspensions. Alkalinization of the external medium elicited by glucose, indicating a H+-glucose symport was observed in 34% of the strains, most of them belonging to the genera Rhodotorula, Hansenula and Candida. H+ uptake coupled to glucose transport was observed only after exhaustion of glucose in growth media. This observation was taken as an indication that, in general, the synthesis of H+-glucose symport is under the control of catabolite repression. Subsequently to the addition of glucose, in most yeasts (82%) acidification was observed. This ability is probably related to the creation of a proton-gradient across the plasma membrane and is generally distributed among yeasts.

Selective Isolation of Fusarium graminearum Mutants Derepressed for Production of beta-Glucosidase.

A strain of Fusarium graminearum produced extracellular beta-glucosidase (beta-d-glucoside glucohydrolase [EC 3.2.1.21]) subject to carbon catabolite repression. Derepressed mutants were selectively isolated by the use of 2-deoxyglucose, a nonmetabolizable catabolic repressor. On each plate of cellobiose and 2-deoxy-glucose inoculated with 10 spores and irradiated with UV light, a few colonies emerged. Comparative growth experiments with one of the derepressed mutants and the parent strain showed that the mutant produced beta-glucosidase in the presence of glucose. Furthermore, the same mutant produced more beta-glucosidase than did the wild-type strain on cellobiose alone.

Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance.

Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo 31P and 13C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism. In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate. Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H+. With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added. Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action. Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where [13C]ethanol was added and formation of labeled acetic acid was detected. The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) [13C]ethanol was added.

Aspects of glucose uptake in Saccharomyces cerevisiae.

A wild-type Saccharomyces cerevisiae strain showed simple saturation kinetics for glucose uptake, with a Km of 4 mM when cells were obtained from exponential growth on glucose, and a similar, single Km of 2 to 8 mM was found under a variety of other growth conditions. Later in growth on glucose, and during ethanol utilization, a second kinetic component was observed, which might reflect either artifacts of membrane alteration or a Km in the molar range.

Misregulation of maltose uptake in a glucose repression defective mutant of Saccharomyces cerevisiae leads to glucose poisoning.

In hex2 mutants of Saccharomyces cerevisiae, which are defective in glucose repression of several enzymes, growth is inhibited if maltose is present in the medium. After adding [14C]maltose to cultures growing with ethanol, maltose metabolism was followed in both hex2 mutant and wild-type cells. The amount of radioactivity incorporated was much higher in hex2 than in wild-type cells. Most of the radioactivity in hex2 cells was located in the low molecular mass fraction. Pulse-chase experiments showed that 2 h after addition of maltose, hex2 cells hydrolysed maltose to glucose, which was partially excreted into the medium. 31P-NMR studies gave evidence that turnover of sugar phosphates was completely abolished in hex2 cells after 2 h incubation with maltose. 13C-NMR spectra confirmed these results: unlike those for the wild-type, no resonances corresponding to fermentation products (ethanol, glycerol) were found for hex2 cells, whereas there were resonances corresponding to glucose. Although maltose is taken up by proton symport, the internal pH in the hex2 mutant did not change markedly during the 5 h after adding maltose. The intracellular accumulation of glucose seems to explain the inhibition of growth by maltose, probably by means of osmotic damage and/or unspecific O-glycosylation of proteins. Neither maltose permease nor maltase was over-expressed, and so these enzymes were not the cause of glucose accumulation. Hence, the coordination of maltose uptake, hydrolysis to glucose and glycolysis of glucose is not regulated simply by the specific activity of the catabolic enzymes involved.(ABSTRACT TRUNCATED AT 250 WORDS)

Utilization of Lactic Acid by Fusarium oxysporum var. lini: Regulation of Transport and Metabolism.

Lactic acid was transported in Fusarium oxysporum var. lini ATCC 10960 by a saturable transport system that had a half-saturation constant of 56.6 +/- 7.5 muM and a maximum velocity of 0.61 +/- 0.10 mmol h g (dry weight) at 26 degrees C and pH 5.0. This transport system was inducible and was not expressed in the presence of a repressing substrate. Evidence is presented that the anionic form lactate was taken up by the cells. Propionic, acetic, pyruvic, and bromoacetic acids but not succinic acid competitively inhibited the transport of lactic acid. Bromoacetic acid, which was not metabolized, was taken up to a steady-state level when intracellular and extracellular concentrations were identical, indicating that the transport system was not accumulative. The enzymatic activity that was physiologically more relevant in the metabolism of lactic acid was lactate: ferricytochrome c oxidase. This enzyme did not exhibit stereospecifity and was induced by lactic acid.

Stress situations induce cyanide-resistant respiration in spoilage yeasts.

AIMS: To investigate the conditions that promote the expression of cyanide-resistant respiration (CRR) in the spoilage yeasts Pichia membranifaciens and Debaryomyces hansenii. METHODS AND RESULTS: CRR was detected by sensitivity of oxygen consumption to salicylhydroxamic acid. It was absent in both yeasts in the early exponential phase, but was triggered by several stress situations. Starvation under aerobic conditions, decreasing pH or incubation of the culture in a narrow temperature range below the maximum temperature for growth promoted the emergence of CRR in both yeasts. In D. hansenii, CRR was also induced by 1.5-2 mol l(-1) NaCl. Although the presence of H2O2 and menadione induced CRR, radical scavengers had no effect on the emergence of CRR. Also, the level of reactive oxygen species did not vary with the CRR activity. CONCLUSIONS: Under aerobic conditions, a respiratory pathway alternative to the cytochrome chain is triggered by stress conditions in P. membranifaciens and D. hansenii. SIGNIFICANCE AND IMPACT OF THE STUDY: The relationship between stress situations and CRR must be taken into account in studies on the performance of spoilage yeasts in the food processing environments where several forms of stress are common.

Glycerol utilization in Fusarium oxysporum var. lini: regulation of transport and metabolism.

Glycerol was transported in the fungus Fusarium oxysporum var. lini by a facilitated diffusion transport system with a half-saturation constant, Ks, of 0.5 mM and a maximum velocity, Vmax, of 0.9 mmol (g dry wt)-1 h-1 at pH 5 and 25 degrees C. 1,2-Propanediol was a competitive inhibitor of glycerol transport, but the cells did not actively accumulate 1,2-propanediol. The transport system was partially constitutive. In cells grown in the presence of glucose, glycerol was not transported, indicating that the synthesis of the system was under glucose repression. Glycerol kinase and NADP(+)-dependent glycerol dehydrogenase activities were present under all physiological conditions tested. A flavin-dependent glycerol phosphate dehydrogenase was induced only when glycerol was the sole energy source in the medium. This enzyme, together with the transport system, constitute the regulated steps in the glycerol metabolic pathway.

Tributyltin oxide affects energy production in the yeast Rhodotorula ferulica, a utilizer of phenolic compounds.

Rhodotorula ferulica, a yeast able to utilize phenolic compounds, was chosen for evaluating the effects of tributyltin oxide (TBTO) on this utilization. TBTO reduced respiratory capacity when vanillic or benzoic acid was the energy source. The ATP level of the cells was severely affected by 2 microM TBTO. The mitochondrial ATPase was strongly inhibited by 0.5 microM TBTO, whereas the activity of the plasma membrane ATPase was not affected by concentrations of TBTO up to 30 microM. Our data support the hypothesis that the target for TBTO action is the mitochondrial ATPase, resulting in a severe disturbance of the yeast utilization of aromatic compounds.

Transport of maltose in Saccharomyces cerevisiae. Effect of pH and potassium ions.

The transport of maltose in Saccharomyces cerevisiae has been generally accepted as a H+-sugar symport, with a stoichiometrical ratio of 1:1. A simultaneous exit of K+ from the cells with the initial uptake of maltose has been reported previously. By using a K+-selective electrode and radioactive maltose, we were able to measure the exit of 1 mol of K+/mol of maltose taken up by the cells in the first 10-15 s. This stoichiometrical ratio is pH-independent. So, uptake of protons in a non-buffered cell suspension or exit of K+ in a buffered one can be used to measure initial rates of maltose uptake. We have used a K+ electrode and a pH electrode to study the effect of external pH and K+ respectively on the kinetic parameters of maltose transport. The following results were obtained: the apparent half-saturation constant for maltose (Km) increased from 5.2 mM at pH 5.8 to 38.0 mM at pH 7.8; the same increase in pH halved the apparent maximum uptake rate (Vmax); K+ had an inhibitory effect, decreasing Vmax. and increasing Km at pH values above 5; K+ had a stimulating effect at pH values below or equal to 4. Under physiological conditions, i.e. lower pH outside, neutral pH inside and much higher [K+] inside the cell, and assuming symmetry of the system, a higher affinity for maltose is to be expected in the outer face of the plasma membrane. This behaviour of the system could explain, by itself, the maintenance of the high concentration of free maltose inside the cell (necessary because of the low affinity of the maltase), without significant back transport to the outside.

Flow microcalorimetry of a respiration-deficient mutant of Saccharomyces cerevisiae.

In aerobic batch cultures in mineral medium with glucose of a respiration-deficient mutant of Saccharomyces cerevisiae, growth parameters were estimated and the heat evolved was measured by a flow microcalorimeter. A growth enthalpy of -163.6 joule per mole of glucose consumed was measured. Under anaerobic conditions, the value was -134.6 joule, closer to the expected for alcoholic fermentation alone. The difference was found to be due to cyanide-resistant respiration under aerobic conditions.