Hiroshi Kuriyama

Clock Control of Ultradian Respiratory Oscillation Found during Yeast Continuous Culture

A short-period autonomous respiratory ultradian oscillation (period approximately 40 min) occurs during aerobic Saccharomyces cerevisiae continuous culture and is most conveniently studied by monitoring dissolved O(2) concentrations. The resulting data are high quality and reveal fundamental information regarding cellular dynamics. The phase diagram and discrete fast Fourier transformation of the dissolved O(2) values revealed a square waveform with at least eight harmonic peaks. Stepwise changes in temperature revealed that the oscillation was temperature compensated at temperatures ranging from 27 to 34 degrees C when either glucose (temperature quotient [Q(10)] = 1.02) or ethanol (Q(10) = 0.82) was used as a carbon source. After alteration of the temperature beyond the temperature compensation region, phase coherence events for individual cells were quickly lost. As the cell doubling rate decreased from 15.5 to 9.2 h (a factor of 1.68), the periodicity decreased by a factor of 1.26. This indicated that there was a degree of nutrient compensation. Outside the range of dilution rates at which stable oscillation occurred, the mode of oscillation changed. The oscillation in respiratory output is therefore under clock control.

Ultradian oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide mediates population synchrony

Saccharomyces cerevisiae showed an ultradian respiratory oscillation during aerobic continuous culture. Analysis of the off‐gas revealed that hydrogen sulphide production also oscillated. Production was first detected at the onset of low respiration and reached a maximum (1.5 µM) prior to minimum respiratory activity. Then H2S concentration fell rapidly to below 0.2 µM before the onset of high respiration. Injection of respiratory oscillation perturbation agents, such as glutathione (50 µM), NaNO2 (50 µM) or acetaldehyde (4.5 mM), transiently increased H2S production above 6 µM. The synchronization properties of H2S were analysed to reveal that changes of oscillation period and amplitude were dependent on H2S concentration in culture. It is concluded that H2S produced during oscillation produces population synchrony by respiratory chain inhibition. Copyright

Synchronization affector of autonomous short‐period‐sustained oscillation of Saccharomyces cerevisiae

When the yeast Saccharomyces cerevisiae was grown under aerobic continuous culture, an autonomous shortperiod‐sustained oscillation appeared. This oscillation was observed in concentrations of various extracellular and intracellular parameters, such as ethanol, acetate, glycogen, dissolved oxygen and intracellular pH. In this work the synchronization affecter of this oscillation was investigated. Ethanol was found not to be the synchronizer of the oscillation because a pulse of ethanol did not affect the phase or period of the oscillation. The oscillation was dependent on the aeration rate, i.e., the oscillation occurred only between 150 and 600 ml min−1. However, the oxygen concentration did not influence synchronization as an upward shift in the oxygen concentration of the gas flow did not affect the sustainability of the oscillation. On the other hand, synchronization was stopped by an enhanced gas flow rate, keeping dissolved oxygen tension at the oscillatory condition, suggesting that synchronization was caused by a volatile compound in the culture. A stepwise increase in carbon dioxide concentration of the gas flow rate ceased synchronization, yet the oscillation seem to continue in each individual cell. Oscillatory behaviour of intracellular pH and carbon dioxide evolution rate showed a phase difference of 90 degrees. Based on these facts it is postulated that carbon dioxide, through the influence of its dissociation on intracellular pH, could be the synchronization affector of the autonomous short‐period‐sustained oscillation of S. cerevisiae.

Involvement of glutathione in the regulation of respiratory oscillation during a continuous culture of Saccharomyces cerevisiae.

Respiratory oscillation occurred during aerobic continuous culture of Saccharomyces cerevisiae. During oscillation, phase-related changes in NAD(P)H and GSH levels occur. Perturbation of oscillation and inhibition of respiration occurred when GSH or GSSG was injected; however, there was a phase delay in perturbation in the case of an injection during high respiration. The perturbation phase delay was not apparent when a combination of DL-buthionine-(S,R)-sulphoximine, GSH and 5-nitro-2-furaldehyde was injected. Perturbation by GSH injection caused the intracellular GSH concentration to increase, the GSSG concentration to decrease and the cessation of ethanol uptake. NAD(P)H during perturbation was inversely related to dissolved oxygen. Perturbation by calcium pantothenate and pyridoxal-HCl caused a period of enhanced respiration before oscillation returned. These results suggest that the NAD+/NADH redox is not directly involved in oscillation control and regulation involves glutathione metabolism. Possible regulation points include alcohol dehydrogenase inhibition and/or respiratory-chain inhibition.

Ultradian metabolic oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide, a population synchronizer, is produced by sulphite reductase.

We have reported that the consecutive cyclic production of H2S resulted in population synchrony of ultradian metabolic oscillation (Sohn et al., 2000 ). In order to understand the origin of H2S and its nature of periodic production, changes of sulphur compounds concentration and responsible enzymes were investigated. The concentrations of extracellular sulphate, intracellular glutathione and cysteine oscillated during metabolic oscillation but only the oscillation of sulphate concentration was out of phase with H2S production. The sulphate concentration in culture directly affected the amplitude and the period of metabolic oscillation: (a) the period of metabolic oscillation shortened from 50 min to 30 min when sulphate concentration in the medium was reduced from 46 mM to 2.5 mM; (b) the metabolic oscillation disappeared under sulphate‐depletion conditions and arose again by the addition of sulphate. Pulse injection of sulphite (10 µM) perturbed metabolic oscillation with a burst production of H2S, while thiosulphate (up to 500 µM) was without apparent effect. Furthermore, addition of S‐adenosyl methionine (100 µM) or azoxybacilin (3 mg/kg) decreased H2S production with perturbation of metabolic oscillation. The results presented here suggest that H2S, a population synchronizer, is produced by sulphite reductase in the sulphate assimilation pathway, and dynamic regulation of sulphate uptake plays an important role in ultradian metabolic oscillation. Copyright

NO+, but not NO⋅, inhibits respiratory oscillations in ethanol-grown chemostat cultures of Saccharomyces cerevisiae

A continuous culture of Saccharomyces cerevisiae strain IFO 0233 growing aerobically at pH 3.4 shows persistent high‐amplitude respiratory oscillations with a period of about 45 min. These robust autonomous cycles are accompanied by changes of product accumulation (acetaldehyde and acetic acid), intracellular pH, and intracellular redox state, as indicated by continuously monitored NADH fluorescence and the glutathione content of cell‐free extracts. Perturbation of the oscillation of dissolved O2 was produced on addition of 100 μM glutathione, >10 nM Na nitroprusside, 8 μM NaNO2, or 10 μM S‐nitrosoglutathione. NO gas, putative NO⋅‐releasing agents, or an inhibitor of NO synthase were ineffective. We suggest that nitrosation by NO+ of a component of a redox switch can account for these data, and we emphasise the different modes of action of the different redox forms of nitrogen monoxide.

Involvement of oxidative stress in the regulation of H2S production during ultradian metabolic oscillation of Saccharomyces cerevisiae

Periodic evolution of H(2)S during aerobic chemostat culture of Saccharomyces cerevisiae resulted in ultradian metabolic oscillation via periodic inhibition of respiratory activity. To understand the nature of periodic H(2)S evolution, we investigated whether oxidative stress is associated with H(2)S production. The cellular oxidative states represented by intracellular level of lipid peroxides oscillated out of phase with the oscillation of dissolved O(2). Pulse addition of antioxidant, oxidative agent or inhibitor of antioxidation enzymes perturbed metabolic oscillation producing changes in H(2)S evolution. Analysis of H(2)S production profiles during perturbation of oscillation revealed that the amount of H(2)S production is closely linked with cellular oxidative states. Based on these results and our previous reports, we suggest that oxidative stresses result in periodic depletion of glutathione and cysteine, which in turn causes stimulation of the sulfate assimilation pathway and H(2)S production.

The effects of pCO2 on yeast growth and metabolism under continuous fermentation

SummaryThe effects of pCO2 were investigated by changing the aeration rate, the purging gas and the total pressure in a chemostat cultivatioa Under glucose supply limitation, an increase in pCO2 from 44 kPa to 195 kPa resulted in 25 % decrease in cell concentration, 8 % increase in ethanol concentration, and 50 % decrease in glycerol concentration. Under oxygen supply limitation, similar dependency of ethanol and glycerol on pCO2 was observed, however, no influence of pCO2 on the cell yield was observed. The change in ethanol yield by pCO2 appeared to be caused by the equilibrium shift of pyruvate dehydrogenase system.

The Gts1 protein stabilizes the autonomous oscillator in yeast.

Continuous cultures of Saccharomyces cerevisiae show a robust autonomous temperature compensated oscillation in many metabolic functions. Respiratory activity, a convenient output to measure, oscillates with a period of 40 min. Deletion of GTS1, whose protein product has homology to the circadian per protein, has been implicated in temporal events within yeast, causes a reduction in periodicity to 18 min (wild‐type period 40–60 min). The dilution rate was steadily increased from 0.04/h to 0.085/h and the oscillation stabilized after four to six dilutions. However, Gts1ps involvement in the maintenance and generation of metabolic synchrony, and in the central oscillating loop, appear to be minimal, as the mutant oscillation was robust and autonomous. Deletion of GTS1 did cause decreased temperature compensation of the period of the oscillation from Q10 = 1.07 for the wild‐type to Q10 = 1.6 for the mutant. Also the degree of nutrient compensation observed for the wild‐type was not observed in the GTS1‐null mutant strain. It is postulated that Gts1p is involved in the mechanism that communicates external conditions, such as temperature, to the central oscillating loop. Copyright

Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

Abstract The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO2) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO2, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol−1, increased from 0.745 to 0.836 with the increase in qO2 from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol−1.