Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains: the comparison of two quantitative methods

The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches was used to identify relative changes in the protein expression levels between the strains. A total of 2388 proteins were relatively quantified, and more than 350 proteins were found to have significantly different expression levels between the two strains of comparison when using the stable isotope labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found, the major reason behind the discrepancy was the lack of reproducible sampling for proteins with low spectral counts. The functional categorization of the relative protein expression differences that occur in Snf1-deficient strains uncovers a wide range of biological processes regulated by this important cellular kinase.     

Growth of Trichosporon cutaneum under oxygen limitation: kinetics of oxygen uptake

The relationship between oxygen concentration and growth rate in the yeast Trichosporon cutaneum was studied. In order to establish the conditions for purely oxygen-limited growth, the cells were first grown in a carbon-limited chemostat, and kinetic parameters determined. The cells were then grown in an oxygen-limited chemostat at different dilution rates yielding different oxygen uptake rates. The steady-state dissolved oxygen tension was found at each dilution rate and the corresponding equilibrium dissolved oxygen tension was found at each dilution rate and the corresponding equilibrium dissolved oxygen concentration determined in the effluent medium. The relationship between oxygen concentration and growth rate followed Monod-type kinetics with an apparent K(O) of 4.38 x 10(-6)M.     

The putative electrogenic nitrate-proton symport of the yeast Candida utilis. Comparison with the systems absorbing glucose or lactate.

Strain N.C.Y.C. 193 of Candida utilis was grown aerobically at 30 degrees C with nitrate as limiting nutrient in a chemostat. The washed yeast cells depleted of ATP absorbed up to 5 nmol of nitrate/mg dry wt. of yeast. At pH 4-6, extra protons and nitrate entered the yeast cells together, in a ratio of about 2:1. Charge balance was maintained by an outflow of about 1 equiv. of K+. Nitrate stimulated the uptake of about 1 proton equivalent during glycolysis or aerobic energy metabolism. Studies with 3,3'-dipropylthiadicarbocyanine indicated that the proton-linked absorption of nitrate, amino acids or glucose depolarized the yeast cells. Proton uptake along with lactate led neither to net expulsion of K+ nor to membrane depolarization.     

Variability of Bacillus thuringiensis under various growth conditions

When a lysogenic culture of Bacillus thuringiensis subsp. galleriae 69-6 was grown under the batch conditions, 93-99% of cells in the population produced R-form colonies and ca. 1% yielded S-form colonies. The amount of spore-forming cells was 99% in R-variants and 8% in S-variants. The quantity of S-variants rose abruptly to 99% when the culture was grown under the chemostat conditions. The number of S-variants increased with the rate and the duration of growth. The process was influenced by growth-limiting factors. Temperate phage variants capable of host culture lysis on solid media (i.e. h-mutants) were not found under the conditions of batch cultivation. However, such phage particles (h-mutants) appeared under the conditions of chemostat. The titre of these phage particles reached 10(8), 10(7) and 10(4) particles per 1 ml at limitation with yeast extract, glucose and phosphorus, respectively. Under the conditions of chemostat, the particles behaved as temperate ones and their growth was not found. Irrespective of the limitation, the phage titre did not correlate with the ratio of R and S-forms in the population. When the growth was limited with phosphorus, the quantity of S-forms increased abruptly while the spontaneous induction of the phage was inhibited. The quantity of cells capable of spore formation decreased in the cultures isolated from the chemostat and grown on MPA: 69-80% of the cells in R-forms and merely 8% in S-forms.     

Influence of aeration during propagation of pitching yeast on fermentation and beer flavor

The effect of yeast propagated at different aeration conditions on yeast physiology, fermentation ability, and beer quality was investigated using three strains of Saccharomyces cerevisiae. It was shown that yeast cells grown under continuous aeration conditions during propagation were almost two times higher as compared with discontinuous aeration conditions. The maximum of cell growth of all samples reached between 36 h and 48 h. The concentration of trehalose was increased under continuous aerated yeasts, whereas glycogen was decreased. It was also observed that the concentration of glycogen and trehalose in yeast cells had no direct effect on subsequent fermentation ability. The effect of yeast propagated under different aeration conditions on subsequent fermentation ability was different from yeast strains, in which the influence will be most pronounced at the first fermentation. Later, the yeasts might regain its original characteristics in the following fermentations. Generally, continuously propagated yeast had a positive effect on beer quality in subsequent fermentation. Hence, the concentration of aroma compounds obtained with yeast propagated under 6 1/h for 48 h aeration was lower than those grown under other aeration conditions in the bottom yeasts; in particular, the amounts of phenylethyl alcohol, ester, and fatty acids were decreased.     

Metabolic engineering under uncertainty--II: analysis of yeast metabolism

Yeast metabolism has been used extensively in scientific investigations and industrial applications. Understanding the properties of the yeast metabolic network is crucial, yet unaccomplished due to its high complexity, the different culture conditions, and the uncertainty associated with kinetic parameters. We recently developed a computational and mathematical framework using Monte Carlo method in which parameter uncertainty can be addressed through large-scale sampling procedure. This framework was applied on the compartmentalized central carbon pathways of Saccharomyces cerevisiae metabolism considering the growth environment of batch and chemostat reactor and integrating information from metabolic flux analysis. Statistical analysis of the results indicates that yeast cells growing in batch culture condition exhibit dramatically different control schemes from those growing in a chemostat. The difference is mainly due to the feedback introduced by the constraints of the chemostat. The control of the enzymes on the rates of the substrate uptake, product excretion, and cell growth and its practical implication are discussed. Clustering of the reaction steps according to the similarity of their responses to enzyme activity perturbations reveals functional coupling of metabolic reactions.     

Practical reactor systems for yeast cell immobilization using biomass support particles

The technique of cell immobilization using porous support particles (biomass support particles) has been successfully applied to yeast cells. Two reactor configurations exploiting the use of these particles have been developed and assessed for use in aseptic yeast fermentations. A liquid-fluidized bed fermenter has been devised for use with particles denser than the fermentation liquor whilst a gas-stirred circulating bed fermenter proved suitable for particles of essentially neutral buoyancy. Both systems have been operated successfully for extended periods of continuous operation. The utilization of biomass support particle technology in such reactors provides a practical and robust system for immobilized cell reactors. This technology offers significant opportunities for further development.     

Glucose transport of Escherichia coli growing in glucose-limited continuous culture.

Dilute cultures of wild-type Escherichia coli K12 and of derivatives impaired in one or other Enzyme-II component of the glucose phosphotransferase system were grown in continuous culture under glucose limitation. Cells harvested from the chemostat took up [U-14C]glucose from 0.1 mM solutions at rates directly related to the rates at which those cells had grown; the activity of the phosphotransferase system in those cells, rendered permeable with optimal accounts of toluene, parallels the ability of the cells to take up glucose. The capacity of these systems was rate-limiting for growth under the negligibly low glucose concentration in the chemostat, but was adequate to account for the stimulation of respiration observed when the cells were presented suddenly with excess glucose.     

Fed-batch cultivation of bakers’ yeast: Effect of nutrient depletion and heat stress on cell composition

The physiology of a commercial strain of bakers' yeast was studied in terms of the cell composition under different growth conditions and of its response to stress. The study comprised fed-batch experiments since this is the system used in bakers' yeast industry. The classical fed-batch fermentation procedure was modified in that the yeast cells were continuously grown to a steady-state at a dilution rate of 0.1/h in order to achieve more or less the same initial starting point in terms of cell composition. This steady-state culture was then switched to fed-batch concomitantly with exposure to stress. The highest amount of trehalose accumulation was achieved when nutrient depletion and heat stress were applied concomitantly. The highest amount of trehalose, 12%, was attained in cells stressed by both nitrogen depletion and heat stress. The protein content remained constant, although with some oscillations, at a value of 30% throughout this dual stress experiment.     

Relationship Between Messenger Ribonucleic Acid and Enzyme Levels Specified by the Leucine Operon of Escherichia coli K-12

The levels of leucine-forming enzymes in Escherichia coli K-12 varied over a several thousand-fold range, depending upon conditions of growth. The highest levels were achieved by growing auxotrophs in a chemostat under conditions of leucine limitation. Under such conditions, enzyme levels were increased 45- to 90-fold relative to cells grown in minimal medium containing leucine (the latter values arbitrarily called 1). Leucine operon-specific messenger ribonucleic acid levels were elevated to about the same extent as enzyme levels in cells grown in a chemostat. Growth in media of greater complexity resulted in progressively lower levels of leucine-forming enzymes, reaching a value of less than 0.02 for growth in a medium containing tryptone broth and yeast extract. The levels of leucine operon-specified enzymes and messenger ribonucleic acid were also measured in strains containing about 25 copies of plasmid pCV1(ColE1-leu) per chromosome. For such strains grown in minimal medium, enzyme levels were proportional to the number of plasmids per cell. Furthermore, they followed the same trends as those described above upon derepression in a chemostat or upon repression following growth in rich media. Leucine messenger ribonucleic acid, measured both by pulse-labeling and hybridization-competition experiments, was roughly proportional to enzyme levels over this entire range. For a plasmid-containing strain grown in a chemostat under conditions of leucine limitation (about 100 plasmids per chromosome), about 27% of pulse-labeled ribonucleic acid was coded for by genes in or adjacent to the leucine operon, and 10% of the total protein was β-isopropylmalate dehydrogenase.     

In vitro gene expression dissected: chemostat surgery for mycobacterium tuberculosis

A unique approach, combining defined and reproducible in vitro models with DNA microarrays, has been developed to study environmental modulation of mycobacterial gene expression. The gene expression profiles of samples of Mycobacterium tuberculosis, from independent chemostat cultures grown under defined and reproducible conditions, were found to be highly correlated. This approach is now being used to study the effect of relevant stimuli, such as limited oxygen availability, on mycobacterial gene expression. A modification of the chemostat culture system, enabling largevolume controlled batch culture, has been developed to study starvation survival. Cultures of M. tuberculosis have been maintained under nutrient-starved conditions for extended periods, with 10(6) - 10(7) bacilli surviving in a culturable state after 100 days. The design of the culture system has made it possible to control the environment and collect multiple time-course samples to study patterns of gene expression. These studies demonstrate that it is possible to perform long-term studies and obtain reproducible expression data using controlled and defined in vitro models.     

Cellular inequivalence in a culture of Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe was grown in the chemostat at D = 0.03, 0.05, 0.1, 0.15 and 0.20 h-1. The dry weight and substrate quantities, the number of cells and their morphological characteristics were determined in the steady state. The curves for the cell number and dry weight demonstrate changes in the coordination between the processes of cell growth and division at various growth rates. The cell division was shown to be asymmetric under the conditions of substrate limitation.     

A portable integrated automatic apparatus for the real-time monitoring of bioluminescence in plants

Real-time monitoring of gene expression by a bioluminescence reporter gene is a powerful method for large-scale, detailed analysis of gene expression in living cells and large-scale screening of mutants. We have developed a portable, compact, integrated automatic bioluminescence-monitoring apparatus that can continuously monitor 960 individual plant seedlings or micro-organism colonies under uniform light conditions at temperatures up to 50 °C. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms of Arabidopsis reporter strain. Using the apparatus, we measured bioluminescence rhythms in the thermophilic cyanobacterium Thermosynechococcus at temperature up to 43 °C. We also monitored the expression of the flowering regulator gene CONSTANS in Arabidopsis as bioluminescence in high time resolution under different photoperiodic conditions. The high-throughput bioluminescence-monitoring apparatus developed here is a powerful tool for real-time monitoring of gene expression and gene function.     

Correlation between the Cellular Content of Mobile Water and the Viability of Lyophilized Yeast Cells

Spin-echo NMR studies showed that lyophilized yeast cells contain isolated mobile water (IMW), whose content varied from 0.25% (of the dry weight of cells) in lyophilized exponential-phase yeast cells to 3.8% in lyophilized lag-phase and stationary-phase yeast cells. The viability rate of yeast cells varied from 20% in a lyophilized preparation of exponential-phase cells to 86% in a lyophilized preparation of early-stationary-phase cells. In a lyophilized preparation of yeast cells grown in a chemostat mode at a constant specific rate, the content of IMW depended on the growth-limiting factor, being minimal in the case of growth limitation by the carbon source. In the latter case, the viability of cells was also minimal. The data obtained show that there is a correlation between the IMW content and the viability of yeast cells in lyophilized preparations.     

Formaldehyde Production by Cells of a Mutant of Candida boidinii S2 Grown in Methanol-limited Chemostat Culture

Formaldehyde production was investigated with cells of a mutant, AOU-1, of a methanol yeast, Candida boidinii S2 grown in methanol-limited chemostat culture. The highest productivity was shown with cells from the culture at a dilution rate of 0.075 hr^-1, when cells had the highest activity of alcohol oxidase and almost minimum activity of formaldehyde dehydrogenase. Under optimal reaction conditions, 950 mm formaldehyde was produced in 10-hr reaction with the cells. By the chemostat culture, not only formaldehyde productivity but also cell productivity was improved in comparison with batch culture. A maximum cell productivity of 0.2 g · liter^-1 · hr^-1 and a cell yield of 47% were obtained.     

Oxidation of an inhibitory substrate by washed cells (oxidation of phenol by Pseudomonas putida)

The specific uptake rate of phenol by washed cells of Pseudomonas putida grown on phenol in steady-state continuous culture at various dilution rates was studied. The Monod-Haldane-type equation was applied to fit the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The values of the kinetic parameters were found to increase monotonically with the phenol concentration in the original chemostat. The relations between the values of kinetic parameters and phenol concentration in the chemostat were described by empirical equations. Then the equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of phenol was proposed. This equation together with the mass balance equations can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.     

Glucose transport in a methylotrophic yeast Hansenula polymorpha

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha . Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system ( K _m for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system ( K _m for glucose 0.05–0.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide- m -chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.     

Kinetic analysis of a trehalase-overexpressing strain grown on trehalose: a new tool for respiro-fermentative transition studies in Saccharomyces cerevisiae

AIMS: The aim was to demonstrate the use of a trehalase-overexpressing Saccharomyces cerevisiae strain grown on trehalose as a valuable tool in the studies of respiro-fermentative transition at a reduced scale. METHODS AND RESULTS: A trehalase-overexpressing strain was cultivated in synthetic medium on trehalose under aerobic conditions. This strain grew at a maximum specific growth rate of 0.16 h(-1) and showed a pure oxidative metabolism. Glucose pulse experiments were carried out in this system in order to quantify the short-term Crabtree effect. These data were then compared with glucose pulse experiments carried out in the conventional way with the wild-type strain in glucose-limited chemostats. Glucose-pulse experiments in aerobic batch cultures grown on trehalose led to a metabolic respiro-fermentative transition similar to the one observed in glucose-limited chemostats. CONCLUSIONS: This cultivation system allowed us to quantitatively mimic at the flask scale the Crabtree effect observed in conventional chemostat studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is of primary interest in S. cerevisiae studies in which: (i) the implementation of oxidative growth is required (as with studies of the Crabtree effect and heterologous protein production); (ii) small-scale culture systems are required (e.g. high-throughput mutant screening and isotopic labelling experiments).