Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts.

To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pH on Adenine and Amino Acid Uptake During Sporulation in Saccharomyces cerevisiae

During the early stages of sporulation in Saccharomyces cerevisiae, the pH of the acetate sporulation medium rises to values of 8.0 or higher. Associated with this rise in pH is a reduced cell permeability to certain precursors of ribonucleic acid (RNA), deoxyribonucleic acid or protein. Uptake of adenine, alanine, and leucine was optimal at pH 5.6 to 6.0, but sporulation was inhibited when the sporulation medium was buffered below pH 7.0. Cellular impermeability can be largely overcome by adjusting the acetate sporulation medium to pH 6.0 for optimal uptake of ¹⁴ C-adenine during short pulses without any apparent effect on sporulation. Sporulating cells pulse-labeled 20 min at pH 6.0 incorporated 40 times more ¹⁴C-adenine into RNA than sporulating cells pulse-labeled at pH 8.0. This increased incorporation can be attributed to a 100-fold increase in labeled adenosine triphosphate in cells pulse-labeled at pH 6.0 where maximum uptake occurs.     

Sporulation of Saccharomyces cerevisiae in the Absence of a Functional Mitochondrial Genome

The role of the mitochondrial system during sporulation of Saccharomyces cerevisiae was studied. Addition of ethidium bromide (EthBr) to cells growing in acetate medium resulted in the quantitative (>98%) conversion of the culture to the petite genotype in one generation. The cells were respiratory active (derepressed) but contained no mitochondrial deoxyribonucleic acid (mtDNA) as demonstrated by analytical ultracentrifugation in CsCl. When transferred to acetate sporulation medium, the culture sporulated. Ascus production was only slightly below that of the control culture. Synthesis of mtDNA occurred during sporulation in the control but not in the EthBr-treated culture. Mitochondrial protein synthesis was virtually eliminated in the EthBr-treated culture. Therefore, completely derepressed cells can sporulate without a functional mitochondrial genetic system. When partially repressed cells were treated with EthBr, no ascus formation was observed after transfer to sporulation medium. Control cultures underwent respiratory adaptation in sporulation medium and then sporulated. Extensive derepression of the respiratory system is thus required for sporulation, and this adaptation is dependent on a functional mitochondrial system. Our results suggest that once the cells are fully derepressed no mitochondrial genetic information has to be expressed during meiosis and ascus formation.     

Protein degradation during yeast sporulation. Enzyme and cytochrome patterns.

The levels of several enzymes have been studied during sporulation of Saccharomyces cerevisia. The specific activities of ribonuclease and aminopeptidase I raised several-fold after transfer of the cells to sporulation medium, whereas the specific activities of phosphofructokinase, glucose-6-phosphate dehydrogenase, tryptophan synthase and pyruvate decarboxylase were not significantly altered. The specific activities of NAD-dependent glutamate dehydrogenase, isocitrate lyase, malate dehydrogenase and fructose bisphosphatase all decreased from the onset of sporulation. The inactivation of these latter enzymes was inhibited by cycloheximide and by inhibitors of energy metabolism. Hexokinase, alcohol dehydrogenase and glutamate oxaloacetate transaminase were partially lost from the cells during the period of ascus maturation. None of the enzyme changes observed proved to be 'sporulation-specific' in that it occurred exclusively in sporulating diploid yeast cells. Therefore it is postulated that the meiotic events and the metabolic changes required for ascospore formation are under separate genetic control in this organism. During sporulation, the cellular content of cytochromes b, c, and aa3 was reduced to 20% or less of that present in vegetative derepressed cells. Since the relative percentage of total to cycloheximide-insensitive mitochondrial protein synthesis was not significantly altered throughout sporulation, and the pattern of mitochondrially synthesized polypeptides was rather similar both in vegetative and in sporulating cells, it appeared that not only degradation but also synthesis and therefore turnover of the mitochondrially coded polypeptides of cytochromes b and aa3 took place during sporulation. The activity ratio of cytochrome c oxidase to F1-ATPase in submitochondrial particles isolated from vegetative cells and from purified asci was almost identical. This indicates that the loss of membrane-bound mitochondrial cytochromes during sporulation is probably due to a nonselective degradation of inner mitochondrial membrane proteins.     

Combined effect of growth medium,age of cells and phase of sporulation on heat resistance and recovery of Hansenula anomala

Effects of two growth media, age of cells and phase of sporulation on heat resistance of Hansenula anomala were determined. Cells were grown on two solid media, McClary's acetate and V8 juice agars, at 21 ° C for 16 days. Heat resistance of cells was determined in 0.06 M potassium phosphate buffer at 48 ° C. Heat-stressed cells were plated on four recovery media: yeast extract-malt extract-peptone-glucose (YMPG), pH 7.0; YMPG, pH 3.5; YMPG containing 6% NaCl, pH 7.0; and YMPG containing 20% sucrose, pH 7.0. The composition of sporulation medium influenced the extent of sporulation and the relative heat resistance of sporulating cells. One-day-old cells were the most sensitive to heat. The heat resistance of cells was generally increased as the incubation time was extended to 16 days. Heat treatment caused a greater increase in sensitivity to NaCl than to sucrose or acid pH in recovery media. Young cells were more sensitive to NaCl than were older cells.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

Nutritional Requirements for Growth and Sporulation of Clostridium perfringens

Two strains of Clostridium perfringens grew in a chemically defined medium consisting of L-tryptophan, L-arginine, L-glutamic acid, L-histidine HCl, L-leucine, DL-threonine, DL-phenylalanine, DL-tryrosine, DL-valine, L-cystine, ascorbic acid, Ca d -pantothenate, pyridoxine, biotin, adenine HCl, glucose, salts and mercaptoacetic acid. Alanine, aspartic acid and methionine were highly stimulatory but not essential for growth. Growth did not occur in the absence of glucose, but other fermentable carbohydrates were not tested. Acetone, isopropyl alcohol, succinic acid, acetic acid, butanol, butyric acid, lactic acid, pyruvic acid, oxaloacetic acid or acetaldehyde did not eliminate the requirement for glucose. Methionine was required for sporulation; one strain also required riboflavin, isoleucine, serine and lysine. Butanol increased the degree of sporulation in a complex thioglycolate medium. Failure of Cl. perfringens to sporulate in inadequately buffered media containing glucose was shown to be caused by the high H-ion concentration developing in the culture medium. In addition, some possible end-products of glucose metabolism such as lactic acid, oxaloacetic acid and acetaldehyde, reduced sporulation in one strain appreciably.     

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Summary Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of ³H-adenine at T₇, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T₄ in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.     

Recombination and hydroxyurea inhibition of DNA synthesis in yeast meiosis

Summary Hydroxyurea (HU) inhibits the premeiotic DNA replication and the meiotic events that follow, namely readiness, recombination commitment, haploidisation, sporulation commitment and ascus formation. Short incubations with HU (2–4 hrs) during the premeiotic replication (i.e. starting between 3 and 6.5 hrs in sporulation medium) allow the resumption of the replication at a normal rate following the removal of the drug. The other meiotic events are similarly delayed by the approximate length of the treatment. In these experiments, intragenic recombination in ade2 reached a higher level than in the controls (x1.3–2.0 in one pair of heteroalleles and x3.0–4.0 in another pair). The recombination response to short HU treatments was not observed for a pair of heteroalleles in ade2 that normally shows a high level of meiotic recombination (750 per 10⁶ cells), nor was the response observed in a pair of heteroalleles in lys2. HU treatments have almost no effect on sporulating cells from 8 hrs onwards. At 7–7.5 hrs the meiotic cells are very sensitive to the drug and even short treatments cause cell death and massive DNA degradation.     

Changes in amino acid permeation during sporulation

Changes in amino acid uptake in Bacillus licheniformis and in the amino acid pools of three Bacillus species were investigated, by use of cells from different stages of the life cycle. B. licheniformis contains catalytic uptake systems for all of the 10 amino acids studied. The apparent maximal velocities of uptake decreased during sporulation but did not fall below the range observed for other microorganisms. In sporulating cells, the apparent affinity constants of the uptake systems for individual amino acids remained about the same as in growing cells, i.e., from 2 x 10(-7)m to 7 x 10(-6)m, whereas, in some cases, the apparent maximal velocities decreased significantly. Because the velocity of uptake showed an atypical dependence on substrate concentration, it was postulated that these cells contain two or more uptake systems for each amino acid. Only one of these systems appeared to be operative at a substrate concentration below 10(-6)m. Working at these low substrate concentrations, catalytic activities producing a net efflux of amino acids were demonstrable in vegetative cells in the presence of chloramphenicol, but these exit systems were lost during sporulation. A pool formed by the addition of radioactive algal hydrolysate will exchange with the external medium in vegetative cells but not in sporulating cells. Glutamic acid and alanine comprise at least 60% of the amino acid pool of B. licheniformis A-5, B. subtilis 23, and B. cereus T during all stages of growth and sporulation. The concentrations of the other amino acids in the pool varied extensively, but reflected, in general, the amino acid turnover known to occur during sporulation.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.     

Two-dimensional protein patterns during growth and sporulation in Saccharomyces cerevisiae.

Proteins synthesized by Saccharomyces cerevisiae in presporulation and sporulation media were compared by using sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast strains. Total cellular proteins were labeled with [35S]methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms and/or fluorograms showed some 700 spots per gel. Nine proteins were synthesized by a/alpha cells which were specific to vegetative, log-phase conditions. During incubation in sporulation medium, sporulating (a/alpha) cells synthesized 11 proteins not present in vegetatively growing cell. These same 11 proteins, however, were synthesized by nonsporulating (a/a and alpha/alpha) cells on sporulation medium as well. Nonsporulating diploids (a/a and alpha/alpha) were also examined with the electron microscope at various times during their incubation in sporulation medium. Certain cellular responses found to be unique to meiotic yeast cells in previous studies were exhibited by the nonsporulating controls. The degree to which all cell types (a/alpha, a/a, and alpha/alpha) were committed to sporulation was also determined by shifting cells from sporulation medium to vegetative medium. Some commitment to the meiotic pathway was observed in both the a/alpha and the a/a, alpha/alpha cells.     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

Induction of multispored asci in two-spored strains of Saccharomyces cerevisiae by amitrole.

Although growth of two yeast strains characterized by consistent production of two diploid spores per ascus was inhibited in complex presporulation media containing amitrole, a fraction of the cells produced were able to form asci with more than two spores after transfer to acetate sporulation medium. Cells grown in a defined presporulation medium containing amitrole did not acquire this ability. The increase in spore numbers per ascus is attributed either to the induction by amitrole in growth medium of cells with more than one nucleus or to the restoration of normal meioses in the multispored asci.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation.

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.     

Comparative Metabolism of Vegetative and Sporulating Cultures of Clostridium thermosaccharolyticum

Cultures of Clostridium thermosaccharolyticum, under conditions of restricted growth achieved by slow feeding of glucose, showed a high degree of sporulation. Analysis of the end products showed an accumulation of ethyl alcohol in addition to butyrate and acetate, whereas, in the nonsporulating cultures, acetate and butyrate were the principal products. Incorporation of uniformly labeled (14)C-glucose by sporulating cells was three to four times higher than by nonsporulating cells. The efficiency of acetate assimilation into the lipid fraction of sporulating cells was at least two times higher than that of glucose. When starch was used as the carbon source, the growth rate was reduced; sporulation occurred, and the end products and carbon distribution were similar. Alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were preferentially formed by sporulating cells. In vegetative cells, the formation of these enzymes was repressed if the glucose concentration in the medium was increased. The change in enzyme activity appeared to be related to a morphological change in the cells and indicated an altered metabolic pattern for sporulating cells.     

Influence of pH on the rate of ribosomal ribonucleic acid synthesis during sporulation in Saccharomyces cerevisiae.

The rate of synthesis of ribosomal RNA (rRNA) is much slower during sporulation than during vegetative growth of yeast. If sporulating cells are transferred from normal incubation conditions at pH 8.8 to the same medium adjusted to pH 7.0, the rate of rRNA synthesis increased to approach that observed in vegetative cells. The response to the pH change is quite rapid, occurring within 10 min. THE PH-dependent, rate-limiting step appears to be in the processing of 35S ribosomal precursor RNA to the final 26S and 18S RNA species. A similar pH effect also was found for the rate of protein synthesis. However, no change in respiration was observed when the pH was lowered. These results indicate that the observed differences in rate of rRNA synthesis in vegetative and sporulating cells are a consequence of pH and are not intrinsic to sporulation. The results also support the correlation between rRNA processing and protein synthesis.     

Acetate-Glyoxylate Medium for the Sporulation of Saccharomyces cerevisiae

S ummary : Studies on ascospore formation by Saccharomyces cerevisiae on sodium acetate agar with and without an addition of sodium glyoxylate showed that the rate of ascus formation was increased when glyoxylate was present. The final proportion of asci was not affected but, since cell multiplication (almost threefold) before ascus formation occurred only on the acetate-glyoxylate medium, on the basis of the number of asci/unit of inoculum the effect of glyoxylate was even more marked. If CO₂ was removed from the atmosphere around the cells, sporulation was strongly inhibited when the medium contained only acetate, but there was less inhibition when glyoxylate was present as well. Ascospores formed on the acetate-glyoxylate medium changed their staining properties after the third day. The majority then stained with safranin but not with malachite green.