Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae.

We have investigated the physiological conditions under which meiosis and the ensuing sporulation of Saccharomyces cerevisiae are initiated. Initiation of sporulation occurs in response to carbon, nitrogen, phosphorus, or sulfur deprivation, and also, when met auxotrophs are partially starved for methionine, but not after starvation of other amino acid auxotrophs. It also occurs after partial starvation of pur or gua auxotrophs for guanine but not after starvation of ura auxotrophs for uracil. Under all these sporulation conditions the concentrations of both guanine nucleotides (GTP) and S-adenosylmethionine (SAM) decrease whereas those of other nucleotides show no trend. We show that the decrease of guanine nucleotides is essential for the initiation of meiosis and sporulation: when a gua auxotroph, also lacking one of the two SAM synthetases, is starved for guanine but supplemented with 0.1 mM methionine, GTP decreases while SAM slightly increases and yet the cells sporulate.

Optimal extraction conditions for high-performance liquid chromatographic determination of nucleotides in yeast.

Different extraction methods of nucleotides from the yeast Saccharomyces cerevisiae were compared. A new extraction solution--formic acid saturated with 1-butanol--was found to be more effective than the commonly used solutions of trichloroacetic acid, perchloric acid, or formic acid alone. Using this solution the optimal extraction conditions were established. Nucleotide recovery was evaluated by adding standard nucleotides to the extraction medium and carrying them together with the cells through the whole extraction procedure. Nucleotides were separated and quantitated by high-performance liquid chromatography on an anion-exchange column.

Initiation of meiosis and sporulation of Saccharomyces cerevisiae by sulfur or guanine deprivation.

Homothallic Saccharomyces cerevisiae, growing exponentially in a synthetic acetate medium, could be initiated to undergo meiosis and subsequent sporulation by removal of sulfur from the medium or by partial purine deprivation of purine auxotrophs or, most efficiently, by guanine deprivation of a guanine auxotroph. In contrast, partial uracil deprivation of uracil auxotrophs did not cause sporulation. Under any of the above and other sporulation conditions, the intracellular concentrations of GTP and, usually at some time later, S-adenosylmethionine (SAM) decreased; the concentrations of the other nucleoside triphosphates decreased under some but increased under other sporulation conditions. The addition of 1 mM methionine or, more effectively, of SAM or the combination of adenine plus methionine greatly increased the intracellular concentration of SAM and reduced or prevented sporulation, even when GTP decreased. However, differentiation can be inhibited by an excess of many metabolites which do not specifically control the initiation process; in particular, SAM is known to inhibit yeast metabolism (e.g., transamination). Therefore, we cannot yet decide whether the deficiency of GTP or SAM (or related compounds) serves as a signal for the initiation of meiosis/sporulation.

Initiation of yeast sporulation of partial carbon, nitrogen, or phosphate deprivation.

In this paper we show that partial deprivation of a carbon source, a nitrogen source, or phosphate in the presence of all other nutrients needed for growth initiates meiosis and sporulation of Saccharomyces cerevisiae homothallic strain Y55. For carbon deprivation experiments, cells were grown in synthetic medium (pH 5.5) containing an excess of one carbon source and then transferred to the same medium containing different concentrations of the same carbon source. In the case of transfer to different acetate concentrations, the log optical density at 600 nm increased at the previous rate until the cells had used up all of the acetate, whereupon the cells entered a stationary phase and did not sporulate. The same was observed with ethanol. In contrast, at different concentrations of dihydroxy-acetone or pyruvate, cells grew at different rates and sporulated optimally at intermediate concentrations (50 to 75 mM). The response to galactose was similar but reflected the presence of a low-affinity galactose transport system and the induction of a high-affinity galactose transport system. Cells could also sporulate when a glucose medium ran out of glucose, apparently because they initiated sporulation during the subsequent lag period and then used the produced ethanol as a carbon source. For phosphate deprivation experiments, cells growing with excess ethanol or pyruvate and phosphate were transferred to the same medium containing limiting amounts of phosphate. First, they used up the intracellular phosphate reserves for rapid growth, and then they sporulated optimally when an intermediate concentration (30 muM) of phosphate had been added to the medium. For nitrogen deprivation experiments, cells grown with excess acetate, ethanol, or pyruvate and NH(4) (+) were transferred to the same medium from which all nitrogen had been removed. These cells sporulated well in acetate medium but poorly in ethanol and pyruvate media. However, the sporulation frequency in the latter media could be increased greatly by adding intermediate concentrations (1 mM) of the slowly metabolizable amino acids glycine, histidine, or phenylalanine. If one assumes that the sporulation response to partial deprivation of carbon-, nitrogen-, or phosphorus-containing compounds reflects control by a single metabolite, the intracellular concentration of this metabolite may decide at the START position (G1 phase) of the cell cycle whether a/alpha cells enter mitosis or meiosis.

Morphological stages of Bacillus subtilis sporulation and resistance to fusidic acid.

During spore development of Bacillus subtilis both protein synthesis and sporulation become resistant to the antibiotic fusidic acid. This resistance develops at the time when asymmetric prespore septa are formed. Simultaneously ribosomes lose their ability to bind fusidic acid, as demonstrated by their affinity chromatography with the immobilized drug. Mutants resistant to fusidic acid during growth are oligosporogenous; their sporulation development is blocked before septum formation. These results indicate that normal ribosomes are needed for prespore septation sporulation; only after septation can protein synthesis be maintained, throughout the development period, by fusidate resistant ribosomes.

Conditions controlling commitment of differentiation in Bacillus megaterium.

The developmental stage at which cells of Bacillus megaterium are committed to continue differentiation, i.e., sporulation, depends on both the previous growth medium and the new medium to which the cells are transferred for the commitment test. The latest "stage of no return," after which cells continue differentiation, no matter how rich in nutrients the medium, is reached as soon as the forespore is completely surrounded by a double membrane.

Adenosine 5'-triphosphate release and membrane collapse in glycerol-requiring mutants of Bacillus subtilis.

Glycerol-requiring mutants of Bacillus subtilis could not sporulate in nutrient sporulation medium even when additional glycerol was added from the beginning of growth. Sporulation could be partially restored either by the frequent addition of small amounts of glycerol during the developmental period or by the single addition of both 10 mM glycerol and 10 mM malate. But sporulation could be completely restored by the addition of 50 mM glycerol-phosphate from the beginning. At the end of growth of the glycerol mutants in nutrient sporulation medium, the cell membrane collapsed and separated from the cell wall, and much of the cellular adenosine 5'-triphosphate was released into the medium. These observations were made in two glycerol mutants, one derived from strain 168 containing glycerol-teichoic acid in the cell wall and the other derived from strain W23 containing ribitol-teichoic acid.

Deficiencies or excesses of metabolites interfering with differentiation.

Auxotrophic mutants of Bacillus subtilis need much higher concentrations of the required adenine, nicotinic acid, riboflavin, thiamine, or tryptophan for optimal sporulation than for maximal growth. Acetate can partially replace thiamine, indicating the importance of the pyruvate dehydrogenase system for differentiation. A glycerol-requiring mutant can sporulate only if its cells contain a small concentration of L-alpha-glycerol phosphate during development. This can best be achieved by excess (>/=5 mM) of extracellular alpha-glycerol phosphate, which enters B. subtilis very slowly. The results show that both biosynthetic and catabolic enzymes are often needed to maintain the precise balance of metabolites required for differentiation. Mutants unable to catabolize fructose 6-phosphate, glucose 6-phosphate, or alpha-glycerol phosphate do not sporulate as long as these compounds accumulate inside the cells; their development is blocked before prespore septa have formed.

Developmental block in citric acid cycle mutants of Bacillus subtilis.

Mutants deficient in different enzymes of the citric acid cycle can be subdivided into two groups according to the frequency at which they produce heat-resistant spores in nutrient sporulation medium. However, the majority of cells can develop in this medium only to the axial filament stage I of sporulation; aconitase and isocitrate dehydrogenase mutants need the addition of glutamate to reach this stage.

Abnormal septation and inhibition of sporulation by accumulation of L- -glycerophosphate in Bacillus subtilis mutants.

Accumulation of l-alpha-glycerophosphate, in cells of Bacillus subtilis mutants lacking the nicotinamide adenine dinucleotide-independent glycerophosphate dehydrogenase activity, suppresses both growth and sporulation. After growth has stopped, the cells slowly develop one and later more asymmetric septa that are thicker than normal prespore septa and apparently contain too much cell wall material to allow further membrane development into forespores or spores. l-Malate prevents accumulation of glycerophosphate and restores sporulation of the mutant. Glucose or gluconate cannot resotre sporulation, because they still effect glycerophosphate accumulation via de novo synthesis. If that accumulation is blocked in a double mutant, which is unable to make glycerophosphate from or to metabolize it into Embden-Meyerhof compounds, then nonsuppressing amounts of glucose or gluconate can restore sporulation.

Growth, sporulation, and enzyme defects of glucosamine mutants of Bacillus subtilis.

Two glucosamine (GCA)-requiring mutants have been isolated which grow on glucose minimal or nutrient sporulation medium only in the presence of either GCA or acetyl-GCA. They lack the l-glutamine-d-fructose-6-phosphate aminotransferase (EC 2.6.1.13), which is repressible by GCA and whose activity in the standard strain decreases after cessation of growth. But the mutants can grow on GCA as sole carbon and ammonia source, because GCA induces the synthesis of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10). With respect to sporulation, the GCA-requiring mutants are in a serious dilemma, as GCA represses the onset of massive sporulation and yet a small amount of GCA-6-phosphate derivatives is necessary to allow sporulation. When GCA is continuously provided in small quantities, sporelike particles are produced which contain little or no spore cortex but a normal spore coat. Apparently, GCA derivatives are needed especially for cortex formation. Many of the sporelike particles can produce colonies after octanol, but not after heat treatment. When they are purified by treatment with lysozyme and sodium dodecylsulfate, they do not show the decrease in optical density at 600 nm typical of germination nor do they produce offspring.