Inhibiton by sulfanilamide of sporulation in Saccharomyces cerevisiae.

The antimetabolite sulfanilamide inhibits sporulation in Saccharomyces cerevisiae strain AP1. Cells exposed to sulfanilamide at various times during the sporulation process become progressively insensitive to the drug, although accumulation of sulfanilamide by the cells increases with time. Vegetative growth of AP1 is practically unaffected by sulfanilamide; pregrowth of the cells in the presence of the drug does not prevent sporulation. Thus, inhibition is confined to the meiotic phase of the cell cycle. Sensitivity to sulfanilamide is independent of pH. Increasing the time cells are exposed to sulfanilamide results in a progressive reduction of ascus formation; however, the inhibition is reversible since sporulation can occur in cells exposed to the drug for greater than 24 h. The drug arrests the cells at a point before commitment to sporulation, since yeast cells exposed to sulfanilamide for 12 h do not complete the sporulation process when returnedto vegetative medium, but resume mitotic growth instead. Meiotic nuclear division is largely prevented by sulfanilamide, and synthesis of RNA and protein is severely retarded. DNA synthesis is inhibited up to 50%; glycogen synthesis is approximately 90% inhibited. Other yeast strains showed varying sensitivity to sulfanilamide; homothallic strains were generally less affected.     

Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii

The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differentiate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chytridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to induce complete sporulation in B.?emersonii. We concluded that the determination of growth or sporulation in this microorganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation.     

Synthesis of beta-glucanases during sporulation in Saccharomyces cerevisiae: formation of a new, sporulation-specific 1,3-beta-glucanase.

A biphasic synthesis of 1,3-beta-glucanase occurred when cells of Saccharomyces cerevisiae AP-1 (a/alpha) were incubated in sporulation medium. The capacity to degrade laminarin increased very slowly during the first 7 h but at a much faster rate thereafter. Changes occurring during the first period were not sporulation specific since the moderate increase in activity against laminarin was insensitive to glutamine and hydroxyurea and also took place in the nonsporulating strain S. cerevisiae AP-1 (alpha/alpha). However, the changes taking place after 7 h must be included in the group of sporulation-specific events since they were inhibited by glucose, glutamine, and hydroxyurea and did not occur in the nonsporulating diploid. Consequently, only when the cells had been incubated for at least 7 h in sporulation medium did full induction of activity against laminarin take place upon shift to a medium which favored vegetative growth. Changes in the relative proportions of the vegetative glucanases, namely, endo- and exo-1,3-beta-glucanase, and the formation of a new sporulation-specific 1,3-beta-glucanase account for the observed events and are the consequence of the expression of the sporulation program.     

Synchronous Growth and Sporulation of Bacillus megaterium

Filtration of late log-phase cultures of Bacillus megaterium ATCC 19213 grown on defined sucrose salts medium (SS) or SS plus glutamate medium (SSG) through nine layers of Whatman no. 40 filter paper in a fritted-glass disc Büchner funnel resulted in filtrates containing cells which showed synchronous growth and proceeded to sporulation. SS cells completed one synchronous division after filtration; sporulation ensued after the cessation of growth. SSG cells completed two synchronous divisions and sporulation occurred during the second division. A high degree of synchrony of vegetative growth of SSG cells was evident by the stepwise pattern of growth, by the doubling of cell numbers at each division, the high division index, and by the rapid formation of sporulation cell types and homogeneity of cell types in the filtered cultures when compared with asynchronous cultures. Because the described system gives both good growth and sporulation synchrony, the method should be useful in delineating early events in sporulation and their regulation.     

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.     

Sporulation Synchrony of Saccharomyces cerevisiae Grown in Various Carbon Sources

Sporulation of several strains of Saccharomyces cerevisiae grown in a variety of carbon sources that do not repress the tricarboxylic acid cycle enzymes was more synchronous than the sporulation of cells grown in medium containing dextrose which does repress those enzymes. Dextrose-grown cells showed optimal sporulation synchrony when inoculated into sporulation medium from early stationary phase when the dextrose in the medium is exhausted. Logarithmic-phase cells grown in either non-fermentable carbon sources (acetate and glycerol) or a fermentable carbon source that does not repress tricarboxylic acid cycle enzymes (galactose) sporulated more synchronously than the early stationary-phase dextrose cells. Attempts were made to sporulate cells taken from both complex and semidefined media. The semidefined acetate medium failed to support the growth of a number of strains. However, cells grown in the complex acetate medium, as well as both complex and semidefined glycerol and galactose media, sporulated with better synchrony than did the dextrose-grown cells.     

Chromosome segregation in<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis, a Gram-positive bacterium commonly found in soil, is an excellent model organism for the study of basic cell processes, such as cell division and cell differentiation, called sporulation. In B. subtilis the essential genetic information is carried on a single circular chromosome, the correct segregation of which is crucial for both vegetative growth and sporulation. The proper completion of life cycle requires each daughter cell to obtain identical genetic information. The consequences of inaccurate chromosome segregation can lead to formation of anucleate cells, cells with two chromosomes, or cells with incomplete chromosomes. Although bacteria miss the classical eukaryotic mitotic apparatus, the chromosome segregation is undeniably an active process tightly connected to other cell processes as DNA replication and compaction. To fully understand the chromosome segregation, it is necessary to study this process in a wider context and to examine the role of different proteins at various cell life cycle stages. The life cycle of B. subtilis is characteristic by its specific cell differentiation process where, two slightly different segregation mechanisms exist, specialized in vegetative growth and in sporulation.     

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis.

To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.     

Changes in regulation of ribosomal protein synthesis during vegetative growth and sporulation of Saccharomyces cerevisiae.

When diploid Saccharomyces cerevisiae cells logarithmically growing in acetate medium were placed in sporulation medium, the relative rates of synthesis of 40 or more individual ribosomal proteins (r-proteins) were coordinately depressed to approximately 20% of those of growing cells. These new depressed rates remained constant for at least 10 h into sporulation. If yeast nitrogen base was added 4 yh after the beginning of sporulation to shift the cells back to vegetative growth, the original relative rates of r-protein synthesis were rapidly reestablished. this upshift in the rates occurred even in diploids homozygous for the regulatory mutation rna2 at the restrictive temperature for this mutation (34 degrees C). However, once these mutant cells began to bud and grow at 34 degrees C, the phenotype of rna2 was expressed and the syntheses of r-proteins were again coordinately depressed. At least one protein whose rate of synthesis was not depressed by rna2 in vegetative cells did have a decreased rate of synthesis during sporulation. Another r-protein whose synthesis was depressed by rna2 maintained a high rate of synthesis at the beginning of sporulation. These data suggest that the mechanism responsible for coordinate control of r-protein synthesis during sporulation does not require the gene product of RNA2 and thus defines a separate mechanism by which r-proteins are coordinately controlled in S. cerevisiae.     

Metabolism of myo-inositol during sporulation of myo-inositol-requiring Saccharomyces cerevisiae.

We investigated the sporulation properties of a series of diploid Saccharomyces cerevisiae strains homozygous for inositol auxotrophic markers. The strains required different amounts of inositol for the completion of sporulation. Shift experiments revealed two phases of inositol requirement during sporulation which coincided with the two phases of lipid synthesis found by earlier workers. Phase I was at the beginning and during premeiotic deoxyribonucleic acid synthesis; phase II immediately preceded the appearance of mature asci. Of the inositol taken up by sporulating cells, 90% was incorporated into inositol phospholipids. By two-dimensional thin-layer chromatography, eight compounds were resolved, one of which was sporulation specific. The majority of the inositol phospholipids were, however, identical to those found in vegetatively growing cells. In the absence of inositol, the cells did not sporulate but, after a certain time, were unable to return to vegetative growth. These nonsporulating cells did, however, incorporate acetate into lipids and double their deoxyribonucleic acid content in the premeiotic phase. We believe that it is this lack of coordination of biosynthetic events which causes inositol-less death on sporulation media without inositol.     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

Yeast physiology — a micro-synopsis

An outline of the main features of yeast physiology is given which focusses mainly — but not exclusively — upon Saccharomyces cerevisiae. The metabolic processes of the cell related to carbon flux, energy production and the formation of reducing equivalents (NADH and NADPH) are discussed for both aerobic and anaerobic conditions. Mechanisms are described by which metabolic processes can be controlled. A brief account of the life cycle of Saccharomyces is given explaining the difference between sporulation and vegetative growth. The Crabtree and Pasteur effects are defined, discussed and explained in relation to glucose metabolism and ethanol formation.     

The use of step enzymes as markers during meiosis and ascospore formation in Saccharomyces cerevisiae.

The activities of ornithine aminotransferase, sucrase and acid and alkaline phosphatases have been studied throughout sporulation in Saccharomyces cerevisiae. The same enzymes were monitored during synchronous vegetative growth. Each of these enzymes has been demonstrated to increase in a 'step' manner during both growth and sporulation. Alkaline phosphatase increased in a two-step manner whereas the others increased in a single step. The times of increase of these enzymes formed a similar sequence during both sporulation and growth. It has been proposed that these enzymes are under a common mechanism of control during growth and sporulation and that the sequence of enzyme appearance may be used as markers of the sporulation process.     

Rapamycin specifically interferes with the developmental response of fission yeast to starvation.

Rapamycin is a microbial macrolide which belongs to a family of immunosuppressive drugs that suppress the immune system by blocking stages of signal transduction in T lymphocytes. In Saccharomyces cerevisiae cells, as in T lymphocytes, rapamycin inhibits growth and cells become arrested at the G1 stage of the cell cycle. Rapamycin is also an effective antifungal agent, affecting the growth of yeast and filamentous fungi. Unexpectedly, we observed that rapamycin has no apparent effect on the vegetative growth of Schizosaccharomyces pombe. Instead, the drug becomes effective only when cells experience starvation. Under such conditions, homothallic wild-type cells will normally mate and undergo sporulation. In the presence of rapamycin, this sexual development process is strongly inhibited and cells adopt an alternative physiological option and enter stationary phase. Rapamycin strongly inhibits sexual development of haploid cells prior to the stage of sexual conjugation. In contrast, the drug has only a slight inhibitory effect on the sporulation of diploid cells. A genetic approach was applied to identify the signal transduction pathway that is inhibited by rapamycin. The results indicate that either rapamycin did not suppress the derepression of sexual development of strains in which adenylate cyclase was deleted or the cyclic AMP-dependent protein kinase encoded by pka1 was mutated. Nor did rapamycin inhibit the unscheduled meiosis observed in pat1-114 mutants. Overexpression of ras1+, an essential gene for sexual development, did not rescue the sterility of rapamycin-treated cells. However, expression of the activated allele, ras1Val17, antagonized the effect of rapamycin and restored the ability of the cells to respond to mating signals in the presence of the drug. We discuss possible mechanisms for the inhibitory effect of rapamycin on sexual development in S. pombe.     

EVIDENCE OF A ROLE OF HETEROCYSTS IN THE SPORULATION OF A BLUE-GREEN ALGA

Sporulation of vegetative cells adjacent to heterocysts is prevented by detachment of the two cell types, probably without impairment of the capacity of those vegetative cells to sporulate. Apparently, therefore, heterocysts are in part responsible for the sporulation of vegetative cells attached to them.     

On the formation of crystal proteins during sporulation in Bacillus thuringiensis var. thuringiensis

The sporulation potential of Bacillus subtilis as a function of position in the cell cycle was determined by transferring cells from growth medium to sporulation medium at various times during growth. Growth was induced by incubating heat-activated spores in rich medium or by diluting stationary phase vegetative cultures with fresh growth medium. The results supported earlier observations that sporulation potential is cell cycle dependent. The rise in sporulation potential was studied by exposing cultures to the inhibitors of cell wall and protein synthesis, vancomycin and chloramphenicol. The delay in the appearance of the peak of sporulation potential caused by these inhibitors compared with the reported lack of effect of nalidixic acid, indicates that the appearance of sporulation potential requires synthesis of a macromolecular component other than deoxyribonucleic acid. The effect of nalidixic acid in preventing the decline of the sporulation potential was compared with the effect of high temperature on a mutant temperature sensitive for the initiation of DNA replication. It was found that prevention of chromosome completion with nalidixic acid maintained a high sporulation potential, whereas prevention of chromosome re-initiation in the temperature sensitive mutant did not affect the decline in sporulation potential as the cells enter stationary phase.Abbreviations NAL Nalidixic acid - HPUra 6-(p-hydroxyphenylazo)-uracil - VAN Vancomycin - CAM Chloramphenicol - BHI Brain heart infusion broth - c.f.u. Colony forming units     

Variation of (1 leads to 3)-beta-glucanases in Saccharomyces cerevisiae during vegetative growth, conjugation, and sporulation.

The total (1 leads to 3)-beta-glucanase activities associated with cell extracts and cell walls of Saccharomyces cerevisiae were measured during vegetative growth, conjugation, and sporulation. Using a system of column chromatography, we resolved (1 leads to 3)-beta-glucanase activity into six different enzymes (namely, glucanases I, II, IIIA, IIIB, IV, and V). The contributions of the individual enzymes to the total activity at the different stages of the life cycle were determined. Total glucanase activity increased during exponential growth and decreased in stationary resting-phase cells. Glucanase IIIA was the predominant enzyme in stationary resting-phase cells. Glucanases I, II, IIIB, and IV were either absent or present at low levels in stationary phase cells, but their individual activities (in particular, glucanase IIIB activity) increased substantially during exponential growth. Total (1 leads to 3)-beta-glucanase activity did not change significantly during conjugation of two haploid mating strains, S. cerevisiae 2180A and 2180B, and no notable changes were detected in the activities of the individual enzymes. Sporulation was accompanied by a rapid increase and then a decrease in total glucanase activity. Most of the increase was due to a dramatic rise in the activity of glucanase V, which appeared to be a sporulation-specific enzyme. Glucanase activity was not derepressed by lowering the glucose concentration in the growth medium.