Macromolecule Synthesis and Breakdown in Relation to Sporulation and Meiosis in Yeast

The time course of synthesis and breakdown of various macromolecules has been compared for sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast cells transferred to potassium acetate sporulation medium. Both types of cells incorporate label into ribonucleic acid and protein. The gel electrophoresis patterns of proteins synthesized in sporulation medium are identical for sporulating and nonsporulating diploids; both are different from electropherograms of vegetative cells. Sporulating and nonsporulating strains differ with respect to deoxyribonucleic acid synthesis; no deoxyribonucleic acid is synthesized in the latter case, whereas the deoxyribonucleic acid complement is doubled in the former. Glycogen breakdown occurs only in sporulating strains. Breakdown of preexisting vegetative ribonucleic acid and protein molecules occurs much more extensively in sporulating than in nonsporulating cells. A timetable of these data is presented.     

Cannibalism: a social behavior in sporulating Bacillus subtilis

A social behavior named cannibalism has been described during the early stages of sporulation of the Gram-positive Bacillus subtilis. This phenomenon is based on the heterogeneity of sporulating populations, constituted by at least two cell types: (1) sporulating cells, in which the master regulator of sporulation Spo0A is active, and (2) nonsporulating cells, in which Spo0A is inactive. Sporulating cells produce two toxins that act cooperatively to kill the nonsporulating sister cells. The nutrients released by the dead cells into the starved medium are used for growth by the sporulating cells that are not yet fully committed to sporulate, and as a result, sporulation is arrested. This review outlines the molecular mechanisms of the killing and immunity to the toxins, the regulation of their production and other examples of killing of siblings in microorganisms. The biological significance of this behavior is discussed.     

Lipid Synthesis During Sporulation of Saccharomyces cerevisiae

Lipid synthesis was studied in both sporulating (diploid) and nonsporulating (haploid) cells of Saccharomyces cerevisiae. Two phases of lipid synthesis occur in diploid cells transferred to sporulation medium. Phase I, which occurs during the first 12 h of exposure to sporulation medium, was also observed in the haploid strains. Phase II, occurring from the 20th to the 25th h, coincided with the appearance of mature asci and was observed only in the diploid cells. The majority of phospholipid synthesis took place during period I, whereas neutral lipid synthesis occurred during both periods. Phospholipid synthesis was virtually identical in both type and quantity in the sporulating and nonsporulating strains.     

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.     

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.     

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.     

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.     

Order of expression of genes affecting septum location during sporulation of Bacillus subtilis.

The mean volumes of stationary-phase cells of wild-type and asporogenous mutants of Bacillus subtilis have been measured. Mutants blocked at stage 0 of sporulation either produced cells that had the same volume as the developing sporangium or they divided to produce cells of one-half this volume. The order of expression of the genes affected by the mutations in these strains was determined by biochemical characterization and by construction of double sporulation mutants. Mutants that produced small cells were blocked at an earlier stage of sporulation than those that produced large cells. It is suggested that the following dependent sequence must occur before the formation of the prespore spetum: (i) the initiation of sporulation, (ii) a signal to block the final central division site, and (iii) a signal to activate a polar septum site.     

Specific alteration of the 30S ribosomal subunits of Bacillus subtilis during sporulation.

Active 30S ribosomal subunits were isolated from vegetative and sporulating cells of Bacillus subtilis. Both subunits were able to function in polyuridylic acid of phage phie messenger ribonucleic acid-dependent protein synthesis in vitro. The sporulation 30S subunits were highly active in polyuridylic acid-dependent polyphenylalanine synthesis but showed a reduced activity in the presence of natural messenger ribonucleic acid as compared with their vegetative counter-parts. The reduced activity was independent of the source of 50S particles and initiation factors (vegetative or sporulation). The alteration of the 30S sporulation subunits appears to be related to the sporulation process, since the same subunits isolated from stationary-phase cells of an asporogenic mutant did not show any impairment in protein synthesis in vitro.     

Thiomethylation of tyrosine transfer ribonucleic acid is associated with initiation of sporulation in Bacillus subtilis: effect of phosphate concentration.

The thiomethylation of Bacillus subtilis tyrosine transfer ribonucleic acid (tRNATyr) (i6A) has been shown to occur during the slowing-down of growth. The extent of this modification in stationary-phase cells grown in defined medium has been determined in parallel with the sporulation frequency. We observed that the presence of phosphate repressed sporulation and also inhibited the thiomethylation of tRNATyr (i6A) of B. subtilis W168. These effects were partially eliminated by decreasing the glucose concentration until it was growth limiting. In the case of strain W23S, in which sporulation is insensitive to glucose repression, sporulation and tRNATyr thiomethylation were not inhibited by nonlimiting concentrations of phosphate. These results suggest that both sporulation and tRNATyr hyper-modification share some common regulatory process.     

Development of a Membrane-Bound Respiratory System Prior to and During Sporulation in Bacillus cereus and Its Relationship to Membrane Structure

Bulk membrane fragments were prepared from cells of Bacillus cereus ATCC 4342 harvested at different stages of growth and sporulation and examined for enzymes involved in electron transport functions. The presence of succinate: DCPIP oxidoreductase (EC 1.3.99.1), succinate: cytochrome c oxidoreductase (EC 1.3.2.1), NADH:DCPIP oxidoreductase (EC 1.6.99.1), NADH:cytochrome c oxidoreductase (EC 1.6.2.1), succinate oxidase [succinate: (O(2)) oxidoreductase, EC 1.3.3.1], and NADH oxidase [NADH:(O(2)) oxidoreductase, EC 1.6.3.1] were demonstrated in membrane fragments from vegetative cells, early and late stationary-phase cells, and in cells undergoing sporulation. During the transition from a vegetative cell to a spore, there was a significant increase in the levels of enzymes associated with energy production via the electron transport system. Cytochromes of the a, b, and c type were detected in all membrane preparations; however, there was a marked increase in the level of cytochromes by the end of vegetative growth which remained throughout sporulation; there were no qualitative changes in the cytochromes throughout growth and sporulation. Sporulation was inhibited by cyanide, stressing the significance of the electron transport system. Enzyme activities were partially masked in washed membrane fragments; however, unmasking (stimulation) was achieved by sodium deoxycholate, sodium dodecyl sulfate, or Triton X-100. The degree of enzyme masking was less in vegetative cell membrane fragments than in membranes prepared from stationary-phase or sporulating cells. Results indicate the development of a membrane-bound electron transport system in B. cereus by the end of growth and prior to sporulation, which results in an increased masking of a number of enzymes associated with the terminal respiratory system of the cell.     

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.     

Effect of alkylhydroxybenzenes, microbial anabiosis inducers, on the structural organization of Pseudomonas aurantiaca DNA and on phenotypic dissociation induction

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas auraniaca and the phenotypic dissociation of this bacterium. The addition of 0.1-0.3 mM hexylresorcinol (C6-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their elastoviscosity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their elastoviscosity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C1-AHB). The DNA SC from actively proliferating cells were characterized by smaller elastoviscosity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C6-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C1-AHB produced a similar effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C6-AHB had a breaking point within the submillimolar (10(-4) M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable genotype and dissociate into distinct variants upon inoculation on solid media.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens.

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

Yeast Hsp70 RNA levels vary in response to the physiological status of the cell.

Yeast Hsp70 genes constitute a multigene family in which at least five of the nine members are heat inducible. Hsp70 RNA levels also vary dramatically during stationary arrest and sporulation. During growth to stationary phase, SSB1-SSB2 and SSC1 RNAs decreased in abundance as cell density increased. In contrast, SSA1-SSA2 RNA levels increased before the diauxic shift and then decreased as cells approach stationary phase. SSA3 RNA was detected only after the diauxic shift and accumulated to high levels as cells entered stationary phase. This accumulation was reversed by addition of glucose. Studies with cyr1 mutants indicated that SSA3 RNA accumulation is stimulated by decreasing intracellular cyclic AMP concentrations. When cells were incubated in sporulation medium, most Hsp70 RNAs, with the exception of SSA1-SSA2 RNA, decreased in abundance. This finding contrasted with the SSA1-SSA2 pattern observed during growth to stationary phase. SSA3 RNA was not detected during growth in acetate-based medium but accumulated after several hours. SSA3 RNA accumulation was higher in sporulating cells than in nonsporulating cells and was reversed by addition of glucose.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

Changes in penicillin-binding proteins during sporulation of Bacillus subtilis

The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined in samples collected at various times from sporulating cultures and compared with the PBPs in a presporulation sample. Large increases in vegetative PBPs 2B and 3 and the appearance of at least one new PBP (42,000 daltons) occurred at reproducible times during sporulation. In some strains a second new PBP (60,000 daltons) was also produced. By comparing the PBP activities in sporulating cells and two spo0 mutants we have classified these changes as sporulation-related events rather than the consequences of stationary-phase aging. The other vegetative PBPs (PBPs 1, 2A, 4, and 5) decreased during sporulation, but not in sufficient amount or at the appropriate time to account for the appearance of the new proteins. A possible connection between specific PBP changes and the penicillin-sensitive stages of sporulation is suggested.