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At least 19 recordsLinked to original sources
PubMed1979

Genetic instability of sporulation-associated characters in a Bacillus subtilis mutant: relationship between sporulation, segregation and the synthesis of extracellular enzymes (kinetic studies).

In the genetically unstable, protease-overproducing 'medusa (M) strains of Bacillus subtilis, segregation of stable, wild-type-like B cells occurred mainly during sporulation. After the end of the exponential growth phase, a small fraction of M cells sporulated quickly and formed M spores, while the majority of the cells, after a 'critical period', gave rise to B segregants which sporulated after a delay. Segregation occurred without cell division. Delayed sporulation, segregation and protease overproduction are related. Similar but more complex results were obtained with the highly unstable TD strains. Sporulation and the kinetics of protease overproduction were also followed in several stable segregants. Depending on the strain, either the rate of protease production or both the rate and time course were affected. The results are interpreted in terms of sequential activation and de-activation of sporulation genes. The production of the alkaline and the neutral proteases was, in general, under common genetic control. In some strains alpha-amylase was also overproduced.

Alleles↗
PubMed1975

Promotion of sporulation by caffeine pretreatment in Saccharomyces cerevisiae. I. Metabolism of nucleic acids and protein during sporulation.

Cells cultured in the presence of caffeine had high sporulation ability. The sporulation-promotive effect of caffeine was studied, special attention being paid upon changes in nucleic acid metabolism. When transferred to a sporulation medium, the breakdown of RNA, the synthesis of protein, RNA and DNA, commitment to sporulation and the appearance of mature asci took place in caffeine-treated cells significantly earlier than in control cells. Commitment to sporulation occurred before the completion of premeiotic DNA synthesis in both caffeine-treated and control cells.

Caffeine↗
PubMed1976

Promotion of sporulation by caffeine pretreatment in Saccharomyces cerevisiae. II. Changes in ribonuclease activity during sporulation.

Changes in RNase activity during sporulation of a homothallic diploid strain of Saccharomyces cerevisiae were measured in caffeine-treated and non-treated cells. 1. In caffeine-treated cells soon after the transfer to the sporulation medium a significant increase in RNase activity was observed; in control cells the rise of RNase activity was less and started after a lag period of 5 h. The final activity of RNase activity was about twice as high in caffeine-treated cells as in control cells. 2. Increase in RNase activity during sporulation was sensitive to cycloheximide in control cells, but insensitive in caffeine-treated cells. 3. RNases from vegetative cells and from sporulating ones are different in their Km values. Relation of the changes in RNase activity to premeiotic DNA synthesis is discussed.

Caffeine↗
PubMed1978

The pattern of sporulation in Anabaena circinalis and comments on the role of heterocysts in sporulation in blue-green algae.

Cells between two intercalary heterocysts differentiate at random into spores in A. circinalis. One or more cells, which fail to transform into spores, are present between the two adjacent spores, and these cells disorganize later. A critical C:N ratio regulates sporulation and heterocyst formation. During sporulation the reductive ability of the heterocyst gradually diminishes. It is concluded on the basis of this and other evidence that sporulation is regulated by interactions between heterocysts and vegative cells which are manifested in diverse patterns in different species of blue-green algae.

Ammonium Chloride↗
PubMed1975

Genetic mapping of sporulation operons in Bacillus subtilis using a thermosensitive sporulation mutant.

A thermosensitive sporulation mutant was used to determine the order of sporulation operonsin the urs region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-SPO-96(SpoII)-spo-85(SpoV)-spo-279(SpoII)-furA-ura-cysC-spo-NG1.67(SpoIII). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972).

Alkaline Phosphatase↗
PubMed1977

Development of bacteriophage phi29 in sporulating and non-sporulating cells of bacillus subtilis 168.

Infection by bacteriophage phi29 of Bacillus subtilis 168 and of its asporogenous mutant spoOA-3NA has been studied in exponential and stationary phases. As first observed with phage phie infections, the burst-size decreases during the stationary phase much more rapidly in wild type than in mutant cells. In addition, the two strains are shown to differ even during growth in their response to phage phi29 infection. During a short period in the exponential phase, no phage production occurs when infected bacteria (whether spo+ or spo-) are incubated in their growth medium, but phage is produced when incubation takes place after transfer to fresh medium. From these and other unexpected findings it is concluded that any causal relation between sporulation and phage development must be considered with caution. Phage infection of spo+ cells at the end of the growth period does not affect the time required for mature spore formation.

Bacillus subtilis↗
PubMed1978

Spore location patterns in sporulating doublets of Bacillus cereus and Bacillus megaterium, derived from single doublet isolates with differing sporulation geometry.

The distribution of spore loci in pairs of Bacillus cereus and B. megaterium showed different degrees of polarization from random location towards location at the old end of the sporangium. When individual doublets containing both spores at either the old or new ends of the sporangia were isolated by micromanipulation, subsequent culture showed the same spore location patterns.

Bacillus megaterium↗
PubMed1979

Relationship of glycolytic intermediates, glycolytic enzymes, and ammonia to glycogen metabolism during sporulation in the yeast Saccharomyces cerevisiae.

To identify the factors which control glycogen synthesis in Saccharomyces cerevisiae, we have studied the regulation of glycogen metabolism during sporulation, since in vivo glycogen has been reported to undergo significant changes in concentration during this process. We examined the concentration of a number of key glycolytic intermediates and enzymes in strains that sporulate at different rates and those that are deficient in sporulation. There were no significant changes found in the adenylate energy charge or cyclic AMP levels throughout sporulation. Although significant alterations occurred in the levels of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, and ATP during sporulation, only the fourfold increase in fructose-1,6-bisphosphate appeared to correlate with glycogen synthesis in all of the strains examined. Only limited changes occurred in the level of a number of glycolytic and gluconeogenic enzymes which were examined during this process. Intracellular glucose content underwent a dramatic 30- to 40-fold increase in sporulating cells. Comparison of strains with different rates of sporulation demonstrated that this increase in glucose content coincides with the time of glycogen degradation in each strain. Both the increase in glucose content and the degradation of accumulated glycogen were not observed in nonsporulating alpha/alpha strains, or in cells incubated in NH(4) (+) supplemented sporulation medium. Although glucose appears to be the direct product of glycogen degradation, a 10-fold increase in a nonspecific alkaline phosphatase occurs at this time, which may be degrading phosphorylated sugars to glucose. All of the strains examined released extracellular glucose while suspended in acetate sporulation medium. It is concluded that most of the changes in the glycolytic pathway that occur during sporulation, with the exception of glycogen degradation and the concomitant increase in intracellular glucose pools, are a response to the transfer to sporulation medium and are independent of sporulation-specific processes. Inhibition of sporulation with ammonium ions resulted in a different pattern of change in all of the glycolytic intermediates examined, including a twofold increase in cyclic AMP levels. Ammonia did not interfere with glycogen synthesis, but prevented sporulation-specific glycogen degradation. The levels of the glycolytic enzymes examined were not affected by ammonia.

Adenine Nucleotides↗
PubMed2025

Identification of sporulation genes in Bacillus anthracis highlights similarities and significant differences with Bacillus subtilis.

The molecular basis of endospore formation in the model gram-positive bacterium Bacillus subtilis has been investigated for over half a century. Here, using high throughput and classical genetic approaches, we performed a comparative analysis of sporulation in the human pathogen Bacillus anthracis. A transposon-sequencing screen identified >150 genes required for B. anthracis sporulation. As anticipated, many of the genes that are critical for sporulation in B. subtilis were also required for B. anthracis sporulation. However, we identified >50 genes that are important for sporulation in B. anthracis but not in B. subtilis, and 22 B. anthracis sporulation genes that are absent from the B. subtilis genome. To validate the hits from our screen, we generated an ordered transposon-mutant library using Knockout Sudoku. Cytological analysis of a subset of the canonical sporulation-defective mutants revealed similar but not identical phenotypes in the pathogen compared to the model. We investigated several of the newly identified sporulation genes, with an in-depth analysis of one, ORF 04167, renamed ipdA. Sporulating cells lacking ipdA are blocked in the morphological process of engulfment, generating septal bulges. An AlphaFold-Multimer screen and a classical genetic enrichment revealed that IpdA is a secreted inhibitor of the polysaccharide deacetylase PdaN. Our data support a model in which induction of IpdA at the onset of sporulation inhibits deacetylation of the cell wall peptidoglycan (PG), enabling the sporulation-specific PG hydrolases to catalyze engulfment. Altogether, our studies reveal that B. subtilis is an excellent model for endospore formation in B. anthracis, while underscoring the importance of direct analysis in B. anthracis. The suite of tools that we have generated will catalyze the molecular dissection of sporulation and other cell biological processes in this important human pathogen.

Bacillus anthracis↗
PubMed1977

Isolation and characterization of fusidic acid-resistant, sporulation-defective mutants of Bacillus subtilis.

Fusidic acid-resistant, sporulation-defective mutants were isolated from Bacillus subtilis 168 thy trp. About two-thirds of the fusidic acid-resistant (fusr) mutants were defective in sporulation ability and fell into three classes with respect to sporulation character. The representative mutants FUS426 and FUS429 were characterized in detail. FUS426 [fusr spo (Ts)], a temperature-sensitive sporulation mutant, grew well at 30 and 42 degrees C but did not sporulate at 42 degrees C. FUS429 [fusr spo (Con)], conditional sporulation mutant, grew and sporulated normally in the absence of fusidic acid, but its sporulation and growth rates decreased in the presence of fusidic acid, depending on the concentration of the drug. Although electron microscopic observation showed that both mutants were blocked at stage I of sporulation, the physiological analyses indicate that these mutants belong to the SpoOB class. Both mutants formed a thickened cell wall as compared with that of the parental strain. Genetic and in vitro protein synthesis analyses led to the conclusion that the sporulation-defective character of mutants FUS426 and FUS429 resulted from an alteration in elongation factor G caused by a single lesion in the fus locus. The possible role of elongation factor G in sporulation is discussed.

Bacillus subtilis↗
PubMed1976

Correlation among turnover of nucleic acids, ribonuclease activity and sporulation ability of Saccharomyces cerevisiae.

The turnover of nucleic acids and changes in ribonuclease activity during sporulation of Saccharomyces cerevisiae were studied. In the sporulating strains, 37-58% of vegatatively synthesized RNA were degraded during the sporulation process. The degree of degradation of vegetative RNA was proportional to the sporulation ability. In the non-sporulating strains, the degradation of vegetative RNA was less than 28% in the sporulation medium. Accompanied by the degradation of vegetative RNA, a ribonuclease activity increased several times during sporulation. We have found a close relation among the sporulation rate, the degree of the degradation of vegetative RNA and the increase in ribonuclease activity in the sporulation medium, using cells of which sporulation ability was repressed by changing the age or carbon source in various degrees.

Carbon↗
PubMed1976

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.

Aminopeptidases↗
PubMed1979

Changes in sporulation potential during the growth cycle of Bacillus subtilis.

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.

Bacillus subtilis↗
PubMed1977

Efficient sporulation of yeast in media buffered near pH6.

Diploid cells of Saccharomyces cerevisiae underwent meiosis and sporulation when placed in 1% potassium acetate sporulation medium. In unbuffered sporulation medium the pH rose very rapidly, reaching pH 8.4 after 2 h of sporulation. Under these conditions, the uptake of radioactive adenine and lysine was extremely limited, and ascus formation was insensitive to inhibitors such as 5-fluorouracil and canavanine. By using several different buffers, we showed that an increase in the pH of sporulation media was not necessary for sporulation to occur. Spore viability and the kinetics of ascus and prototroph formation were normal for cells sporulated in several types of media buffered as low as pH 5.5. Incubation of sporulating cells below pH 6.5 did cause separation of small but viable buds from their mother cells. With sporulating cells buffered below pH 6.5, the incorporation of radioactive adenine and lysine was greatly enhanced and cells became sensitive to inhibition by 5-fluorouracil and canavanine.

Acetates↗
PubMed1979

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.

Culture Media↗
PubMed1979

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.

Alkaline Phosphatase↗
PubMed1979

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Spectinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc 1-11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1-11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.

Bacillus subtilis↗