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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↗
PubMed1975

RNA polymerase from sporulating Bacillus subtilis. Purification and properties of a modified form of the enzyme containing two sporulation polypeptides.

A new form of DNA-dependent RNA polymerase termed enzyme III has been purified from sporulating cells of Bacillus subtilis. In addition to the subunits of core RNA polymerase (beta', beta, alpha, and omega), enzyme III contains sporulation-specific polypeptides of 85,000 (P85) and 27,000 (P27) daltons. P85 corresponds to an RNA polymerase-binding protein previously identified by precipitation of RNA polymerase from crude extracts of sporulating cells with antibody directed against core enzyme. Both P85 and P27 co-purified with RNA polymerase highly purified by gel filtration, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation. Enzyme III bound more tightly to phosphocellulose and sedimented more rapidly during zone centrifugation than did RNA polymerase lacking the sporulation polypeptides. RNA polymerase containing P85 and P27 transcribed B. subtilis DNA about 4.5 times more actively than did core RNA polymerase, although both enzymes exhibited similar activities with poly(dA-dT) and phage phie DNA as templates. Enzyme III and core RNA polymerase also differed in their response to increasing concentrations of Mg2+ and KCl.

Bacillus subtilis↗
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↗
PubMed1976

Ethanol sensitivity of sporulation in Bacillus subtilis: a new tool for the analysis of the sporulation process.

The growth rate of Bacillus subtilis is lowered but the final cell yield is unchanged when certain concentrations of ethanol are present in the culture medium. At the concentration allowing growth at half-maximal rate, practically no spores are formed. Blockage of spore formation generally occurs at stage 0-I. Sensitivity to ethanol of the capacity to form spores is limited, in a nonsynchronized culture, to a period of at most 45 min around t1. Postexponential events such as excretion of certain enzymes and modification of ribonucleic acid polymerase are altered or suppressed in the presence of ethanol, possibly as the results of a physical change upon the cell membrane. In effect, ethanol is turning wild-type cells into phenocopies of spoO mutants.

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↗
PubMed1976

Protein degradation and proteinases during yeast sporulation.

During ascospore formation in Saccharomyces cerevisiae, at least 60-70% of the pre-existing vegetative protein was broken down at a rather constant rate until the time mature asci appeared. Under the same conditions in a non-sporulating haploid derived from the same strain the rate of protein degradation, although initially comparable to that of sporulating cells, decreased much more rapidly. Proteins synthesized at different times during sporulation had approximately the same degradation rates as the vegetative proteins. Similar rates of degradation were observed for the vegetative proteins in all fractions obtained from cell homogenates by differential centrifugation. Protein breakdown after transfer to sporulation medium was blocked by uncouplers and inhibitors of energy metabolism, and was partially inhibited by cycloheximide. Polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, of the proteins extracted from vegetative cells and from isolated asci and ascospores revealed that ascus formation was accompanied by a shift of the cellular proteins to a lower molecular weight. From several proteinase inhibitors tested, only tosyl-p-lysine chloromethylketone slightly reduced the rate of ascus formation. During sporulation the total activity of proteinase A increased more than twofold with a maximum at 18 h after transfer to sporulation medium. Total proteinase B activity showed a striking increase in the first hours after transfer to sporulation medium and after that remained constant throughout sporulation. The levels of carboxypeptidase Y and of the proteinase B inhibitor were not significantly altered during sporulation. The proteinases and the proteinase B inhibitor were present within the mature ascospore. The proteinases from both vegetative and sporulating cells were eluted with the same ionic strength from DEAE-Sephadex, and they were undistinguishable in their sensitivity to different proteinase inhibitors. No additional proteolytic activities could be detected in sporulating cells using 3H-labelled denatured yeast protein as a substrate.

Carboxypeptidases↗
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↗
PubMed1976

Repression of sporulation in Bacillus subtilis by L-malate.

L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis. When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h. The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed. Sporulation was not repressed when malate was added to the culture at t4 or later. A mutant was selected for ability to sporulate in the presence of malate. This strain could also sporulate in the presence of glucose. The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme. This indicates a defect in the metabolism of malate in the mutant. A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate. A model for the regulation of sporulation by malate is presented and discussed. Citric acid cycle intermediates other than malate did not affect sporulation. In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block. The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate.

Alcohol Oxidoreductases↗