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.     

Protein degradation during sporulation ofBacillus megaterium: Effect of actinomycin D

The first acceleration of protein degradation in cells ofBacillus megaterium was found at the stage 0–I of sporulation, the second one at the stage II–III, where the sporulation process became irreversible. These accelerations were reduced by actinomycin D inhibiting RNA and protein syntheses by more than 95%. In the presence of the antibiotic, only 8% of prelabeled proteins were degraded. Actinomycin D did not lower either the concentration of ATP or the proteolytic activity in the homogenate prepared from sporulating cells. This indicates that the inhibition of protein catabolism by actinomycin D was not owing to the absence of ATP or proteolytic enzymes. Actinomycin probably inhibited an unknown step preceding the proteolytic attack of the protein molecules during sporulation, because it had no significant effect on proteolysis during vegetative growth.     

Factors modulating differentiation of spores: Characterization of mutants defective in sporulation in the cyanobacterium Anabaena doliolum (AdS strain)

Summary Mutants of Anabaena doliolum (AdS strain) altered with respect to the time of initiation and degree of sporulation were isolated following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine and hydroxylamine. The non-sporulating mutant showed a high phycocyanin (Pc): chlorophyll a (chl a) ratio (ca. 7.2) as compared to sporulating strains (Pc:chl a, 4.7–5.3). Also this strain seemed to have higher RNA pools per unit of genomic material as reflected in a higher RNA:DNA ratio. The data suggest that degradaton of phycocyanin and controlled RNA synthesis are prerequisites for sporulation. Mutants exhibiting non-sporulation and delayed initiation of sporulation accumulated more nitrogen through nitrogen fixation, probably indicating nitrogenase function over an extended vegetative phase.     

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

Hydrolytic Enzymes and Sporulation in Bacillus intermedius

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II–IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.     

Ribonucleoprotein particle appearing during sporulation in yeast.

During sporulation of Saccharomyces cerevisiae, most strains accumulate an unmethylated 20S RNA. Contrary to previous reports, this sporulation 20S RNA is distinct from the short-lived methylated 20S RNA precursor of 18S rRNA. This RNA species was found in a cytoplasmic 32S ribonucleoprotein particle consisting of one single-stranded 20S RNA molecule and 18 to 20 identical protein subunits of molecular weight 23,000. The ribonucleoprotein particle was resistant to ribonuclease digestion, although purified 20S RNA was ribonuclease sensitive. Both the RNA and the protein of the 32S ribonucleoprotein particle were only synthesized under conditions that induce sporulation. The accumulation of 20S RNA depended on continued protein synthesis but was actinomycin D insensitive, despite a high guanine-plus-cytosine content. Synthesis of 20S RNA stopped when cells were removed from sporulation conditions and placed in growth medium.     

Conditional Mutants of Meiosis in Yeast

Three temperature-sensitive mutants, spo1-1, spo2-1, and spo3-1, were characterized with respect to their behavior in sporulation medium at a restrictive temperature. The time of expression of the functions defective in the mutants was determined by temperature-shift experiments during the sporulation process. In addition, each mutant was examined for the following: (i) its ability to undergo the nuclear divisions of meiosis; (ii) deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis; (iii) protein turnover; and (iv) colony-forming ability after exposure to sporulation medium. Mutant spo1-1 is defective in a function which confers a temperature-sensitive period which extends over 32% of the sporulation cycle. The temperature-sensitive period of mutant spo2-1 occupies 34% of the cycle, whereas the temperature-sensitive period of mutant spo3-1 extends over 2% of the sporulation cycle. Cytological evidence indicates that all three mutants initiate but do not complete the meiotic nuclear divisions. The DNA content of sporulation cultures of mutants spo1-1 and spo3-1 did not increase to the wild-type level; DNA synthesis in spo2-1 was normal. All three strains exhibit a loss of colony-forming ability during incubation in sporulation medium at the restrictive temperature. RNA and protein synthesis and protein turnover occur in the mutants.     

Phenol degradation by immobilized cold-adapted yeast strains of Cryptococcus terreus and Rhodotorula creatinivora

Three strains were isolated from hydrocarbon-polluted alpine habitats and were representatives of Cryptococcus terreus (strain PB4) and Rhodotorula creatinivora (strains PB7, PB12). All three strains synthesized and accumulated glycogen (both acid- and alkali-soluble) and trehalose during growth in complex medium containing glucose as carbon source and in minimal salt medium (MSM) with phenol as sole carbon and energy source. C. terreus strain PB4 showed a lower total accumulation level of storage compounds and a lower extracellular polysaccharides (EPS) production than the two R. creatinivora strains, PB7 and PB12. Biofilm formation and phenol degradation by yeast strains attached to solid carriers of zeolite or filter sand were studied at 10°C. Phenol degradation by immobilized yeast strains was always higher on zeolite compared with filter sand under normal osmotic growth conditions. The transfer of cells immobilized on both solid supports to a high osmotic environment decreased phenol degradation activity by all strains. However, both R. creatinivora PB7 and PB12 strains maintained higher ability to degrade phenol compared with C. terreus strain PB4, which almost completely lost its phenol degradation activity. Moreover, R. creatinivora strain PB7 showed the highest ability to form biofilm on both carriers under high osmotic conditions of cultivation.     

Degradation and replenishment of the labile protein fraction in non-growing cells of the asporogenic mutant ofBacillus megaterium

Kinetics of degradation of labelled proteins was followed in two asporogenic mutants ofBacillus megaterium during incubation in a sporulation medium. Both the mutant producing exocellular protease (KM 1prn ⁺) and the mutant not producing the enzyme (KM 12prn) were found to contain a labile protein fraction, whose proportion decreases with prolonged time of labelling and whose half-life is about 1 h. Most proteins were relatively stable and were degraded at a rate of 1 %/h and 2 %/h in strains KM 1 and KM 12, respectively (half life 70–80 h and 35–40 h in strains KM 1 and KM 12, respectively). The intracellular proteolytic activity of the KM 12 mutant remains practically the same during incubation in the sporulation medium or slowly increases. The labile protein fraction practically disappears from the cells after a 3.5-h incubation. When such a culture is then subjected to a shift-up and transferred again to the sporulation medium, the rate of protein turnover temporarily increases. The temporary increase of the turnover rate is caused by a partial replenishment of the labile protein fraction rather than by an accelerated degradation of the relatively stable fraction. The intracellular proteolytic activity does not increase under these conditions. The wild sporogenic strain ofB. megaterium also contains the labile protein fraction. Its half protein life is 1 h or less. However, the second protein fraction is degraded much more rapidly than in the asporogenic mutants and its half life is 6–7 h.     

Synthesis of Ribosomal Ribonucleic Acid During Sporulation of Bacillus subtilis

The incorporation of radioactive uracil into 50s and 30s ribosomal subunits and ribosomal ribonucleic acid (rRNA) was studied during the growth cycle of different sporogenic and asporogenic strains of Bacillus subtilis. It was found that partially synchronized cultures of the strains examined incorporated labeled uracil into the two ribosomal subunit species and rRNA during sporulation and during the stationary phase of the asporogenic strains. Kinetic studies have shown that, compared to vegetative cells, the percentage of uracil incorporated into the ribosomal subunits of cells taken 30 min after the end of exponential growth was decreased by about 25 to 35%. This decrease, however, appeared to be a general characteristic of stationary-phase cells and seems to depend on the nature of the sporulation medium and to some extent on the nature of the strain but not on the sp(+) or sp(-) phenotype of the strain. Moreover, by use of actinomycin D it was shown that the labeled uracil incorporated, in the presence of the drug, during the sporulation period was located in the ribosomal subunits (stable RNA). Based on these results, we concluded that during sporulation ribosomal genes are transcribed and consequently rRNA continues to be synthesized, although to a lesser extent than during vegetative growth. These results are discussed in the light of those obtained by Hussey et al.     

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Summary Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of ³H-adenine at T₇, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T₄ in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.     

Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens.

The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined. Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium. The rate of RNA synthesis decreased as sporulation progressed. Deoxyadenosine increased uptake of [14C]uracil and [14C]thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml. Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation. The rate of protein breakdown during vegetative growth was 1%/h. During sporulation this rate increased to 4.7%/h. When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h. If added at 3 h the rate decreased to 2.1%/h. The role of proteases in this process is discussed.     

Early-blocked sporulation mutations alter expression of enzymes under carbon control in Bacillus subtilis

Summary The physiological roles of the gene subset defined by early-blocked sporulation mutations (spo0) and their second-site suppressor alleles (rvtA11 and crsA47) remain cryptic for both vegetative and sporulating Bacillus subtilis cells. To test the hypothesis that spo0 gene products affect global regulation, we assayed the levels of carbon- and nitrogen-sensitive enzymes in wild-type and spo0 strains grown in a defined minimal medium containing various carbon and nitrogen sources. All the spo0 mutations (except spo0J) affected both histidase and arabinose isomerase levels in an unexpected way: levels of both carbon-sensitive enzymes were two- to six-fold higher in spo0 strains compared to wild type, when cells were grown on the derepressing carbon sources arabinose or maltose. There was no difference in enzyme levels with glucose-grown cells, nor was there a significant difference in levels of the carbonindependent enzymes glutamine synthetase and glucose-6-phosphate dehydrogenase. This effect was not due to a slower growth rate for the spo0 mutants on the poor carbon and nitrogen sources used. The levels of carbon-sensitive enzymes were not simply correlated with sporulation ability in genetically suppressed spo0 mutants, but the rvtA and crsA suppressors each had such marked effects on wild-type growth and enzyme levels that these results were difficult to interpret. We conclude that directly or indirectly the spo0 mutations, although blocking the sporulation process, increase levels of carbon-sensitive enzymes, possibly at the level of gene expression.     

Efficient expression of a 100-kilodalton mosquitocidal toxin in protease-deficient recombinant Bacillus sphaericus.

The expression of the 100-kDa mosquitocidal toxin (Mtx) during vegetative growth and sporulation in nine different mosquito-larvicidal strains of Bacillus sphaericus has been analyzed. In five out of the nine strains the 100-kDa toxin was found to be expressed predominantly in the vegetative phase of growth, and in all nine strains the level of the toxin in sporulated cells was very low or undetectable. Strains in four out of the six DNA homology groups of B. sphaericus produced intracellular and extracellular proteases, which degraded the 100-kDa toxin, during sporulation. The 100-kDa toxin gene was expressed by using its native promoter on a multicopy number plasmid in B. sphaericus 1693 (protease negative) and B. sphaericus 13052 (protease positive). High levels of the 100-kDa toxin were produced in vegetative cells of both strains as well as in sporulated cells of protease-negative strain 1693, which is in contrast to the low levels of the 100-kDa toxin produced in sporulated cells of protease-positive strain 13052. Thus, the small amount of the 100-kDa toxin in sporulated cells of the nine mosquito-larvicidal strains is probably due to degradation of the 100-kDa toxin synthesized during vegetative growth by a protease(s) produced during sporulation. B. sphaericus 1693 transformed with the 100-kDa toxin gene was as toxic to mosquito larvae during both vegetative growth and sporulation as the natural high-toxicity strains of sporulated B. sphaericus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrient conditions determine the localization of Bacillus thuringiensis Vip3Aa protein in the mother cell compartment

Vip3Aa was first identified as a protein secreted during the vegetative growth phase of Bacillus thuringiensis (Bt) bacteria and which shows high insecticidal toxicity against lepidopteran insect pests (Estruch et al., 1996). Bt strains formulated as bio-insecticides only had low amounts of Vip3Aa secreted to the medium. Here, we report that Vip3Aa proteins produced by three different Bt strains, including an industrial strain, were indeed not secreted to the culture solution when grown in sporulation medium, but were retained in the mother cell compartment. In order to further investigate the Vip3Aa secretion and location, we grew the strains in rich medium. We found that in rich medium, a fraction of Vip3Aa was secreted, suggesting that Vip3Aa secretion is nutrient-dependent. Regardless of the growth conditions, we found that Vip3Aa retained in cell pellets exhibited high toxicity against Spodoptera frugiperda larvae. Hence, we speculate that the accumulation of Vip3Aa protein in the mother cell compartment under sporulation conditions could still be used as an efficient strategy for industrial production in commercial Bt strains.     

Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to 98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.     

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.     

少根根霉产孢能力、变种与发酵产物的相关性

[目的]探究少根根霉不同生态环境、不同地理位置、不同产孢能力和不同分类学变种的发酵产物多样性及相关性,为工业生产提供指导.[方法]代表性的68株少根根霉菌株于糯米培养基中进行液态发酵,利用高效液相色谱法测定各种发酵产物浓度,计算发酵产物间的Pearson相关系数,通过多因素方差分析和主成分分析解析各类菌株与发酵特性的相...