Role of acetate metabolism in sporulation of Saccharomyces carlsbergensis.

Several aspects of the role of acetate metabolism in the sporulation of Saccaromyces carlsbergensis were investigated. Experiments in which the development of the respiratory system was either stimulated by growth on sugars to which the cells have to adapt, or inhibited by chloramphenicol suggested a correlation between respiratory development and sporulation. In cells in which the respiratory system has been repressed during growth, mitobhondrial protein synthesis and derepression are prerequisites for sporulation. When derepression is complete, sporulation no longer depends on mitochondrial protein synthesis. Incorporation experiments with acetate showed that this compound is an important source of intermediates for biosynthetic processes that occur during sporulation. Its incorporation into macromolecular fractions is tightly coupled to sporutlation.     

Sporulation of Saccharomyces cerevisiae in the Absence of a Functional Mitochondrial Genome

The role of the mitochondrial system during sporulation of Saccharomyces cerevisiae was studied. Addition of ethidium bromide (EthBr) to cells growing in acetate medium resulted in the quantitative (>98%) conversion of the culture to the petite genotype in one generation. The cells were respiratory active (derepressed) but contained no mitochondrial deoxyribonucleic acid (mtDNA) as demonstrated by analytical ultracentrifugation in CsCl. When transferred to acetate sporulation medium, the culture sporulated. Ascus production was only slightly below that of the control culture. Synthesis of mtDNA occurred during sporulation in the control but not in the EthBr-treated culture. Mitochondrial protein synthesis was virtually eliminated in the EthBr-treated culture. Therefore, completely derepressed cells can sporulate without a functional mitochondrial genetic system. When partially repressed cells were treated with EthBr, no ascus formation was observed after transfer to sporulation medium. Control cultures underwent respiratory adaptation in sporulation medium and then sporulated. Extensive derepression of the respiratory system is thus required for sporulation, and this adaptation is dependent on a functional mitochondrial system. Our results suggest that once the cells are fully derepressed no mitochondrial genetic information has to be expressed during meiosis and ascus formation.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

UBI4, the polyubiquitin gene of Saccharomyces cerevisiae, is a heat shock gene that is also subject to catabolite derepression control

Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position ?542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress.     

UBI4, the polyubiquitin gene of Saccharomyces cerevisiae , is a heat shock gene that is also subject to catabolite derepression control

 Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position −542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress. Received: 3 June 1996 / Accepted: 20 August 1996     

Derepression of mitochondria and their enzymes in yeast: regulatory aspects

We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate:cytochrome c reductase and NADH:cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for l-malate, l-glutamate, and l₈-isocitrate. Cytochromes b, c₁, and aa₃ are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected.We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4%; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa₃, b, and c₁; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than 6-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression.The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.     

Acetate Utilization and Macromolecular Synthesis During Sporulation of Yeast

Acetate utilization and macromolecule synthesis during sporulation (meiosis) of Saccharomyces cerevisiae were studied. When diploid cells are transferred from glucose nutrient medium to acetate sporulation medium at early stationary phase, respiration of the exogenously supplied acetate proceeds without any apparent lag. At the completion of ascospore development, 62% of the acetate carbon consumed has been respired, 22% remains in the soluble pool, and 16% is incorporated into lipids, protein, nucleic acids, and other cell components. Measurements of the rate of protein synthesis during sporulation reveal two periods of maximal synthetic activity: an early phase coincidental with increases in deoxyribonucleic acid, ribonucleic acid, and protein cellular content and a later phase during ascospore formation. Experiments in which protein synthesis was inhibited at intervals during sporulation indicate that protein synthesis is required both for the initiation and completion of ascus development.     

Effect of mitochondrial functions on synthesis of yeast cytochrome c

The effects of the mitochondrial protein synthesis inhibitor chloramphenicol and the mitochondrial F0 adenosine triphosphatase inhibitor oligomycin on the synthesis of nucleus-encoded cytochrome c protein were studied. Both inhibitors stimulated cytochrome c protein synthesis in the derepressed state (growth in media containing 2% raffinose) but had no effect on the synthesis of the cytochrome c protein in the repressed state (growth in media containing 5% glucose). Oligomycin uncoupled the synthesis of the apoprotein from its processing into the hemoprotein. Neither antibiotic had a significant effect on the rate of glucose repression of cytochrome protein synthesis. The kinetics of cytochrome c derepression and the effects of these two antibiotics on these kinetics were also studied. Cells were derepressed by transfer from glucose- to faffinose-containing media, and the rate of cytochrome c synthesis increased from the repressed to the derepressed level during the second hour of derepression. Chloramphenicol delayed this derepression, but after 5 h the rate of cytochrome c protein synthesis increased to twice the rate of synthesis in uninhibited cells. On the other hand, oligomycin inhibited derepression of cytochrome c. These results are discussed with respect to the effects of mitochondrial function in the derepressed and repressed states and during the processes of repression and derepression of cytochrome c.     

Effect of pH on Adenine and Amino Acid Uptake During Sporulation in Saccharomyces cerevisiae

During the early stages of sporulation in Saccharomyces cerevisiae, the pH of the acetate sporulation medium rises to values of 8.0 or higher. Associated with this rise in pH is a reduced cell permeability to certain precursors of ribonucleic acid (RNA), deoxyribonucleic acid or protein. Uptake of adenine, alanine, and leucine was optimal at pH 5.6 to 6.0, but sporulation was inhibited when the sporulation medium was buffered below pH 7.0. Cellular impermeability can be largely overcome by adjusting the acetate sporulation medium to pH 6.0 for optimal uptake of ¹⁴ C-adenine during short pulses without any apparent effect on sporulation. Sporulating cells pulse-labeled 20 min at pH 6.0 incorporated 40 times more ¹⁴C-adenine into RNA than sporulating cells pulse-labeled at pH 8.0. This increased incorporation can be attributed to a 100-fold increase in labeled adenosine triphosphate in cells pulse-labeled at pH 6.0 where maximum uptake occurs.     

Microbody-marker Enzymes during Transition from Phototrophic to Organotrophic Growth in Euglena

Transfer of Euglena gracilis Klebs Z cells from phototrophic to organotrophic growth on acetate results in derepression of the key enzymes of the glyoxylate cycle, malate synthase and isocitrate lyase, which appear coordinately regulated. The derepression of malate synthase and isocitrate lyase was accompanied by increased specific activities of succinate dehydrogenase, fumarase, and malate dehydrogenase, but hydroxypyruvate reductase activity was unaltered.     

Metabolic rates during sporulation of Saccharomyces cerevisiae on acetate

We have quantified yeast carbon and oxygen consumption fluxes and estimated anabolic fluxes through glyoxylate and gluconeogenic pathways under various conditions of sporulation on acetate. The percentage of sporulation reached a maximum of 55% to 60% after 48 h in sporulation medium, for cells harvested from logarithmic growth in acetate minimal medium. When cells were harvested in the stationary phase of growth before transfer to sporulation medium, the maximum percentage of sporulation decreased to 40% along with the occurrence of meiosis as could be judged by counting of bi- and tetra-nucleated cells. In both experiments, the rates of acetate and oxygen consumption decreased as a function of time when exposed to sporulation medium. Apparently, the decrease of metabolic rates was not due to alkalinization. By systematically varying the cell concentration in sporulation medium from 1.4×10⁷ to 20×10⁷ cell ml^-1, the percentage of sporulating cells was found to decrease in parallel with the rate of acetate consumption. When the sporulation efficiency attained under the different experimental conditions was plotted as a function of the rate of acetate consumption, a linear correlation was found. Anabolic fluxes estimation revealed a decrease of the rate through gluconeogenic and glyoxylate pathways occurring during sporulation progression. The pattern of metabolic fluxes progressively evolved toward a predominance of more oxidative catabolic fluxes than those exhibited under growth conditions. The results obtained are discussed in terms of a characteristic pattern of metabolic fluxes and energetics, associated to the development of yeast sporulation.Abbreviations DAPI 4,6-diamidino-2-phenylindole - dw dry weight - OD₅₄₀ optical density at 540 nm - SEM standard error of the mean - RQ respiratory quotient     

Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. In Streptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are bound in vitro and in vivo by topoisomerase I, TopA, which is the only topoisomerase I homolog found in S. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion of parB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes.     

Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ~20 μm h⁻¹, which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.     

Inhibition of sporulation in the water mold Blastocladiella emersonii by antipain

Blastocladiella emersonii express two different types of caseinolytic activities during the process of sporulation. They can be distinguished in vitro on the basis of their sensitivity to antipain. The alkaline protease activity is inhibited by antipain and PMSF, whereas the second enzyme, denoted here as the caseinolytic activity, is not inhibited by antipain but is sensitive to PMSF and concanavalin A. In vivo, antipain blocks sporulation when added to cultures during the first 60 min of sporulation, but if added 90 min after sporulation is induced, it is biologically ineffective. In both cases, antipain enters the cells and decreases the rate of total protein degradation by 60%. The antisporulation effect of antipain cannot be reversed by washing the cells. The ability of cells which have been pretreated with antipain to sporulate can be recovered, but only after a period of growth. These data provide evidence for the critical role of the alkaline protease for a limited period of time during the initial phases of sporulation in Blastocladiella. A hypothesis based on the processing of preformed proteins by the alkaline protease as a key control mechanism for sporulation is presented.     

Physiology of Sporeforming Bacteria Associated with Insects: Metabolism of Bacillus popilliae Grown in Third-Instar Popillia japonica Newman Larvae

The timing and relative participation of concurrent pathways of carbohydrate metabolism as well as the extent of terminal respiratory activity were determined by radiorespirometry with 14-C substrates and by enzyme assays for vegetative and sporulating cells of the bacterium Bacillus popilliae cultured in whole, intact Popillia japonica (Japanese beetle) larvae. During vegetative proliferation, the pentose phosphate pathway predominates in the bacterial cells with minor involvement of the Embden-Meyerhof-Parnas pathway. As the cells proceed through sporulation, pentose phosphate and Embden-Meyerhof-Parnas activity remains constant. No tricarboxylic cycle activity is evident during growth and sporulation of B. popilliae. The results demonstrate (i) predominantly aerobic metabolism for carbohydrate assimilation within in vivo sporulating cells, (ii) a major contrast to the metabolism of other aerobic sporeforming bacteria that exhibit derepression of tricarboxylic acid cycle enzymatic activity at the onset of sporulation, and (iii) no causal necessity of the cycle to B. popilliae sporogeny.     

Einfluß des Phasenstatus der Vorkulturzellen auf die Ascosporenbildung von Saccharomyces cerevisiae

Summary Cells from three growth phases were examined for their ability to sporulate: cells from a) phase II (first phase of exponential growth with glucose as carbon source), b) phase III (second lag-phase during adaptation to oxidative metabolism), and c) phase IV (second phase of almost exponential growth with ethanol as carbon source). 1. Cells from phase III showed the best sporulation ability because they reached the highest percentage of asci and also of 4-spored asci. 2. Cells of phase II exhibited the highest and those of phase IV the lowest rate of sporulation (Fig. 3). 3. The longer the cells remained in the presporulation medium the more abbreviated was the time in the sporulation culture before the first asci appeared, and this abbreviation was just equal to the time of elongation in the preculture. This clearly demonstrates the different degree of respiratory adaptation. — After transfer to the sporulation medium O₂-consumption arose to a steep maximum within the first 10 hours followed by medium values which dropped again rapidly at the onset of ascospore formation (Fig. 4). Only during the time of high and medium O₂-consumption there was an increase in dry weight reflecting the assimilation of acetate. In cells of phase II compared with those of phase IV this assimilation of acetate showed the same delay as the onset of sporulation, whereas full capacity of respiration was reached much sooner.     

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.     

Methylglyoxal-mediated Gpd1 activation restores the mitochondrial defects in a yeast model of mitochondrial DNA depletion syndrome

Human MPV17, an evolutionarily conserved mitochondrial inner-membrane channel protein, accounts for the tissue-specific mitochondrial DNA depletion syndrome. However, the precise molecular function of the MPV17 protein is still elusive. Previous studies showed that the mitochondrial morphology and cristae organization are severely disrupted in the MPV17 knockout cells from yeast, zebrafish, and mammalian tissues. As mitochondrial cristae morphology is strictly regulated by the membrane phospholipids composition, we measured mitochondrial membrane phospholipids (PLs) levels in yeast Saccharomyces cerevisiae MPV17 ortholog, SYM1 (Stress-inducible Yeast MPV17) deleted cells. We found that Sym1 knockout decreases the mitochondrial membrane PL, phosphatidyl ethanolamine (PE), and inhibits respiratory growth at 37 ?C on rich media. Both the oxygen consumption rate and the steady state expressions of mitochondrial complex II and super-complexes are compromised. Apart from mitochondrial PE defect a significant depletion of mitochondrial phosphatidyl-choline (PC) was noticed in the sym1? cells grown on synthetic media at both 30 ?C and 37 ?C temperatures. Surprisingly, exogenous supplementation of methylglyoxal (MG), an intrinsic side product of glycolysis, rescues the respiratory growth of Sym1 deficient yeast cells. Using a combination of molecular biology and lipid biochemistry, we uncovered that MG simultaneously restores both the mitochondrial PE/PC levels and the respiration by enhancing cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase 1 (Gpd1) enzymatic activity. Further, MG is incapable to restore respiratory growth of the sym1?gpd1? double knockout cells. Thus, our work provides Gpd1 activation as a novel strategy for combating Sym1 deficiency and PC/PE defects.