Induction of multispored asci in two-spored strains of Saccharomyces cerevisiae by amitrole.

Although growth of two yeast strains characterized by consistent production of two diploid spores per ascus was inhibited in complex presporulation media containing amitrole, a fraction of the cells produced were able to form asci with more than two spores after transfer to acetate sporulation medium. Cells grown in a defined presporulation medium containing amitrole did not acquire this ability. The increase in spore numbers per ascus is attributed either to the induction by amitrole in growth medium of cells with more than one nucleus or to the restoration of normal meioses in the multispored asci.     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

Variation in yeast mitochondrial activity associated with asci

An increase in mitochondrial membrane potential (DeltaPsim) and mitochondrially produced 3-hydroxy (3-OH) oxylipins was experienced in asci of the nonfermentative yeasts Galactomyces reessii and Lipomyces starkeyi and the fermentative yeasts Pichia farinosa and Schizosaccharomyces octosporus. Strikingly, asci of Zygosaccharomyces bailii showed no increase in mitochondrial activity (DeltaPsim and oxylipin production). As expected, oxygen deprivation only inhibited ascus formation in those yeasts with increased ascus mitochondrial activity. We conclude that ascus formation in yeasts is not always dependent on mitochondrial activity. In this case, fermentation may provide enough energy for ascus formation in Z. bailii.     

Regulation of spindle pole function by an intermediary metabolite

Spore formation in the yeast Saccharomyces cerevisiae depends on a modification of spindle pole bodies (SPBs) at the onset of meiosis II that allows them to promote de novo membrane formation. Depletion of the environmental carbon source during sporulation results in modification of only one SPB from each meiosis II spindle and formation of a two-spored ascus, called a nonsister dyad (NSD). We have found that mutants impaired in the glyoxylate pathway, which is required for the conversion of acetate to glucose, make NSDs when acetate is the primary carbon source. Wild-type cells make NSDs when the carbon source is glycerol, which is converted to glucose independently of the glyoxylate pathway. During NSD formation in glycerol, only the two SPBs created at the meiosis I/II transition ("daughters") are modified. In these conditions, the SPB components Mpc70p and Spo74p are not recruited to mother SPBs. Moreover, cooverexpression of Mpc70p and Spo74p suppresses NSD formation in glycerol. Our findings indicate that flux through the glyoxylate pathway during sporulation regulates modification of mother SPBs via recruitment of Mpc70p and Spo74p. These results define a cellular response in which the accumulation of an intermediary metabolite serves as a measure of biosynthetic capacity to regulate the number of daughter cells formed.     

Preferential Occurrence of Nonsister Spores in Two-Spored Asci of SACCHAROMYCES CEREVISIAE: Evidence for Regulation of Spore-Wall Formation by the Spindle Pole Body

Yeast cells subjected to a reversible thermal arrest of meiosis yielded progressively fewer spores per ascus as the arrest was extended. Dissection of two-spored asci by a newly developed method that prevents selection of false asci revealed that the spores were not a random sample of the haploid meiotic products. Most, if not all, pairs of spores contain nonsister products of the reductional division. Electron microscopic examination of the meiotic cells revealed the cytological basis for this bias. All four spindle pole bodies (SPBs) present at the second meiotic division normally gain a structural modification (the outer plaque) upon which the initiation of the prospore wall occurs. In the formation of a two-spored ascus, only one spindle pole body on each meiosis II spindle was so modified. These observations suggest that the morphogenesis of spores is regulated at meiosis II by limiting the number of SPBs gaining the outer plaque. The enhancement of spore yield upon addition of fresh medium suggests that this morphogenetic regulation responds more directly to nutrient deprivation arising during the thermal arrest, rather than to elevated temperature per se.     

Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.     

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.     

Incubation of excised apothecia enhances ascus maturation of Sclerotinia sclerotiorum

Synchronized maturation of ascospores of Sclerotinia sclerotiorum is desirable for establishing a transformation system, conducting genetic analyses of the pathogen, defining the precise epidemiological roles of ascospores and screening plant germplasm for resistance. In general, fresh apothecia collected from germinated sclerotia contained primarily immature or discharged asci. This study was undertaken to investigate whether maturation of asci and ascospores could be enhanced by incubation of excised apothecia and to determine the effects of factors such as temperature, excision time, light and ventilation on maturation of asci and ascospores in excised apothecia. Maturation of asci was compared between intact and excised apothecia that were incubated under similar conditions. Results demonstrated that temperature was an important factor affecting ascus maturation of S. sclerotiorum during incubation of excised apothecia, and the optimum temperature was around 21 C. After incubation at 21 C for 30 h, the percentage of undischarged mature asci in excised apothecia increased up to 70-80%. This increase was accompanied by a significant increase in ascospore production of up to 5 x 10(5) ascospores per apothecium. Detailed time course studies indicated that mature asci peaked at 30-36 h of postexcision incubation. Mature asci and the number of ascospores were higher in open incubation than in closed incubation, suggesting that accumulation of volatile substances was not required for ascus/ascospore maturation during postexcision incubation and ventilation could enhance the maturation process. Light also did not affect the maturation of asci during the incubation of excised apothecia. Germination rates for ascospores from excised apothecia under various treatments were similar to those from untreated apothecia but declined slightly with time postexcision. The incubation of excised apothecia promoted ascus maturation compared with intact apothecia.     

Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts.

To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

The release of elongated, sheathed ascospores from bottle-shaped asci in Dipodascus geniculatus

Yeasts use different mechanisms to release ascospores of different lengths from bottle-shaped asci. Round to oval-shaped ascospores are enveloped in oxylipin-coated compressible sheaths, enabling ascospores to slide past each other when they reach the narrowing ascus neck. However, more elongated ascospores do not contain sheaths, but are linked by means of oxylipin-coated interlocked hooked ridges on the surfaces of neighboring ascospores, thereby keeping them aligned while they are pushed towards the ascus tip by turgor pressure. In this study, we found elongated, oxylipin-coated sheathed ascospores in Dipodascus geniculatus that are released effectively from bottle-shaped asci without alignment. This is possible because the ascus neck and opening have a diameter that is the same as the length of the ascospore, thus allowing the ascospores to turn sideways without blocking the ascus when they are released. We found that increased concentrations of acetylsalicylic acid inhibit both ascospore release and 3-hydroxy oxylipin production in this yeast, thereby implicating this oxylipin in sexual reproduction.     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed "naked" nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei from meiosis. In 2--4 spored asci usually four products of meiosis in form of enclosed and free nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.     

Morphology,development and cytology of Taphrina maculans Butler

Morphology, development and nuclear behavior of the ascogenous stroma and asci in the infection spots have been described inTaphrina maculans Butler. The fungus forms subcuticular and intercellular mycelium in the leaf tissues and the ascogenous layers originate through division of the subcuticular hyphal cells in the infection sites. Germination of ascogenous cells starts with their elongation in the uppermost layer forming asci and ascospores without formation of stalk cells. Meiosis of the fusion (diploid) nucleus occurs in the young ascus as in otherTaphrina species devoid of stalk cells. The haploid chromosome complement in this species consists of 3 chromosomes (n=3). All the cells in the stromatic layer are potential ascogenous cells and ascus formation continues, until all of them are exhausted in the infection spot. Eight ascospores are normally formed in each ascus, but multi-plication of ascospores may occurin situ later. Three morphologically distinct types of ascus opening are encountered, which are apparently not correlated with prevalent environment. Multiplication of ascospores after their discharge from mature asci occurs by budding proceded by a mitotic division of the spore nucleus. Blastospores (budded cells) germinate into short hyphae and binucleate condition of cells originates by mitotic division of the nucleus. Occurrence of giant cells containing 2 nuclei is often observed. Possible origin of Uredinales fromTaphrina-like ancestors has been indicated due to their close resemblance.     

Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,growth state and acetate metabolism

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes.Abbreviations IL Isocitrate lyase [threo-D_s-isocitrate glyoxylate-lase, E.C. 4.1.3.1.] - MS malate synthase [l-malate glyoxylate-lyase (CoA-acetylating), E.C. 4.1.3.2.]     

Preferential Ascus Discharge during Cross Maturation in SORDARIA BREVICOLLIS

Crosses involving spore color mutants of Sordaria brevicollis all showed a decline in the frequency of second division asymmetric asci (2:2:2:2's) as the cross matured. This decline was due to the preferential maturation and/or discharge of these asci. The proportion of spindle overlap and recombinational asci within the group did not change as shown by ascus dissection. The preferential discharge was also found to occur in two-point crosses where the asci did not contain wild-type spores.     

Studies of sulfate utilization by algae

Summary Several aspects of amino acid metabolism were studied in the fruiting myxobacterium Myxococcus xanthus. Alanine and aspartate aminotransferases were detected at significant levels in vegetative cells and myxospores. In contrast, glutamate dehydrogenase, alanine dehydrogenase and aspartase were not detectable in the same preparations, which is consistent with the fact that inorganic nitrogen is not required for growth. The data presented suggest that the aminotransferases demonstrated provide for the synthesis of nonessential amino acids and concomitantly, oxidizable substrates.Isocitrate lyase activity was found in glycerol induced myxospores, but not in vegetative cells grown on two per cent Casitone medium. The emergence of isocitrate lyase in myxospores would indicate a metabolic shift toward the biosynthesis of compounds not required during vegetative growth. However, the presence of isocitrate lyase activity in vegetative cells grown in defined medium suggests that the amino acids present in the growth medium contribute to the formation of pyruvate and acetate and that glyoxylate enzymes are subject to repression when cells are grown on Casitone medium. Also, that expression of glyoxylate enzymes is not specific to myxospore formation.Based on a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree in Microbiology, August, 1968.     

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora Crassa

In previous work, the asd-1 (ascus development) gene of the filamentous fungus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-1 gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-1 protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-1 crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-1 asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-1 protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

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.     

FACTORS CONTROLLING THE SPORULATION OF YEASTS. I. THE PRESPORULATION PHASE

SUMMARY: Yeasts tend to dissociate into mixtures of cell types with different powers of sporulation; hence single cell isolates are recommended for sporulation studies. The ability of yeasts to produce 4-spored asci can be improved by single cell selection. Cells from actively fermenting cultures sporulate much better than those grown under aerobic conditions. Sporulating ability depends on fermentation 'age', reaching a maximum when 85–90% of the CO₂ has been evolved. Carbon dioxide assimilation in the presporulation phase appears essential for maximal sporulation, but complete anaerobiosis in this phase is detrimental to sporulating ability. Malt wort cultures of a baker's yeast have given remarkably constant figures, in successive tests, for sporulation; but some batches of wort have an adverse effect on sporulating ability. The same yeast, grown on Lodder-Rij's synthetic medium containing 4 or 8% (w/v) of glucose, is capable of 80% sporulation (proportion of cells forming asci) on sodium acetate agar, comparable to that obtainable with malt wort cultures. Sporulation is depressed by excess storage of fat, while storage of glycogen does not affect sporulating ability.     

Metschnikowia kunwiensis comb. nov., the teleomorph of Candida kunwiensis

The teleomorph of Candida kunwiensis Hong, Bae, Herzberg, Titze, Lachance, Metschnikowia kunwiensis, is described. Repeated attempts to obtain ascospore formation succeeded using modified V8 sporulation media and extended incubation times. The asci are ovoid, with only a small protrusion caused by the spore(s). The species is diplontic, possibly homothallic, with one or two ascospores per ascus. Aside from having atypical ovoid asci, the acicular shape of the spores is characteristic of the genus Metschnikowia. The type strain is CBS 9676(T).     

Effect of yeast extract,peptone, and certain nitrogen compounds on sporulation ofSaccharomyces cerevisiae

Summary Aeration of cells for 24 hrs. previous to placing them in 0.1% sodium acetate solution diminished sporulation, but this decrease was overcome by the addition of 0.1% yeast extract to the acetate solution. Cells starved by growth on Czapek solution agar +0.03% peptone formed very few ascospores in acetate solution. The addition of yeast extract or peptone in low content to the acetate solution increased the yields. However, the cells did not form as many ascospores as well-nourished cells in acetate solution.A comparison was made of the sporulation of cells from basal presporulation medium containing, separately, 18 nitrogen sources. In general, nitrogen sources that supported growth gave cells that sporulated well. Tyrosine and tryptophan were exceptions.Cells multiplied in basal medium with the nitrogen source deleted formed no asci in 0.1% acetate solution. When nitrogen sources were added to the acetate solution, many stimulated sporulation. Yields of asci in these sporulation cultures were, however, lower than the yield obtained from well-nourished cells in 0.1% acetate solution.Based on a thesis submitted byJ. H. Tremaine in May, 1953, to McMaster University in partial fulfilment of the requirements for the degree of Master of Science.