Induction of sporulation in food-spoilage yeasts

Four yeasts, Hansenula anomala, Kluyveromyces fragilis, Lodderomyces elongisporus and Saccharomyces cerevisiae, were cultured in two presporulation media at 30 ° C. Media consisted of yeast extract — peptone — acetate and yeast extract — peptone — dextrose broths. Except for K. fragilis, the test yeasts reached a high degree of sporulation when transferred to acetate- and ethanol-supplemented sporulation media. The percentage of S. cerevisiae cells forming asci was as high as 79% after 24 h incubation. H. anomala and L. elongisporus sporulated more rapidly in ethanol- compared to acetate-containing medium. Within test parameters, the concentration of acetate or ethanol, _pH, and incubation temperature (25 ° C and 30 ° C) did not substantially influence the extent of sporulation.     

EFFECT OF CARBON AND NITROGEN NUTRITION ON REPRODUCTION IN LEPTOSPHAERULINA BRIOSIANA

Reproduction of Leptosphaerulina briosiana was studied on modified Richard's medium supplemented with seven carbon compounds, six amino acids, five inorganic nitrogen compounds or with urea. The fungus grew on each of the media, but it did not reproduce on all. Ascostromata formed on each of the carbon sources, but the formation of asci and ascospores and the ejection of ascospores varied with the carbon source and the isolate of the fungus. Ascostromata formed on all the nitrogen sources except arginine and ammonium sulfate. Formation of asci and ascospores and ejection of the ascospores varied with the nitrogen source and the isolate of the fungus. The fungus grew and produced ascostromata on Richard's medium supplemented with each of six vitamins, but asci and ascospores were not formed.     

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.     

Sporulation in Hansenula wingei Is Induced by Nitrogen Starvation in Maltose-Containing Media

The sexually agglutinative yeast Hansenula wingei lives in association with bark beetles that inhabit coniferous trees. This yeast was induced to sporulate by malt extract, which contains a high percentage of maltose (50%) and a low percentage of nitrogen (0.5%). A solution of 1.5% maltose without any growth factors also induced ascosporogenesis in H. wingei. Thus, only a carbon source is required for sporulation as in Saccharomyces. However, potassium acetate did not induce sporulation in H. wingei as it does in S. cerevisiae. Instead, disaccharides (such as maltose, sucrose, or cellobiose) promote sporulation better than either monosaccharides (such as dextrose, fructose, or mannose) or respiratory substrates (such as ethanol or glycerol). The specificity of disaccharides in promoting sporulation in H. wingei may be considered an adaptation since these disaccharides are present in the natural environment of this yeast. In addition, the specificity of disaccharides may be related to the induction of the disaccharidase because cells precultured on dextrose sporulate well on maltose, but cells precultured on maltose sporulate poorly on maltose. When (NH₄)₂SO₄ was added at a low concentration (3 mM) to synthetic sporulation medium (1.5% maltose solution), sporulation was abolished, whereas other salts and nitrogen sources inhibited to a lesser extent and vitamins and trace elements had no effect. Oxygen was required for sporulation, as expected for an obligate aerobe. Maximal sporulation was achieved in 2% malt extract broth at high cell density (10⁹ cells per ml), pH 5, and 25°C. By using these optimal physiological conditions and hybrid strains selected from an extensive genetic breeding program, about 30% asci (10% tetrads) were obtained routinely. Thus, the genetics of cell recognition in this yeast can now be studied.     

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.     

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.     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: II. INFLUENCE OF CARBON-NITROGEN RATIO OF CULTURE MEDIUM

1. Several factors affecting sporulation of a wild yeast, Hansenulasaturnus, especially carbon sources and the carbon-nitrogenratio of sporulation medium were studied.
2. The sporulationis stimulated at a certain definite C/N ratioof glucose medium.
3. Several carbon sources such as ethanol, acetate, lactate,glycerol,succinate, glucose, gluconate and citrate are utilizedby theorganism both for growth and sporulation.
4. The numberof spores in an ascus depends on the C/N ratio ofthe medium.An increase in the ratio stimulates the yield of2-and 3-sporedasci, especially of the former. One-spored ascibecome abundantas this ratio decreases.
5. Lysine promotes sporulation in anacetate medium, and its presencein a large amount in glucosemedium also stimulates sporulation,while a small amount isinhibitory. When lysine was employedas the sole nitrogen source,most of the asci were 1-spored.
6. It is discussed that sporulationof yeast is induced by a balanceof metabolism, rather thanby one definite "sporulation substrate".

¹ Present address: Laboratory of Microbiology, Department ofAgriculture, T{circumflex}hoku University, Sendai. (Received May 23, 1961; )     

Production of poly(l-lactide)-degrading enzyme by Amycolatopsis orientalis for biological recycling of poly(l-lactide)

Efficient production of poly(l-lactide)(PLA)-degrading enzyme was achieved by addition of 0.1% (w/v) silk fibroin powder into a liquid culture medium of an actinomycete, Amycolatopsis orientalis, without other complex nitrogen sources, such as yeast extract and peptone. Scaled-up production of the enzyme in a 5-l jar fermenter showed the possibility of producing this enzyme on an industrial scale at low production cost. The extracellular PLA-degrading enzyme showed potent degrading activity, which is effective for biological recycling of PLA, i.e., 2,000 mg/l of PLA powder was completely degraded within 8 h at 40°C using 20 mg/l purified enzyme. An optically active l-lactic acid with 600 mg/l was obtained as degradation product of PLA without undesirable racemization.     

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus have been studied. When concentrations of the carbon source in the medium were lower than 100 g/l, it was found to be favorable to the mating of vegetative cells, both haploid and diploid. The addition of nitrogen and sulfur sources to the medium influenced the life phases of haploid cells and partially stabilized the vegetative growth of diploid cells. Enrichment of the nutrient medium with potassium, vitamins, and microelements was shown to be necessary for the formation and maturation of conjugated ascospores. Microelements, vitamins, and phosphorus in excessive amounts activated conjugation but did not provide for the distinct phases of formation of unconjugated asci and spores in the diploid cells. Possible reasons for the unstable diplophase in the yeast P. tannophilus are discussed.     

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.     

Nitrite as an Energy-Conserving Electron Sink for the Acetogenic Bacterium <Emphasis Type="Italic">Moorella thermoacetica</Emphasis>

Nitrite served as an energy-conserving electron acceptor for the acetogenic bacterium Moorella thermoacetica. Growth occurred in an undefined (0.1% yeast extract) medium containing 20 mM glyoxylate and 5 mM nitrite and was essentially equivalent to that observed in the absence of nitrite. In the presence of nitrite, acetate (the normal product of glyoxylate-derived acetogenesis) was not detected during growth. Instead, growth was coupled to nitrite dissimilation to ammonium, and acetogenesis was limited to the stationary phase. Furthermore, membranes from glyoxylate-grown cells under nitrite-dissimilating conditions were deficient in the b-type cytochrome that is typically found in the membranes of acetogenic cells. Unlike glyoxylate, other acetogenic substrates (fructose, oxalate, glycolate, vanillin, and hydrogen) were not growth supportive in the undefined medium containing nitrite, and glyoxylate-dependent growth did not occur in a nitrite-supplemented, basal (without yeast extract) medium. Glyoxylate-dependent growth by Moorella thermoautotrophica was not observed in the undefined medium containing nitrite. Received: 1 April 2002 / Accepted: 9 July 2002     

A novel role of Dma1 in regulating forespore membrane assembly and sporulation in fission yeast

In fission yeast Schizosaccharomyces pombe, a diploid mother cell differentiates into an ascus containing four haploid ascospores following meiotic nuclear divisions, through a process called sporulation. Several meiosis-specific proteins of fission yeast have been identified to play essential roles in meiotic progression and sporulation. We report here an unexpected function of mitotic spindle checkpoint protein Dma1 in proper spore formation. Consistent with its function in sporulation, expression of dma1(+) is up-regulated during meiosis I and II. We showed that Dma1 localizes to the SPB during meiosis and the maintenance of this localization at meiosis II depends on septation initiation network (SIN) scaffold proteins Sid4 and Cdc11. Cells lacking Dma1 display defects associated with sporulation but not nuclear division, leading frequently to formation of asci with fewer spores. Our genetic analyses support the notion that Dma1 functions in parallel with the meiosis-specific Sid2-related protein kinase Slk1/Mug27 and the SIN signaling during sporulation, possibly through regulating proper forespore membrane assembly. Our studies therefore revealed a novel function of Dma1 in regulating sporulation in fission yeast.     

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)     

Extractability of polyhydroxyalkanoate synthesized by <Emphasis Type="Italic">Bacillus flexus</Emphasis> cultivated in organic and inorganic nutrient media

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86–100% compared to those grown in yeast extract or peptone (32–56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.     

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.     

Production of an extracellular maltase by thermophilic Bacillus sp. KP 1035.

Production of extracellular maltase was studied with thermophilic Bacillus sp. KP 1035, which was selected as the organism producing the highest levels of maltase. The final enzyme yield was increased by maltose, peptone, and yeast extract but reduced by succinate and fumarate. Maximum enzyme production was achieved at 55 degrees C and at an initial pH of 6.2 to 7.0 on a medium containing 0.3% maltose, 1% peptone, 0.1% meat extract, 0.3% yeast extract, 0.3% KH2PO4, and 0.1% KH2PO4. Maltase was synthesized in cytoplasm and accumulated as a large pool during the logarithmic growth phase, which preceded sporulation. At the end of this phase, the enzyme appeared in the culture broth, and its accumulation increased in parallel with a rise in the extracellular protein level. Maltase was stable for 24 h at 60 degrees C over a pH range of 5.6 to 9.0 and retained 95% of the original activity after treatment for 20 min at 70 degrees C at pH 6.8.     

Production of an extracellular maltase by thermophilic Bacillus sp. KP 1035.

Production of extracellular maltase was studied with thermophilic Bacillus sp. KP 1035, which was selected as the organism producing the highest levels of maltase. The final enzyme yield was increased by maltose, peptone, and yeast extract but reduced by succinate and fumarate. Maximum enzyme production was achieved at 55 degrees C and at an initial pH of 6.2 to 7.0 on a medium containing 0.3% maltose, 1% peptone, 0.1% meat extract, 0.3% yeast extract, 0.3% KH2PO4, and 0.1% KH2PO4. Maltase was synthesized in cytoplasm and accumulated as a large pool during the logarithmic growth phase, which preceded sporulation. At the end of this phase, the enzyme appeared in the culture broth, and its accumulation increased in parallel with a rise in the extracellular protein level. Maltase was stable for 24 h at 60 degrees C over a pH range of 5.6 to 9.0 and retained 95% of the original activity after treatment for 20 min at 70 degrees C at pH 6.8.     

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.]     

Sporulation of Streptomyces venezuelae in submerged cultures

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.     

Regulation of differentiation of the dimorphic fungusPaecilomyces viridis by nitrogen sources,antibiotics and metabolic inhibitors

The effect of nitrogen and carbon sources, vitamins, antibiotics and metabolic inhibitors on growth and differentiation ofPaecilomyces viridis was investigated. Sodium nitrate,l-asparagine,l-proline and peptone were found to be suitable nitrogen sources for mycelial growth (M) in a synthetic medium with glucose.Paecilomyces viridis could also grow slowly in a synthetic medium containing benzylpenicillin or bacitracin as the only nitrogen sources and very slowly even in a medium with polymyxin as the nitrogen source. Ammonium salts, area,l-arginine,d, l-aspartic acid andl,-serine were found to support intensive sporulation. Partially yeast-like growth (Y) was facilitated by NaNO₂, (NH₄)₂SO₄, NH₄NO₃, urea,d, l-alanine,l-arginine,d, l-aspartic acid,l-cysteine,l-glutamic acid andl-serine. Partially yeastlike growth could be observed in a medium with peptone and at an initial pH of 2. The following compounds appear as suitable carbon sources for mycelial growth:d-glucose,d-galactose,d-mannose, maltose, sucrose, chitin andd-mannitol. No changes in morphology could be detected on any of the 25 used carbon sources in a synthetic medium with NaNO₃. Yeast-like growth was induced by the antibiotics azalomycin F, cyanein (brefeldin A), griseofulvin and monorden (radicicol). After removal of the antibiotics, mycelial growth was restored. Sporulation was stimulated by chloramphenicol, 2-deoxy-d-glucose, furancarboxylic acid and stipitatic acid. Deformation of phialides was observed after treatment with actinomycin D, amphotericin B, boromycin, citrinin, cycloheximide, cytochalasin D, fungicidin and scopathricin. Microcyclic conidiation or growth of phialides directly from conidia were induced by cycloheximide, desertomycin, ethidium bromide and 5-fluorouracil.