SPORULATION OF THE YEAST, HANSENULA SATURNUS: I. EFFECT OF MAGNESIUM AND CALCIUM IONS

Sporulation in a strain of the wild yeast, Hansenula saturnus,was investigated. The yeast was found to form spores even indistilled water. The sporulation rate (percentage of ascus-bearingindividuals) in this case was found to be markedly affectedby the cell concentration adopted in the test. The addition of inorganic nutrients to the sporulation mediumstimulates sporulation. The yeast requires either magnesiumor calcium for growth and sporulation. Higher concentrationsof these ions are required for sporulation than for growth.In both cases magnesium is effective at more dilute concentrationsthan calcium. Under the conditions of the experiments, in which the yeastforms a pellicle, the sporulation rate in the pellicle far exceedsthat in the sediment. The effects of environmental factors on the sporulation wasconsidered in relation to growth. It was found that, under theconditions of poor growth in the sporulation culture, no exogenousmagnesium and calcium are required for sporulation. In suchcases, the yeast cells are inferred to have an endogenous stockof magnesium and calcium enough for the sporulation. ¹ Present address: Laboratory of Microbiology, Department ofAgriculture, Tôhoku University, Sendai. (Received May 4, 1961; )     

Salt Secretion in Aeluropus litoralis (Willd.) Parl.

The effect of ion composition and concentration in the rootmedium on salt secretion by Aeluropus litoralis was investigated.The presence of a high ionic concentration in the medium stimulatedthe secretion process. The sodium concentration in the secretedfluid was found to be always higher than its concentration inthe medium. A positive correlation was found between the outersodium chloride concentration and the amount of sodium secretedand/or leaf contents. Sodium secretion exhibited a high efficiencyin excluding excess sodium from leaftissues. Sodium retentionin the leaves occurred in relatively low rates. The secretion mechanisms were found to be highly selective tosodium, opposing potassium and calcium. In contrast, potassiumand calcium were retained in the leaves to a greater degreethan sodium. Antagonistic relationships between sodium and potassiumand sodium and calcium were observed in secretion. The secreted fluid contains also various organic substances.Several interpretations to the results in connection with theproposed hypotheses to the mechanism of salt secretion werediscussed.     

Influence of Calcium on Formation and Reduction of Protochlorophyllide in the Pigment Mutant C-2A' of Scenedesmus obliquus

The levels of protochlorophyllide and protochlorophyll of pigmentmutant C-2A' of Scenedesmus obliquus grown in darkness dependupon the calcium concentration in the growth medium. In thepresence of calcium both the protochlorophyllide and protochlorophylllevels decrease upon irradiation whereas the amount of photoreducedchlorophyllide increases. In contrast to light-dependent protochlorophyllide reduction,the activity of light-independent protochlorophyllide reductionis higher in calcium free cultures compared to those grown inthe presence of calcium. It is discussed whether calcium actsdirectly on the activity of the protochlorophyllide oxidoreductaseor stabilizes the newly formed chlorophyllide. (Received September 1, 1989; Accepted February 19, 1990)     

Sporulation of Spheroplasts in Saccharomyces cerevisia

Spheroplasts of Saccharomyces cerevisiae sporulated in 0.1 Mpotassium acetate solution which contained 0.8 M sorbitol ormannitol as the osmotic stabilizer. The appearance of matureasci in both spheroplasts and intact cells was retarded by theaddition of the osmotic stabilizer. Sporulation was repressedmarkedly when 0.6 M KCl was used as the osmotic stabilizer inthe sporulation medium. The germination rate of the spores formedin spheroplasts was 97%. Tetrad analysis showed that meiosiswas normal during the sporulation of spheroplasts. (Received September 5, 1980; Accepted November 29, 1980)     

Calcium and malate are sporulation-promoting factors of Physarum polycephalum

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime mold Physarum polycephalum. The most important requirement for sporulation of Physarum is a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25 degrees C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state of Physarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.     

The production of extracellular thermostable neutral proteinase and α-amylase byBacillus stearothermophilus

Summary Extracellular thermostable neutral proteinase was produced byBacillus stearothermophilus strains NCIB 8924 and NRRL B-3880 growing at 55°C. The formation and stabilization of this proteinase was found to be dependent on the concentration of free calcium ions. Therefore, procedures that removed free calcium ions from the medium, such as the use of phosphate buffer, resulted in a lower production of proteinase. The calcium-deficient proteinase was denaturated or adsorbed by calcium phosphate compounds. During the sterilization procedure of the culture medium, the CaCO₃ precipitation, caused by the removal of CO₂, influenced the amount of proteinase produced in a phosphate buffered medium made with tap water. An improved medium without phosphate buffer was used for 10 and 300 l batch cultivations and the calcium requirement for proteinase formation by the two strains was determined.     

Effect of actinomycin D on viability,sporulation and nucleotide pool ofBacillus megaterium

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50 % drop of the concentration of GTP inBacillus megaterium cells immediately after their transfer to the sporulation medium were observed. Actinomycin D, in concentrations inhibiting RNA synthesis by 95%, blocked the rise of the (p)ppGpp pool and caused an instant several-fold increase of the GTP level. When the cells were exposed to actinomycin D in the sporulation medium for a 1-h period (time 0–1 h, 1–2 h or 2.20–3.20-h), they were able to form colonies on nutrient agar after being kept, in addition for 1–2 h in the sporulation medium free of the antibiotic. The ability of sporulation was, however, markedly limited. The share of cells that could sporulate increased when the irreversible sporulation phase was reached.     

On the importance of calcium and magnesium ions in yeast sporulation

Irrespective of the nutritional conditions, the sporulation frequency of wild and industrially used yeasts on agar or agarose plates has been found to vary from one experiment to another. An analysis of agar- and agarose-extracts by ion-exchange column chromatography proved that the amount of calcium and/or magnesium ions contained in the agar was a factor in the fluctuation of sporulation frequency. Furthermore, these two cations enhanced the formation of four-spored asci. When calcium or magnesium ions were added to a nutrition-deprived medium solidified with agarose containing no detectable calcium and magnesium ions, wild and industrially used sake yeasts efficiently sporulated with a frequency of 10–40%. A strictly controlled sporulation condition suitable for the analysis of meiosis and sporulation of yeast cells was constructed by using calcium and/or magnesium ions and highly purified agarose.     

Regulation of Free Calcium Concentration in the Pitchers of the Carnivorous Plant Sarracenia purpurea: A Model for Calcium in the Higher Plant Apoplast?

The pitcher of the carnivorous plant Sarracenia purpurea L.contains an entrapped body of liquid within which its prey isdigested. Free calcium in the pitcher is derived from eitherthe pitcher walls or from prey falling into the pitcher; inthe absence of exogenous (prey-derived) calcium it will dependon the active and passive calcium regulatory properties of thepitcher walls and may to some extent therefore mimic calciumin the apoplast of plant cells. Using a calcium-specific electrode,the free calcium concentration of the pitchers of Sarraceniaplants was investigated and the effect of adding a variety ofconcentrations of calcium in water determined. The mean pitcherfree calcium concentration in vivo was 2.3 x 10^–5 M±2.5x 10^–5 M; when pitchers were washed and filled with watercontaining lower calcium concentrations, the concentration inthe pitcher water rose to 1–5 x 10^–5 M. When highercalcium concentrations (up to 1 x 10^–4 M) were added,the pitcher calcium concentration declined to 1–7 x 10^–5M. Concentrations of calcium above 1 x 10^–4 M were alsoreduced, but to a lesser extent. Metabolic inhibition of activeion transport, while inhibiting pitcher acidification, did notinhibit regulation of pitcher free calcium, suggested that itoccurs as a result of calcium exchange sites in the pitcherwalls. The data are discussed in relation to the physiologyof Sarracenia pitchers and to the usefulness of the pitcheras a model for free calcium in the higher plant apoplast. Sarracenia purpurea L., carnivorous plant, pitcher, free calcium, plant apoplast     

Effect of pH on the Membrane Potential and Electrical Resistance of Nitella flexilis in the Presence of Calcium

Effects of pH on the membrane potential and electrical resistanceof Nitella were investigated in a bathing medium with or withoutcalcium. The membrane potential became more negative as theexternal pH was raised, at a faster rate in the presence ofcalcium than in its absence. The value then achieved by thepotential could be reversed by restoring the original pH whilstin a Ca-free medium the cell remained ‘hyperpolarized’.Tenfold changes of the external concentration of potassium broughtabout larger modifications of the membrane potential when thepH of the solution was high and calcium concentration low. Theelectrical resistance was lowest in alkaline and calcium-freesolutions. We conclude that calcium prevents the mediation ofsome changes in the membrane structure by lowering the concentrationof external H⁺ ions, and that the permeability of Nitella topotassium increases with rising pH.     

Effect of external ionic environment on phototaxis of Volvox carteri

The sign of phototaxis in Volvox carteri is temperature-dependent;positive at room temperature and negative at low temperature.Modification of the tactic sign by external ions, pH and chemicalswas studied. The addition of 30 mM potassium ion to the mediumchanged the tactic sign from positive to negative, and a mediumwith a high pH elicited positive phototaxis. An increase inthe potassium or hydrogen ion concentration raised the reversaltemperature of the phototactic sign, and the addition of magnesiumor calcium ion also raised the reversal temperature of the signslightly. Valinomycin, a highly specific ionophore of potassium,raised the reversal temperature. CCCP, DCMU and DCCD, whichdepolarize biological membranes, also raised the reversal temperature.These results show that the sign of phototaxis is determinedby membrane polarization; on depolarization of the membranethe sign of phototaxis changes from positive to negative. Ethanol lowered the reversal temperature, and sodium azide inhibitedboth positive and negative phototaxis. The effects of ethanoland azide indicate that depolarization of the membrane is notthe only factor that induces change in the phototactic sign. (Received July 23, 1979; )     

The Sporulation of Penicillium notatum Westling in Submerged Liquid Culture: II. THE INITIAL SPORULATION PHASE

The initial sporulation time of Penicillium notatum Westlinggrown in shaken submerged culture inoculated with spores orvegetative mycelia is inversely proportional to the logarithmof the inoculum load, with a minimum time of 17.5 hours in theformer and 8 hours in the latter cultures. The development ofcultures is divisible into two stages. Firstly, there is a periodof vegetative growth, the duration of which depends on the inoculumload, after which the culture can be described as mature. Calciumis not required for the development of this maturity. Secondly,the cultures when mature develop phialides and spores if calciumis added to the medium. The development of phialides and thefirst spores takes 6 hours from the time of adding calcium andthis period is not influenced by the inoculum load of the culture.The medium from a mature culture promoted more rapid sporulationof vegetative mycelia placed in it than did a fresh medium,indicating the presence of a sporulation factor(s) in maturemedium. Similar activity was also demonstrated in media whichhad supported growth of any one of five other Penicillium speciesor Aspergillus niger. The nature of the sporulation factor isso far unknown.     

Effects of cholesterol and docosahexaenoic acid on cell viability and (Ca2+)i levels in acutely isolated mouse thymocytes

We investigated the effects of lipids on thymocyte function. The effects of application of cholesterol or docosahexaenoic acid (DHA), a C22, omega‐3 (n‐3) polyunsaturated fatty acid (PUFA), on viability and intracellular calcium concentrations of acutely isolated mouse thymocytes were investigated using flow cytometry. Cholesterol (100 µM) caused significant cell death after 30–60 min whether or not calcium was present in the medium. Cell death was associated with an elevation of intracellular calcium whether or not calcium was present in the extracellular medium. However, the elevation of calcium concentration was not responsible for the cell death since calcium levels in the presence of ionomycin rose higher without significant cell death. DHA had similar actions but was more potent, causing significant cell death and elevation of calcium concentration within 5 min at 1 µM. In the absence of extracellular calcium 1 µM DHA caused 100% cell death within 15 min. Linolenic acid, a C18 omega‐3 fatty acid also caused cytotoxicity at low concentrations whether or not albumin was present, but omega‐6 or saturated C22 fatty acids were much less effective. These observations demonstrate that thymocyte viability is very sensitive to acute exposure to low concentrations of omega‐3 fatty acids. Copyright © 2009 John Wiley & Sons, Ltd.     

Absorption of Copper by Lemna as Influenced by Some Factors Which Nullify the Copper Effect on Flowering and Growth

The effect of copper on flowering and growth of Lemna paucicostata6746 and Lemna gibba G3 in a copper-containing medium is nullifiedby the addition of EDTA, ammonium ions or salicylic acid tothe medium or a decrease in its nitrate concentration. Thesefactors were examined for their effects on the absorption ofcopper by the plants. The addition of EDTA to the medium completelyinhibited the absorption of copper in both species, thus eliminatingthe copper effect. Ammonium ions also inhibited copper absorption,their effectiveness rising with their concentration. Loweringthe nitrate concentration in the medium nullified the coppereffect on flowering in L. paucicostata 6746, and the additionof salicylic acid to the medium also nullified the copper effectin L. gibba G3, both without affecting the absorption of copper. (Received June 7, 1982; Accepted August 27, 1982)     

Uptake and Leakage of Some Ions in Relation to Potassium Uptake Rhythm in Lemna gibba G3

The time courses of the uptake of various inorganic ions fromthe flow medium were compared with the rhythmical potassiumuptake in Lemna gibba G3. The duckweeds in the flow medium absorbed20–40% of magnesium, 30–50% of phosphate, 40–80%potassium and 50–90% of nitrate, the percentage varyingrhythmically, but they constantly absorbed about 20% of calcium.The total anion uptake was 1.5 times greater than the totalcation uptake. The duckweeds absorbed cesium or rubidium aswell as they did potassium. They also absorbed sodium if potassiumwas absent from the medium. Lithium was not absorbed even inthe absence of potassium. Potassium leaked from the duckweed into the potassium-free flowmedium, and the rate of leakage changed diurnally under continuouslight, with maximum leakage occurring at the same circadiantime as when the potassium uptake rhythm in the normal flowmedium drops to the minimum point. The pattern of potassiumleakage in dim light was influenced by the species of monovalentcation in the medium. In darkness, however, the leakage rhythmran for 36 hr irrespective of cation species. (Received December 28, 1981; Accepted May 19, 1982)     

Inhibition of Zeatin-Induced Growth of Cucumber Cotyledons by Sulfite Ions

The inhibitory effects of sulfite ions on zeatin-induced cellexpansion in cotyledons excised from dark-grown seedlings ofcucumber (Cucumis sativus L.) were examined. With 50 µMzeatin the growth rate in white light was about twice that ofthe control. Addition of Na₂SO₃ in the growth medium inhibitedthe zeatin-induced growth of cotyledons. Zeatin-treatment increasedthe osmotic potential in cell sap of cotyledons, while sulfitedecreased it. These treatments had no significant effect onpotassium concentration. Sulfite inhibited the zeatin-inducedincrease in contents of fructose and glucose, but did not affectsucrose content. The relative contents of non-cellulosic constituentsof cell walls fell with the advance of culture. This decreasewas repressed by sulfite, indicating that inhibition of expansiongrowth in cucumber cotyledons by sulfite ions was the resultof alterations in the cell wall structure due to changes inthe cell wall metabolism. (Received June 12, 1984; Accepted October 24, 1984)     

Properties of Alkaline Phosphatase in Cucumber Roots Induced by Calcium Starvation

Calcium deficiency caused an increase in alkaline phosphataseactivity in cucumber roots [Matsumoto and Yamaya (1981) Plant& Cell Physiol. 22: 1137]. The activities of other hydrolasesincluding acid phosphatase, nucleases and proteases, however,were much less affected by the removal of calcium. Nucleosidedi- and triphosphates and inorganic pyrophosphate were effectivelyhydrolyzed by the induced alkaline phosphatase, whereas nucleosidemonophosphate-hydrolyzing activity was basically equal in theroots grown with either complete medium or a medium lackingcalcium. The alkaline phosphatase in cucumber roots was foundin fractions pelleting at 3,000 x g and in the 100,000 x g supernatant.The calcium-starved roots increased their alkaline phosphataseactivity in both fractions. Four isozyme bands of the alkalinephosphatase in the soluble fraction were separated by polyacrylamidegel electrophoresis. One of the isozyme bands showed a prominentincrease with the calcium deficiency, but not in the presenceof cycloheximide. (Received June 24, 1981; Accepted September 3, 1981)     

An Endogenous Rhythm of Spore Discharge in Sordaria fimicola

The sporulation process in Sordaria fimicola has been studiedunder controlled conditions, with particular reference to discharge.Discharge occurred with a circadian periodicity for the greaterpart of the sporulation life of the culture, with peaks at about18.00 hours in either light or darkness. The consistency ofthis phenomenon was confirmed by an analysis of previous work.Fluctuations in light intensity, temperature, relative humidity,carbon dioxide concentration, and vibration were eliminatedas far as possible, as causes of this periodicity. Minor fluctuationsin discharge rate with a period of less than 24 h were probablyrandom. The general nature of the periodicity was shown to be consistentwith some aspects of endogenous rhythms in other organisms.However, spore discharge in S. fimicola is very sensitive tolight, and it is readily entrained to discharge out of phasewith the natural period. The rate of development of two stages of spore formation (delimitationand maturation) were determined, and these were related to therate and periodicity of discharge. From the data it was impossibleto establish at which stage of sporulation the periodicity arose.     

Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells.

Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process. Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent. Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. Finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.