Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

Regulation of trehalose metabolism by Streptomyces griseus spores.

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.     

Metabolism of endogenous trehalose by Streptomyces griseus spores and by spores or cells of other actinomycetes.

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Nongerminating spores used their trehalose reserves slowly when incubated in buffer for several months. In contrast, spores rapidly depleted their trehalose pools during the first hours of germination. Extracts of dormant spores contained a high specific activity of the enzyme trehalase. The level of trehalase remained relatively constant during germination or incubation in buffer. Nongerminating spores of Streptomyces viridochromogenes, Streptomyces antibioticus, and Micromonospora echinospora and nongrowing spherical cells of Arthrobacter crystallopoietes and Nocardia corallina also maintained large amounts of trehalose and active trehalase. These trehalose reserves were depleted during spore germination or outgrowth of spherical Arthrobacter and Nocardia cells into rods.     

Physiology of the cell surface of neurospora ascospores

Summary Acids like hydrogen fluoride, hydrazoic and fluoroacetic have been shown to prevent the germination of ascospores of N. tetrasperma when dormant spores are treated. On the other hand, propionate, cysteine and others are ineffective when used in this way. When activated ascospores were treated, much lower concentrations of the acids were sufficient to poison the spores. As in other systems, these substances are most effective at a p_H below their pK_a.The kinetics of uptake of fluoride by dormant ascospores were studied and shown to be very different from those reported for cations. However, P³² was not absorbed by dormant ascospores, even at p_H 1.5.Respiratory inhibition by azide and fluoroacetate occurred immediately after the spores were activated, but in the case of 5-nitro-2-furfuryl methyl ether no effect was observed until just before germination occurred.These results suggest that a permeability barrier exists in the dormant ascospore which disappears upon germination. Moreover, the dormant spore seems to be permeable to acids of small size but impermeable to those possessing more than 3 methylene groups or of equivalent size.This work was made possible by a grant from the Michigan-Memorial Phoenix Project of the University of Michigan to whom the authors would like to express their gratitude.     

RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum

Abstract RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. Ras Glevels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.     

Changes in the permeability of ascospores of Neurospora tetra-sperma during germination

The respiration and germination of activated ascospores of Neurospora tetrasperma have been shown to be almost completely inhibited by concentrations of ethylene diaminetetraacetic acid (EDTA) as low as 0.0035 M. In contrast, however, dormant ascospores are insensitive to this chelating agent. At any time up to about 150 minutes after activation Ca(++) or Mg(++) can completely reverse this toxicity but Cu(++), Co(++), and Mn(++) only partially reverse it. After this time, the minerals of the Neurospora "minimal" medium taken singly, or in various combinations cannot reverse this effect. Adding EDTA at 120 minutes after activation eliminates the lag period associated with its effect upon respiration. Inhibition occurs even though the cells seem to be impermeable to EDTA. Cationic exchange resins, as another example of a non-penetrating metal-binding agent, gave effects similar to those noted with EDTA. Of the resins used the H(+) form of IR-120 and the Na(+) and K(+) forms of amberlite IRC-50 were the most toxic to activated ascospores. On the other hand, dormant ascospores were entirely unaffected by the resins. The release of Ca(++) from activated ascospores coincided with the period of maximum sensitivity to EDTA. More than 60 per cent of the cell's content of K(+) is released by EDTA-inhibited ascospores. A low pH decreased the effectiveness of EDTA as a poison. The data are consistent with the possibility that non-penetrating metal-binding agents are toxic because of the irreversible removal of essential cations from the cell. The kinetic data for the inhibitory effects, and for the release of Ca(++) establish that the permeability of germinating ascospores to minerals changes drastically as a result of activation.     

High viscosity and anisotropy characterize the cytoplasm of fungal dormant stress-resistant spores

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia--the main airborne vehicles of distribution--are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h(0)/h(+1) = 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.     

Conversion of Intrasporal Trehalose into Reducing Sugars During Germination of Nosema algerae (Protista: Microspora) Spores: A Quantitative Study

ABSTRACT. Carbohydrates were extracted from dormant, stimulated and germinated spores of Nosema algerae . Concentrations of total sugars were measured by the Anthrone test. Non-reducing sugars were quantified by NaOH hydrolysis followed by the Anthrone reaction, and reducing sugars by the Nelson's test. Glucose was measured by the o -toluidine test and a glucose oxidase assay. The concentrations of trehalose in the cytoplasm of the dormant, ungerminated spore was estimated to be in excess of 1.0 M. Trehalose decreased by 70% during the five-minute course of germination. All of the lost trehalose was converted to reducing sugar of which 70–78% was glucose. The osmotic potential increase caused by catabolism of trehalose appears to be sufficient for germination.     

THE REVERSIBLE HEAT ACTIVATION INDUCING GERMINATION AND INCREASED RESPIRATION IN THE ASCOSPORES OF NEUROSPORA TETRASPERMA

The heat activation of Neurospora tetrasperma ascospores is a reversible process, since activated spores may be returned to secondary dormancy by preventing respiration, and these secondarily dormant spores may be induced to germinate by reheating. Activation of the spores brings about a large increase in respiration prior to the germination of the spores. As the spores are reversibly activated or deactivated the rate of respiration is increased or is decreased. By poisoning the cells with iodoacetamide it is possible to prevent all germination without greatly inhibiting this increase in respiration. Precisely with the beginning of germination a secondary rise in respiration occurs. The respiration of the spores is cyanide sensitive. The heat activation has a critical temperature at about 49 to 52°C.; and at a constant temperature within this range, the percentage of the spores activated as plotted against the time, follows an S-shaped population curve.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Some aspects of germination induction in Phycomyces blakesleeanus by an ammonium-acetate pretreatment

Summary Dormancy in the sporangiospores of Phycomyces blakesleeanus can be broken by a short pretreatment (10 min at 30° C) with NH₄-acetate. The effect is partly reversible.Acetate activation is accompanied by a transient rise in trehalase activity, which causes a sharp decrease in the reserve substance trehalose followed by an accumulation of glucose in the surrounding medium. At the same time pyruvate, acetaldehyde, ethyl alcohol and lactate can be detected in the culture medium.CO₂ production by respiration of externally supplied glucose is predominant in dormant and in germinating spores. During acetate treatment most of the CO₂ produced, is supplied by the turnover of endogenous material.High activity of the pentose-phosphate (P-P) pathway occurs in dormant spores, as measured by the C₆/C₁ ratio. Adding acetate results in a sudden rise in the glycolytic Krebs cycle (EMP) pathway. Afterwards, the P-P pathway also increases and it predominates again during the initial phases of germination.     

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207-217. 1963.-It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates.     

Biochemistry of spore germination in Phycomyces

Abstract The constitutionally dormant spores of Phycomyces blakesleeanus can be activated by heat shock or treatment with several monocarboxylic acids. Activation is followed first by a general stimulation of metabolism, e.g. respiration, protein-, RNA- and cell-wall synthesis, and subsequently by nuclear division and germ-tube emergence. Initial germination is not dependent on RNA synthesis and can even start without protein synthesis. The first common effect of different activating treatments is a transient rise in cyclic AMP (cAMP) content, caused by a change in phosphodiesterase activity after heat activation, and by unknown factors during activation by acids. cAMP transiently activates trehalase and glycerol-3-phosphatase in the spores. The activation of these enzymes causes a quick turnover of trehalose into glycerol. During the same period, the water status of the cells is altered so dramatically that perhaps this may explain at least part of the stimulation of metabolism in the germinating spore.     

Properties of Germinating Spores of Dictyostelium discoideum

The process of spore germination in Dictyostelium discoideum consists of three sequential stages: activation of dormant spores, swelling of activated spores, and emergence of myxamoebae from swollen spores. Dormant and activated spores are resistant to heating, freezing, or drying. Drying and freezing, moreover, may maintain the activated state until the spores are returned to normal conditions. Low temperature incubation after heat shock or the presence of an autoinhibitor will return activated spores to the dormant state. The entire spore germination process is aerobic, being inhibited at any point by oxygen deprivation or respiratory poisons. Each spore of this social organism appears to germinate at its own rate and independent of the other spores in the suspension.     

Trehalose metabolism in dormant and activated spores of Phycomyces blakesleeanus Burgeff

Evidence is obtained for the existence of two different localizations of trehalase (,-trehalose glucohydrolase, EC 3.2.1.28) in Phycomyces spores: one inside the cell, and one in the periplasmic region. The latter enzyme is sensitive to 0.1 mol l^-1 HCl treatment and its activity can be regulated by external pH changes. The periplasmic form of the enzyme is involved in the metabolism of added labelled trehalose. This sugar is hydrolyzed externally to glucose which is found mainly in the incubation medium and which is partly absorbed by the spores. During incubation trehalose leaks out from both dormant and activated spores and is subsequently hydrolyzed to glucose. The intracellular trehalase is probably involved in the breakdown of endogenous trehalose in spores. After heat activation the hydrolysis of endogenous trehalose is stimulated even without an important increase in activity of intracellular trehalase. Additional treatments which break dormancy of spores without a significant activation of trehalase are the following: heating of HCl-treated spores and treatment of spores with reducing substances (e.g. Na₂S₂O₄ and NaHSO₃).     

Spore pool glutamic acid as a metabolite in germination

Spore glutamic acid pools were examined in dormant and germinating spores using colorimetric and (14)C analytical procedures. Germination of spores of Bacillus megaterium (parent strain), initiated by d-glucose, was accompanied by a rapid drop in the level of spore pool glutamate, from 12.0 mug/mg of dry spores to 7.7 mug/mg of dry spores after 30 sec of germination. Similar decreases in extractable spore pool glutamate were observed with l-alanine-initiated germination of B. licheniformis spores. On the other hand, glutamate pools of mutant spores of B. megaterium, with a requirement of gamma-aminobutyric acid for spore germination, remained unchanged for 9 min of germination, at which time more than 50% of the spore population had germinated. Evidence for conversion of spore pool glutamate to gamma-aminobutyric acid during germination of spores of B. megaterium (parent strain) was obtained.     

Production of large amounts of acetate during germination of Bacillus megaterium spores in the absence of exogenous carbon sources.

When Bacillus megaterium spores germinate in the absence of an exogenous carbon source, the first minutes of germination are accompanied by production of large amounts (approximately 70 nmol/mg of dry spores) of acetate and much smaller amounts of pyruvate and lactate. The majority of these compounds are excreted into the medium. Exogenous pyruvate and alanine are also converted to CO2 and acetate by germinating spores, presumably by using the pyruvate dehydrogenase that is present in dormant spores. These data suggest that the 3-phosphoglyceric acid stores in the dormant spore and alanine generated by proteolysis early in germination can be catabolized to acetate during germination with production of large amounts of reduced nicotinamide adenine dinucleotide, acetyl coenzyme A, and adenosine 5'-triphosphate.