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.     

Trehalose accumulation in vegetative cells and spores of Myxococcus xanthus.

The disaccharide trehalose is found in the spores and cysts of a variety of organisms. We analyzed developing cells of Myxococcus xanthus for trehalose accumulation. Vegetative cells grown in media with low osmotic strengths contained less than 5 micrograms of trehalose per mg of protein. Spores formed in fruiting bodies accumulated up to 1,100 micrograms of trehalose per mg of protein. Spores formed in liquid culture following the addition of glycerol contained up to 300 micrograms of trehalose per mg of protein. The trehalose contents of both spore types decreased rapidly during the early stages of germination. Trehalase activity was not detected in extracts of dormant or germinating spores. Trehalose accumulation in M. xanthus was also associated with elevated osmotic strength. Vegetative cells accumulated up to 214 micrograms of trehalose per mg of protein when grown in media containing elevated levels of solutes.     

Effects of intracellular trehalose content on Streptomyces griseus spores.

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Growth on media containing excess glucose yielded spores containing up to 25% of their dry weight as trehalose. Spores containing as little as 1% of their dry weight as trehalose were obtained during growth on media containing a limiting amount of glucose. Spores containing low levels of trehalose accumulated this sugar when incubated with glucose. The increase in trehalose content coincided with increases in spore refractility, heat resistance, desiccation resistance, and the time required for spore germination in complex media. Trehalose is accumulated by a wide variety of actinomycetes and related bacteria and may be partially responsible for their resistance properties.     

Purification of Dictyostelium discoideum spores by centrifugation in percoll density gradients with retention of morphological and biochemical integrity

Rapid and reliable methods have been developed for the preparation and purification of dormant spores of the cellular slime mold, Dictyostelium discoideum, using Percoll density gradient centrifugation. Percoll gradients were generated in situ (20,000g; 30 min) with subsequent banding of nondamaged dormant spores at an isopycnic density equal to about 1.12 g/cm³. Examination of the prepared spores by phase-contrast microscopy indicated the absence of stalk cells and other nonspore material and the retention by the spores of their morphological integrity. Biochemical integrity was also retained by the isolated spores as evidenced by their efficiency of germination and the level of endogenous trehalase activity present in crude cell-free spore extracts.     

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe

Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.     

Trehalose as an endogenous reserve in spores of the fungus Myrothecium verrucaria

Mandels, G. R. (U.S. Army Natick Laboratories, Natick, Mass.), Rasma Vitols, and Frederick W. Parrish. Trehalose as an endogenous reserve in spores of the fungus Myrothecium verrucaria. J. Bacteriol. 90:1589-1598. 1965.-Gross analysis of Myrothecium verrucaria spores showed approximately 3% fat, 33% carbohydrate, and 9.5% nitrogen. The water-soluble carbohydrates were trehalose, glucose, mannitol, and an unidentified phosphorylated compound. Water-soluble amino acids include leucine or norleucine (or both), valine, gamma-amino-n-butyric acid, beta-amino-n-butyric acid, ergothionine, glutamic acid, glutamine, glycine, aspartic acid, asparagine, cystine, and cystathionine. Ergosterol was also present. alphaalpha-Trehalose is the major reserve (20% of the dry weight), although approximately 30% of it appeared to be at the spore surface and was released by nonlethal treatment with 0.1 n HCl. Treatment with toluene or exposure to heat sufficient to kill the spores (20 min at 60 C) caused rapid liberation of all of the trehalose. Although spores could utilize exogenous trehalose with no appreciable lag, some stimulus, such as exposure to heat (10 min at 55 C), incubation with azide, or germination on exogenous substrates, was necessary to effect utilization of trehalose reserves. Spores have trehalase, but it is apparently at the spore surface, since it is inactivated by acid treatment which does not kill the spores. The metabolic pathway for utilization of trehalose is not known, but presumably it is not mediated by trehalase. The involvement of mannitol is indicated, since it tends to increase as trehalose decreases, although the changes are not quantitatively equivalent.     

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination.

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.     

Trehalose mobilization during early germination ofPilobolus longipes sporangiospores

Pilobolus longipes spores were activated by either exogenous glucose or 6-deoxyglucose. Trehalose content of glucose-activated spores increased and the substrate for trehalose synthesis was exogenous glucose. Addition of 6-deoxyglucose resulted in mobilization of trehalose, with about 20% of the reserve being consumed in the first hour. Little or no change in trehalase activity occurred during spore activation. Most of the trehalase activity associated with spores could be removed by washing with phosphate buffer. This extracellular enzyme was relatively stable, had a pH optimum of 5.6 and a K_m of about 0.5 mM and was estimated to be 66,000 in molecular weight. The specific activity of the crude enzyme extracts fromP. longipes was not influenced by cAMP, but, under the same conditions, the regulatory trehalase fromSaccharomyces cerevisiae became activated. These experiments indicate that trehalase activity in germinatingP. longipes spores may not be regulated by cAMP-dependent phosphorylation. Instead, the results suggest that trehalose is mobilized by a decompartmentation process.     

Spore Dormancy Mutants of Phycomyces

Rivero, F., and Cerdá-Olmedo, E. 1994. Spore dormancy mutants of Phycomyces. Experimental Mycology 18, 221-229. The spores of the Zygomycete Phycomyces blakesleeanus are called dormant because few of them germinate when placed in a medium that sustains mycelial growth and development. Nearly all the spores germinate after activation, that is, exposure to heat or certain chemicals. We have looked for mutants whose spores would not need activation. Nine mutants formed authentic, but transient spores, which germinated spontaneously in the sporangium. Mutant mycelia had lower alcohol and aldehyde dehydrogenase activities and less glycogen than wild-type mycelia. The spontaneous germination and the metabolic alterations are attributed to the same recessive mutations. No differences were found between mutants and wild type in the cyclic AMP and fructose 2,6-bisphosphate concentrations in immature sporangia and the trehalase activity in the mycelia. In another mutant the spore primordia did not form spores, but remained viable for some time in the sporangium. The mutants were difficult to keep in the laboratory (except as lyophils); this stresses the importance of preventing spore germination in the sporangium.     

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.     

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.     

Evidence that a glucose-mediated rise in cyclic AMP triggers germination ofPilobolus longipes spores

Spores ofPilobolus longipes incubated in phosphate buffer were activated within 5–10 min following the addition of either glucose or 6-deoxyglucose. Cyclic AMP content increased in response to glucose or 6-deoxyglucose, and the increase consistently preceded spore activation. Dibutyryl cyclic AMP also caused activation. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not cause activation, but, when added to spores with a suboptimal level of 6-deoxyglucose, it amplified the signal to produce a large increase in activation. IBMX increased intracellular cyclic AMP levels when it was applied with 6-deoxyglucose, but had no effect when it was applied alone. Phosphodiesterase activities in cell extracts from dormant and activated spores were not significantly different. These results indicate that the rise in cyclic AMP that follows exposure to glucose may play an important role in triggering spore germination.     

Cyclic AMP,phosphodiesterase, and spore activation inPhycomyces blakesleeanus

A soluble cyclic AMP phosphodiesterase was demonstrated in crude extracts ofPhycomyces spores. During an activating heat treatment of the spores the cyclic AMP phosphodiesterase activity was reduced to some 15% of its value in dormant spores. During early germination the activity slowly increased. No difference was found in the behavior of the enzyme from dormant and activated spores during gel filtration and anion exchange chromatography or in its sensitivity toward heat denaturation. After the spores were heated at different temperatures there was a coincidence between germination induction and cyclic AMP phosphodiesterase inactivation. 3-Isobutyl-1-methylxanthine induced an increase in both cyclic AMP concentration and trehalase activity in the spores and led to complete germination of the spores.     

Glycolytic enzymes in resting spores and vegetative mycelia of Entomophthora pyriformis.

Specific activities of 14 enzymes of the Embden-Meyerhof-Parnas and pentose phosphate pathways were determined in extracts of resting spores and vegetative mycelia of Entomophthora pyriformis. All these enzymes were detected in mycelial extracts, whereas only nine were detectable in resting spore extracts. Activities of detectable spore enzymes were much lower than those of the corresponding mycelial enzymes with the exception of triosephosphate dehydrogenase, which had higher activity in spore extracts. The enzyme deficiencies noted point to the inability of either pathway to function in dormant spores.     

Trehalose catabolism in microsporidia Nosema grylli spores

Some differences in trehalose catabolism were found for terrestrial and aquatic microsporidian species (Undeen, Van der Meer, 1999). In microsporidia species from aquatic hosts, the spore extrusion causes the intrasporal trehalose hydrolysis by trehalase that is followed by the drastic rise of reducing sugars (glucose) concentration. On the contrary, in tested terrestrial microsporidian species, total and reducing sugars remain unchanged through the germination. In this study we demonstrate by means of the enzymatic and paper chromatography methods, that in spores of microsporidia Nosema grylli, infecting fat bodies of crickets Gryllus bimaculatus, neither an increase of glucose concentration nor a reduction in intrasporal trehalose content takes place during the spore discharge. In this respect N. grylli is close to other terrestrial species. However, we have revealed in N. grylli spores activity of alpha,alpha-trehalase (EC 3.2.1.28) with acid pH-optimum like it was found by other authors in spores of aquatic microsporidia N. algerae. This result differs from the neutral pH-optimum (7.0) of trehalse of other terrestrial microsporidia N. apis. Concentration of trehalose in N. grylli spores reduces during long-term storage. All attempts to detect an activity of trehalose phosphorylase (synthase) (K phi 2.4.1.64), other potential key enzyme for trehalose catabolism in N. grylli spores have failed. The absence of changes of the sugar content in terrestrial microsporidian spores during the extrusion indicates, that the main physiological role of trehalose hydrolysis by trehalase in these species is catabolism of energy reserves for providing the long-term survival in the environment.     

Effect of mechanical abrasion on the viability, disruption and germination of spores of Bacillus subtilis

AIMS: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. METHODS AND RESULTS: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. CONCLUSIONS: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.     

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.     

The role of osmotic pressure in the germination of Nosema algerae spores

Both the lag period and the time required for the filament and sporoplasm to emerge from Nosema algerae spores were prolonged when germination occurred under hyperosmotic conditions. Polyethylene glycol (PEG) and sucrose inhibited germination, first by preventing eversion of the filament, and then at higher concentrations by preventing stimulation. The size of the spore cases decreased by about 21% following germination, indicating an elastic spore wall and turgor pressure in the dormant spores. Increased pressure during germination was indicated by less osmotically-induced shrinkage in stimulated than in dormant spores and by higher concentration of solutes in the homogenates of germinated than ungerminated spores. These results are consistent with the hypothesis of a pressure increase during germination that is caused by an endogenous increase in solute concentration.     

Glutamic Acid Decarboxylase in Spores of Bacillus megaterium and Its Possible Involvement in Spore Germination

Spores of Bacillus megaterium were examined for glutamic acid decarboxylase (GAD). Although dormant spores showed no GAD activity, spores given sonic treatment and heat-activated spores had high activities when assayed for this enzyme. Several parameters of GAD in heat-activated spores were examined. The effects of KCN, NaN(3), 2,4-dinitrophenol, and KF on GAD activity were examined. Only KCN was an effective inhibitor of GAD activity in heated spores and was also shown to be the only effective inhibitor of GAD activity in vegetative bacteria. Similar patterns of inhibition were obtained with GAD activity and with spore germination, KCN being the only effective inhibitor of both, although at different concentrations. Spore GAD activity in heat-activated spores showed a loss with storage at 4 C; on the other hand, storage at 25 C was not accompanied by a loss, but, to the contrary, showed an increase in GAD activity of about 30%. A comparison of GAD activity at different times during germination, growth, and sporulation showed it to be highest in freshly germinated spores. Although vegetative cells contained GAD activity, the level in log-phase cells was approximately one-half the level obtained with freshly germinated spores. Heat-activated mutant spores with a requirement of gamma-aminobutyric acid for germination gave no GAD activity. GAD activity appeared in mutant spores after germination and increased to levels comparable to parent spores after 9 min of germination.