CHANGES IN THE HEAT-RESISTANCE OF ASCOSPORES OF NEUROSPORA UPON GERMINATION

Lingappa , Yamuna , and A. S. Sussman . (U. Michigan, Ann Arbor.) Changes in the heat-resistance of ascospores of Neurospora upon germination. Amer. Jour. Bot. 46 (9): 671–678. Illus. 1959.—A rapid loss in heat-resistance accompanies activation of ascospores of Neurospora tetrasperma after incubation at 27°C. When activated spores are given a 5-min. “heat-flash” at 65°C. after only 5 min. at 27°C., fully % fail to germinate. Such treatment, if administered 25 min. after activation, results in the complete destruction of the spores. By contrast, when incubation at 27°C. is not interposed, more than ½ of the spores will germinate, even when they have been exposed to 65°C. for 30 min. Similar results were obtained with “heat-flashes” at 50 and 60°C., although exposures of longer duration were required to affect the spores. Conidia respond very differently to “heat-flashes” in that germination is stimulated if they are provided after an incubation period at 27°C. On the other hand, conidia are killed by short exposures to 60°C., so that they are far more susceptible to such treatment than are ascospores. A study of the cardinal temperatures of germination revealed that the maximum is about 44°C. for both conidia and ascospores. The maximum for the growth of two strains of N. tetrasperma and for one of N. crassa is between 40–45°C.; however, another strain of the latter species grows at 45°C. Dry heat was shown to be less effective than wet in activating ascospores. Removal of the exospore of ascospores results in the loss of considerable heat-resistance. In addition, the requirement for heat-activation is considerably mitigated in such spores, suggesting that the exospore, or an associated layer is the locus of the ascospore's heat-resistance.     

Trehalose degradation and glucose efflux precede cell ejection during germination of heat-resistant ascospores of Talaromyces macrosporus

Talaromyces macrosporus forms ascospores that survive pasteurization treatments. Ascospores were dense (1.3 g ml(-1)), relatively dry [0.6 g H(2)O (g dry weight)(-1)] and packed with trehalose (9-17% fresh weight). Trehalose was degraded to glucose monomers between 30 and 100 min after heat activation of the spores. The maximal activity of trehalase was calculated as 400-520 nmol glucose formed min(-1) (mg protein)(-1) as judged by measurements of the trehalose content of spores during germination. During early germination, glucose was released from the cell (10% of the cell weight or more). The intracellular concentration of glucose only peaked briefly. After 160-200 min, the protoplast encompassed by the inner cell wall was ejected through the outer cell wall in a very quick process. Subsequently, respiration of spores increased strongly. The data suggested that trehalose is primarily present for the protection of cell components as glucose is released from the cell. Then, an impenetrable outer cell wall is shed before metabolic activity increases.     

Protein synthesis in germinating Saccharomyces cerevisiae ascospores

The uptake and incorporation of macromolecular precursors in germinating Saccharomyces cerevisiae ascospores were investigated. Addition of cycloheximide at various times during germination revealed that protein synthesis can occur within 20 min after the spores are shifted to glucose-containing media. The time of initiation of uptake and incorporation of several amino acids differed; this can be attributed to differing amino acid pool levels in the spores, as well as differing transport activities. Two-dimensional gel electrophoresis of proteins labeled with [35S]methionine for various 20-min periods after germination began showed at least one protein whose synthesis begins well after the bulk of the proteins.     

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.     

Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos

We employed whole-mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock-induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33 degrees C heatshock. The lowest temperature capable of inducing this pattern was 30 degrees C. Placement of embryos at 22 degrees C following a 1-h 33 degrees C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues.     

REVERSIBLE DEACTIVATION OF NEUROSPORA ASCOSPORES BY LOW TEMPERATURE

Sun, Clare Y., and Alfred S. Sussman. (U. Michigan, Ann Arbor.) Reversible deactivation of Neurospora ascospores by low temperature. Amer. Jour. Bot. 47(7): 589-593. Illus. 1960.—Heat-activated ascospores of Neurospora tetrasperma are reversibly deactivated after incubation at 4°C. for 36–48 hr. Two cycles of deactivation and reactivation are possible although the percentage germination decreases in the last cycle. By contrast, spores held at 20°C., or in glycerol at 4°C., will remain activated for much longer periods of time. If an incubation period at 20°C. greater than 30 min. is interposed before the activated spores are placed at 4°C., germination occurs despite the cold-treatment. Furfural-activated ascospores, when held at 4°C., are deactivated but can be reactivated only by heat, pointing up a difference between ascospores activated by these different means. Although a fraction of the stimulus afforded by heat-sensitization to chemical activators is preserved for 2 days at —20°C., it is dissipated completely after a short time at 4°C. These data are discussed on the basis of the suggestion that the reversible production of a substance initiates a series of steps which lead to germination. Thus, the temperature minimum of the forward reaction is greater than 4°C. whereas the back reaction proceeds at this temperature.     

Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor

Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.     

Spatial pattern of constitutive and heat shock‐induced expression of the small heat shock protein gene family,hsp30, in Xenopus laevis tailbud embryos

We employed whole‐mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock‐induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33°C heatshock. The lowest temperature capable of inducing this pattern was 30°C. Placement of embryos at 22°C following a 1‐h 33°C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues. Dev. Genet. 25:365–374, 1999. © 1999 Wiley‐Liss, Inc.     

Two developmental stages of Neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for recovery.

At the heat shock temperature of 45 degrees C, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of Neurospora crassa. Both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. At the RNA level, however, these two developmental stages responded with different kinetics to elevated temperature. Heat shock RNAs (for hsp30 and hsp83) accumulated and declined more rapidly in spores than in mycelia, and during recovery spores accumulated mRNA that encoded a normal protein (the proteolipid subunit of the mitochondrial ATPase), whereas mycelia showed no increase in this normal RNA (for at least 120 min). Therefore, the resumption of normal protein synthesis in spores may depend upon accumulation of new mRNAs. In contrast, mycelial cells appeared to change their translational preference during continued incubation at elevated temperature, from a discrimination against normal mRNAs to a resumption of their translation into normal cellular proteins, exemplified by the ATPase proteolipid subunit whose synthesis was measured in the heat-shocked cells.     

Nature of Deoxyribonucleic Acid Synthesis and Its Relationship to Protein Synthesis During Outgrowth of Bacillus cereus T

Deoxyribonucleic acid (DNA) synthesis during early outgrowth of spores of Bacillus cereus T (thy(-)) has been examined. (14)C-thymidine incorporated begins 2 to 5 min after germination and continues at a slow rate up to 30 min, after which the rate of (14)C-thymidine incorporation increases considerably. Early DNA synthesis up to 30 min after germination is dependent upon simultaneous protein synthesis. The examination of the stability of proteins synthesized soon after germination shows that they are susceptible to intracellular degradation. The evidence provided here indicates that protein degradation is the cause of observed dependence of DNA synthesis on simultaneous protein synthesis. The DNA synthesis occurring soon after germination is primarily a repair type synthesis which is followed by the onset of normal replication approximately 30 min after germination.     

Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.     

In vivo development of spores of Absidia ramosa.

Approximately 10(6) spores of Absidia ramosa were inoculated intravenously into normal and cortisone pretreated mice. At subsequent time intervals the liver, lungs and kidneys were removed and examined for fungal localization and growth. In normal mice, spore germination and continued hyphal growth was restricted to the kidneys-evidence of germination not being visible until around 30h post inoculation. Cortisone therapy allowed germination of spores in the lung and kidney by 7h but subsequent hyphal growth in the lung was severely restricted compared with the kidney where extensive hyphal growth occurred. Germination of spores in the liver of cortisone treated animals was slow, not becoming apparent until about 40h after inoculation. These results suggest that host defence mechanisms in the form of phagocytosis as well as biochemical inhibitors and/or lack of suitable stimulators are important in preventing germination of introduced fungal spores. Once germination has occurred, it appears that additional as yet undetermined factors play a role in allowing continued growth of the fungus.     

Expression of microinjected hsp 70/CAT and hsp 30/CAT chimeric genes in developing Xenopus laevis embryos

The expression of microinjected chimeric genes containing Drosophila hsp 70 and Xenopus hsp 70 and hsp 30 promoters linked to the reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) was examined during early development of Xenopus laevis. Heat-inducible expression of fusion genes containing either the Drosophila hsp 70 promoter (1100 bp) or the Xenopus hsp 70 promoter (750 bp) was first detectable after the midblastula stage of development. This coincides with the embryonic stage at which the endogenous hsp 70 gene is first heat-inducible. A Xenopus hsp 30/CAT fusion gene containing 350 bp of promoter sequences was also heat-inducible after the midblastula stage unlike the endogenous hsp 30 genes which were not heat-inducible until the early tailbud stage (stage 23-24). Sequences that are present within either the coding or 3' region of the hsp 30 clone do not cause the microinjected hsp 30 gene to be developmentally regulated in a normal manner. Additionally, microinjected hsp 30 gene sequences have no effect on the developmental regulation of endogenous hsp 30 genes which continue to be activated at the tailbud stage of development. Our data suggest, that an inhibitory system, which may control the expression of the endogenous hsp 30 gene during development, does not regulate the expression of the injected hsp 30 gene.     

Spores of microorganisms

Spores ofBacillus cereus were germinated in a germination limited medium (GL-medium) which facilitates only germination but not the postgerminative development of spores. Under these conditions a limited protein synthesis occurs. However, this protein synthesis is stopped after a short time interval. The rate of synthesis of new proteins, as well as their total amount, is influenced by the length of the activation heat shock. Synthesis of the wall material continues for several hours and thick-walled cells with a changed ultrastructure are formed. Synthesis of the diaminopimelic acid (dap) containing material of the cell wall is sensitive to actinomycin D and relatively resistant to chloramphenicol. Similarly, protein synthesis is relatively chloramphenicol-resistant but is fully inhibited by azauracil or spiramycin. Whereas RNA formed in the control culture is partially decomposed after 30 min of incubation, chloramphenicol accelerates its synthesis and prevents its decay. Exudate components apparently stimulate synthesis of ribonucleic acid, proteins and the wall material. The¹⁴C-dap containing material released by prelabelled spores in the form of the exudate during the germination is not re-utilized by the spores germinated in the GL-medium. The results are discussed with respect to the atypical primary synthetic activities of spores under conditions when the postgerminative development is prevented and from the point of view of participation of the germination exudate during these syntheses.     

Evidence for oxygen and carbon dioxide receptors in insect CNS influencing ventilation

We investigated heat activation and germination of Eurotium repens ascospores to follow high pressure inactivation. Activation energy and entropy values strengthen the idea of protein denaturation as the underlying mechanism of heat activation. Preceding activation, germination or a combination of both affected high pressure inactivation in different ways. Activation followed immediately by high pressure treatment led to the most efficient improvement in inactivation. However, a pause after activation caused a partial re-establishment of the spores' stability and less efficient high pressure inactivation. Germination stabilized the spores against high pressure. A combined treatment of activation and germination led to an initially fast inactivation, but compared to high pressure treatment of only activated spores the time course of inactivation was slowed down.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.     

Metabolism and the triggering of germination of Bacillus megaterium. Concentrations of amino acids, organic acids, adenine nucleotides and nicotinamide nucleotides during germination.

A considerable amount of evidence suggests that metabolism of germinants or metabolism stimulated by them is involved in triggering bacterial-spore germination. On the assumption that such a metabolic trigger might lead to relatively small biochemical changes in the first few minutes of germination, sensitive analytical techniques were used to detect any changes in spore components during the L-alanine-triggered germination of Bacillus megaterium KM spores. These experiments showed that no changes in spore free amino acids or ATP occurred until 2-3 min after L-alanine addition. Spores contained almost no oxo acids (pyruvate, alpha-oxoglutarate, oxaloacetate), malate or reduced NAD. These compounds were again not detectable until 2-3 min after addition of germinants. It is suggested, therefore, that metabolism associated with these intermediates is not involved in the triggering of germination of this organism.     

Changes in Saccharomyces cerevisiae ascospore proteins during early germination

Abstract Proteins present in Saccharomyces cerevisiae ascospores and in germinating ascospores were compared using two-dimensional polyacrylamide gel electrophoresis. Yeast ascospores incorporated labeled methionine efficiently facilitating the electrophoretic analysis. Proteins synthesized in the yeast ascospores differed significantly from those proteins found 15 min after the initiation of germination in the ascospores. An immediate transition from ascospore proteins to proteins required for ascospore germination appears likely.