Metabolism and chemical activation ofPhycomyces blakesleeanus spores

Respiratory and other data indicated that acetate was quickly metabolized byPhycomyces spores. Azide prevented metabolism of acetate although it did not inhibit oxygen uptake by dormant spores. Azide also inhibited activation of dormant spores by acetate, suggesting that acetate metabolism was necessary for spore activation. Pyruvate uptake by the spores was very limited in culture medium but could be increased substantially by lowering the pH to 3. However, pyruvate failed to activate dormant spores event at low pH values. Therefore, the lack of pyruvate metabolism could be involved in maintaining spore dormancy. Ammonium salts at pH 9 largely activated the spores. Under such conditions no increase in trehalase activity was found in the spores. Incubations with acetate in the presence of azide yielded a stimulation of trehalase activity without triggering spore activation. Therefore, spore activation and stimulation of trehalase activity seem to be two independent phenomena inPhycomyces spores.     

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.     

Water potential,glycerol synthesis,and water content of germinating Phycomyces spores

During early germination, the sporangiospores of Phycomyces blakesleeanus synthesized large amounts of glycerol. Glycerol started leaking out of the spores after some 20 min germination. Simultaneously the water content of the spores greatly increased. Water uptake was accompanied by disapperance of the phase contrast halo and an increase in spore cross-sectional area which all occurred during the same period between 10 and 30 min germination. When spores were incubated in 0.5 or 1 M sucrose, glycerol accumulated in the spores to much higher concentrations and the increase in cellular water content was greatly reduced and retarded. Glycerol synthesis and the concomitant lowering of spore osmotic potential was not the only mediator of spore swelling since equally important glycerol concentrations loaded into dormant spores did not cause spore water uptake or swelling. Also the swelling of the spores was less affected than water uptake by decreases in ambient water potential. Apparently also cell wall loosening was involved in the swelling phenomenon which might have important implications for cellular metabolism.     

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.     

Activate to Eradicate: Inhibition of Clostridium difficile Spore Outgrowth by the Synergistic Effects of Osmotic Activation and Nisin

Background

Germination is the irreversible loss of spore-specific properties prior to outgrowth. Because germinating spores become more susceptible to killing by stressors, induction of germination has been proposed as a spore control strategy. However, this strategy is limited by superdormant spores that remain unaffected by germinants. Harsh chemicals and heat activation are effective for stimulating germination of superdormant spores but are impractical for use in a hospital setting, where Clostridium difficile spores present a challenge. Here, we tested whether osmotic activation solutes will provide a mild alternative for stimulation of superdormant C. difficile spores in the presence of germinants as previously demonstrated in several species of Bacillus. In addition, we tested the hypothesis that the limitations of superdormancy can be circumvented with a combined approach using nisin, a FDA-approved safe bacteriocin, to inhibit outgrowth of germinated spores and osmotic activation solutes to enhance outgrowth inhibition by stimulating superdormant spores.

Principal Findings

Exposure to germination solution triggered ∼1 log₁₀ colony forming units (CFU) of spores to germinate, and heat activation increased the spores that germinated to >2.5 log₁₀CFU. Germinating spores, in contrast to dormant spores, became susceptible to inhibition by nisin. The presence of osmotic activation solutes did not stimulate germination of superdormant C. difficile spores exposed to germination solution. But, in the absence of germination solution, osmotic activation solutes enhanced nisin inhibition of superdormant spores to >3.5 log₁₀CFU. The synergistic effects of osmotic activation solutes and nisin were associated with loss of membrane integrity.

Conclusions

These findings suggest that the synergistic effects of osmotic activation and nisin bypass the limitations of germination as a spore control strategy, and might be a novel method to safely and effectively reduce the burden of C.difficile spores on skin and environmental surfaces.     

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.     

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 fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.     

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.     

Superdormant Spores of Bacillus Species Have Elevated Wet-Heat Resistance and Temperature Requirements for Heat Activation

Purified superdormant spores of Bacillus cereus, B. megaterium, and B. subtilis isolated after optimal heat activation of dormant spores and subsequent germination with inosine, d-glucose, or l-valine, respectively, germinate very poorly with the original germinants used to remove dormant spores from spore populations, thus allowing isolation of the superdormant spores, and even with alternate germinants. However, these superdormant spores exhibited significant germination with the original or alternate germinants if the spores were heat activated at temperatures 8 to 15°C higher than the optimal temperatures for the original dormant spores, although the levels of superdormant spore germination were not as great as those of dormant spores. Use of mixtures of original and alternate germinants lowered the heat activation temperature optima for both dormant and superdormant spores. The superdormant spores had higher wet-heat resistance and lower core water content than the original dormant spore populations, and the environment of dipicolinic acid in the core of superdormant spores as determined by Raman spectroscopy of individual spores differed from that in dormant spores. These results provide new information about the germination, heat activation optima, and wet-heat resistance of superdormant spores and the heterogeneity in these properties between individual members of dormant spore populations.Spores of Bacillus species are formed in sporulation and are metabolically dormant and extremely resistant to a variety of stress factors (31, 32). While spores can remain dormant for long periods, if given the proper stimulus, they can rapidly “return to life” in the process of spore germination followed by outgrowth (30). Since spores are generally present in significant amounts on many foodstuffs and growing cells of a number of Bacillus species are significant agents of food spoilage and food-borne disease (32), there is continued applied interest in spore resistance and germination. While dormant spores can be killed by a treatment such as wet heat, this requires high temperatures that are costly and detrimental to food quality. Consequently, there has long been interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. However, this strategy has been difficult to apply because of the significant heterogeneity in germination rates between individual spores in populations. One reflection of this heterogeneity is the extremely variable lag times following addition of germinants but prior to initiation of germination events; while these lag times can vary from 10 to 30 min for most spores in populations, some spores have lag times of many hours or even many days (2, 12, 13, 15, 25). The spores that are extremely slow to germinate have been termed superdormant spores, and populations of superdormant spores have recently been isolated from three Bacillus species, and their germination properties characterized (9, 10). These superdormant spores germinate extremely poorly with the original germinants used to remove dormant spores from spore populations, thus allowing superdormant spore isolation, and also poorly with a number of other germinants, in particular, germinants that target nutrient germinant receptors different than those activated to isolate the superdormant spores. However, the superdormant spores germinate reasonably well with mixtures of nutrient germinants that target multiple germinant receptors. All reasons for spore superdormancy are not known, but one contributing factor is the number of nutrient germinant receptors in the spore''s inner membrane that trigger spore germination by binding to nutrient germinants (9). The levels of these receptors are most likely in the tens of molecules per spore (24), and thus stochastic variation in receptor numbers might result in some spores with such low receptor numbers that these spores germinate very poorly (23). Indeed, 20- to 200-fold elevated levels of at least one nutrient germinant receptor greatly decreases yields of superdormant spores of Bacillus subtilis (9).Spores of Bacillus species generally exhibit a requirement for an activation step in order to exhibit maximum germination (17). Usually this activation is a sublethal heat treatment that for a spore population exhibits an optimum of 60 to 100°C depending on the species. Spores are also extremely resistant to wet heat, generally requiring temperatures of 80 to 110°C to achieve rapid spore killing, with the major factor influencing the wet-heat resistance of spores of mesophilic strains being the spore core''s water content, which can be as low as 30% of wet weight as water in a fully hydrated spore (8, 19, 27, 28, 31). Invariably, increases in core water content are associated with a decrease in spore wet-heat resistance (8, 19, 22, 25). While spore populations most often exhibit log-linear kinetics of wet-heat killing, the observation of tailing in such killing curves at high levels of killing is not uncommon, suggesting there is significant heterogeneity in the wet-heat resistances of individual spores in populations (27, 28). While there has been no comparable work suggesting that there is also heterogeneity in the temperature optima for heat activation of individual spores in populations, this certainly seems possible and indeed was suggested as one cause of spore superdormancy, as yields of superdormant spores from spore populations that are not heat activated are much higher (9, 10). Consequently, the current work was initiated to test the hypothesis that superdormant spores require heat activation temperatures that are higher than those of the original dormant spores. Once this was found to be the case, the wet-heat resistance and core water content of the superdormant and original dormant spores were compared, and the environment of the spore core''s major small molecule, pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) was assessed by Raman spectroscopy of individual spores.     

Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 micrograms/ml (26 microM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of beta-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Glycerol formation after the breaking of dormancy of Phycomyces blakesleeanus spores. Role of an interconvertible glycerol-3-phosphatase

The breaking of dormancy of Phycomyces blakesleeanus spores by a heat shock was followed by a transient production of glycerol, which culminated within 5-10 min and was terminated at 20 min. Extracts of spores contained a magnesium-dependent glycerol-3-phosphatase active on both L-glycerol 3-phosphate and dihydroxyacetone phosphate but having more affinity for the first substrate than for the second. In extracts from dormant spores, the phosphatase was profoundly inhibited by physiological concentrations of inorganic phosphate, which induced cooperativity for the substrate, whereas the enzyme from heat-activated spores was much less inhibited and this difference in kinetic properties persisted after gel filtration of the enzymic preparation. When measured at 1 mM phosphate and 0.1 mM glycerol 3-phosphate, the phosphatase activity was undetectable in dormant spores, increased sharply during the heat treatment and the following 5 min at 25 degrees C, then fell again to a low value by 20 min. A similar transient activation of the enzyme was observed following the breaking of dormancy by incubation of the spores in the presence of 0.1 M ammonium acetate. Incubation of a cell-free extract or of the partially purified glycerol-3-phosphatase in the presence of ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase released the enzyme from inhibition by phosphate and endowed it with the same kinetic properties as did the heat treatment of the spores. It appears therefore most likely that phosphorylation of glycerol-3-phosphatase by cyclic-AMP-dependent protein kinase causes its activation and that this transient process explains the equally transient formation of glycerol by the spores after the heat shock.     

Survival of Shigella in Sewage: II. Effect of Glycerol on Shigella flexneri and Shigella Bacteriophage1

Glycerol (30%) inhibited or delayed the adsorption of Shigella bacteriophage on its host organism, S. flexneri II; glycerol also inhibited or delayed the burst of phage, whether or not adsorption was carried out in the presence of glycerol. Studies of the mechanisms of these effects showed that viscosity and osmotic shock probably were not responsible for either phenomenon. The inhibition of adsorption, however, was proportional to the concentration of glycerol, and appeared to be a function of the hydroxyl groups on the glycerol molecule. The inhibition of burst seemed to be related to the osmotic pressure outside the bacterial cells.     

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.     

Viability and physiological state transitions of <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> sporangiospores in tempe starter culture

The viability and various physiological characteristics of individual sporangiospores of Rhizopus oligosporus in tempe starter cultures that had been stored for 8, 10, 16 and 30 months were examined by flow cytometry in combination with fluorescent dyes. Besides live, dead, and dormant spores we distinguished a category of sublethally damaged spores. Results indicated that the shelf-life of tempe starters was not limited by the death of spores, but by sublethal damage to spores as well as by dormancy which can be overcome by resuscitation, respiratory activation. During storage, the number of dormant and sublethally damaged spores increased: the longer the starter cultures were stored, the less dormant spores could still be activated. In contrast, the transition from sublethally damaged (spores that are not able to transform cFDA and emit green fluorescence except by activation treatment) to activated spores did not decrease with longer storage. However, after very long (30 months) storage, sublethally damaged spores could still be activated but could not germinate anymore. The shelf-life of spores in tempe starter is related to the physiological state of spores being sublethally damaged; a mechanism of physiological state transitions of R. oligosporus sporangiospores is proposed.     

Mathematical Model of Thermal Destruction of Bacillus stearothermophilus Spores

The experimental survival curves of Bacillus stearothermophilus spores in aqueous suspension, for six constant temperatures ranging from 105 to 130°C, displayed an initial shoulder before a linear decline. To interpret these observations, we supposed that, before the heat treatment, the designated spore suspension contained a countable and mortal N₀ population of activated spores and an M₀ population of dormant spores which remained masked during spore counting and had to be activated before being destroyed by heat. We also hypothesized that the mechanisms of both activation and destruction are, at constant temperature, ruled by first-order kinetics, with velocity constants k_A and k_D, respectively. Mathematical analysis showed that this model could represent not only our experimental survival curves, but also all other shapes (linear and biphasic) of survival curves found in the literature; also, there is an inherent symmetry in the model formulation between the activation and destruction reactions, and we showed that the dormancy rate (τ = M₀/N₀) is the only parameter which permits a distinction between the two reactions. By applying the model to our experimental data and considering that the dormancy rate is not dependent on the treatment temperature, we showed that, for the studied suspension, the limiting reaction was the activation reaction.     

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.     

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.