Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Cell viability and protein turnover in nongrowingBacillus megaterium at sporulation suppressing temperature

InBacillus megaterium, a temperature that suppresses sporulation (43°C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40–41°C). Here we show that, when cells grown at 35°C and transferred to a sporulation medium, were subjected to shifts between 35°C and the sporulation suppressing temperature (SST, 43°C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35°C for 2–3 h (T₂–T₃), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T₄–T₅. Protein turnover rate increased again after T₂ and up to T₈ 60–70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T₃ no asymmetric septa were observed and the amount of heat-resistant spores at T₈ was by 4–5 orders lower than at 35°C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T₃ but then its velocity sharply dropped and the amount of degraded protein at T₈ corresponded to slightly more than one-half of that found at 35°C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T_2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35°C. A later (T₄) shift to SST had no effect on the sporulation process.     

Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.     

Elevated content of hsp 70 does not induce tolerance of sporulation to higher temperature

The temperature permissive for sporulation (up to 42°C) inBacillus megaterium is by 4–5°C lower than that for its growth (up to 46–47°C). The ability ofB. megaterium cells to synthesize and degrade stress proteins under incubation in the sporulation medium was therefore investigated. The higher level of hsp 70, a typical stress protein induced by a temperature shock in postexponential growth phase, did not increase the permissive temperature of sporulation. The hsp 70 protein did not undergo a rapid turnover and its portion in the soluble protein fraction did not drop for at least 6 h at a temperature that was nonpermissive for sporulation (43.5°C). On the other hand, the elevated level of hsp 70 could not bring about the inhibition of sporulation as it was retained in the cells even after a shift of the temperature to 35°C, permitting sporulation of the culture.     

Temperature Effects on the Onset of Sporulation by Phytophthora ramorum on Rhododendron ‘Cunningham's White’

The effect of temperature and moist period on the onset of sporangia production by Phytophthora ramorum on Rhododendron ‘Cunningham's White’ was examined with misted detached leaves held in humid chambers. Following wound inoculation with sporangia, leaves were pre‐incubated at 20°C for either 24 or 72 h prior to placement at six different temperatures (4, 10, 15, 20, 25 and 30°C). The overall mean moist period required for first occurrence of sporulation over all six temperatures was 3.24 days with the 24‐h pre‐incubation time, compared with 1.49 days for the 72‐h pre‐incubation time. Following 24 h pre‐incubation at 20°C and at an incubation temperature of 15°C, sporangia were first collected from leaves following a 24 h incubation. At 10 and 20°C, sporangia were first collected after 48 h, whereas at 4, 25 and 30°C, sporangia were first collected after 3 days. Following 72 h pre‐incubation at 20°C, sporulation generally occurred within 1 day, even at temperatures such at 4 and 30°C that are suboptimal for sporulation. The highest levels of P. ramorum sporulation were observed at 20°C. P. ramorum formed sporangia on host tissue under moist conditions within the same time frame reported for P. phaseoli, P. palmivora and P. nicotianae, but substantially more slowly than certain other species such as P. infestans. Quantifying moisture and temperature conditions for initiation of sporangia production provides knowledge which leads to a greater understanding of the epidemic potential of P. ramorum.     

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.     

Life from the ashes: survival of dry bacterial spores after very high temperature exposure

We found that spores of Bacillus amyloliquefaciens rank amongst the most resistant to high temperatures with a maximum dry heat tolerance determined at 420 °C. We found that this extreme heat resistance was also maintained after several generations suggesting that the DNA was able to replicate after exposure to these temperatures. Nonetheless, amplifying the bacterial DNA using BOXA1R and (GTG)₅ primers was unsuccessful immediately after extreme heating, but was successful after incubation of the heated then cooled spores. Moreover, enzymes such as amylases and proteases were active directly after heating and spore regeneration, indicating that DNA coding for these enzymes were not degraded at these temperatures. Our results suggest that extensive DNA damage may occur in spores of B. amyloliquefaciens directly after an extreme heat shock. However, the successful germination of spores after inoculation and incubation indicates that these spores could have a very effective DNA repair mechanism, most likely protein-based, able to function after exposure to temperatures up to 420 °C. Therefore, we propose that B. amyloliquefaciens is one of the most heat resistant life forms known to science and can be used as a model organism for studying heat resistance and DNA repair. Furthermore, the extremely high temperature resistivity of these spores has exceptional consequences for general methodology, such as the use of dry heat sterilization and, therefore, virtually all studies in the broad area of high temperature biology.

     

Climatic factors influencing spore production in Alternaria brassicae and Alternaria brassicicola

Sporulation in A. brassicae and A. brassicicola on naturally-infected leaf discs of oilseed rape and cabbage required humidities equal to or higher than 91.5% and 87% r.h. respectively. The optimum temperatures for sporulation were 18–24°C for A. brassicae and 20–30°C for A. brassicicola at which temperatures both fungi produced spores in 12–14 h. Above 24°C sporulation in A. brassicae was inhibited. At sub-optimal temperatures sporulation times for A. brassicicola were significantly longer than for A. brassicae with the differences increasing with decrease in temperature. Interrupting a 16-h wet period at 20°C with a period of 2 h at 70% or 80% r.h. did not affect sporulation in either fungus but a dry interruption of 3–4 h inhibited sporulation in both. Exposure of both fungi to alternating wet (18 h at 100% r.h., 20°C) and dry periods (6 or 30 h at 5565% r.h., 20°C) did not affect the concentration of spores produced in each wet period. Sporulation times were not affected by either the host type of the age of the host tissue. White light (136 W/m²) inhibited sporulation in A. brassicae with the degree of inhibition increasing with increasing light intensity. The effect of light on sporulation in A. brassicicola was not tested.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

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.     

Inducing sporulation in different strains of alternaria solani

Summary Ultraviolet light induced abundant sporulation in two, out of the three strains ofAlternaria solani studied. Scraped cultures produced larger number of spores than unscraped ones. Ten seconds' exposure was found optimum for maximum sporulation. The optimum temperature of incubation subsequent to irradiation was 20°C. Young cultures were more responsive to ultraviolet light than the older ones. However, old cultures were more tolerant to a greater time of exposure than the younger ones. Intense ultraviolet light greatly reduced or even completely inhibited sporulation whereas low intensity of ultraviolet light was less effective in inducing sporulation. More irradiations than one greatly enhanced sporulation which reached its maximum with four irradiations. Spore length was considerably influenced by the age of the mycelium, temperature of incubation and the intensity and number of irradiations.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Effect of Temperature on Germination of Spores from Some Bacillus and Clostridium Species

The effect of temperature on germination of spores of Bacillus subyilis, B. megaterium. B. cereus, Clostridium sporogenes, Cl. butyricum and Cl. bifermentans was studied. At lower temperatures (+5°C to +10°C) the three Glostridium species germinated to a less extent than the three Bacillus. species. The optimum temperature for germination of the six species varied between +35°C and +45°C. The Clostridium species were more tolerant to heat than the Bacillus species.     

Control of Postharvest Diseases of Sweet Cherry with Ethanol and Hot Water

Complete inhibition of the germination of spores of Penicillium expansum occurred after 10 s exposure to 40% ethanol or more at ambient temperature, while spores of Botrytis cinerea were completely inhibited by 30% ethanol or more. Mortality of the spores of P. expansum and B. cinerea in heated 10% ethanol was higher than in water at the same temperatures. Immersion of naturally inoculated fruit in 20, 30, 40, or 50% ethanol reduced the decay present after storage for 10 days at 20°C similarly and by approximately 60–85%. Immersion of fruit that had been inoculated with the spores of P. expansum and B. cinerea reduced decay by both pathogens after storage for 30 days at 0°C and 5 days at 20°C when 30% or higher concentrations of ethanol were used. The incidence of decay after immersion in water alone for 30 s at 24, 50, 55, or 60°C was 57.7, 44.7, 46.2, and 35.7%, respectively, while 10% ethanol at these temperatures the decay incidence to 52.2, 33.9, 32.8, or 14.7%, respectively. Water treatments at 50, 55, or 60°C alone were not effective against P. expansum, while their efficacies were significantly increased by the addition of 10% ethanol. The most effective treatment was immersion in 10% ethanol at 60°C. Ethanol treatments at 20, 30, 40, or 50% and water treatments at 55 or 60°C significantly reduced natural fungal populations on the surfaces of fruit in all of the experiments. Addition of 10% ethanol to water significantly increased the efficacy of water in reducing the fungal populations at elevated temperatures. None of these treatments caused surface injuries to the fruit or adversely affected stem colour.     

Sulfidic acetate mineralization at 45°C by an aquifer microbial community: key players and effects of heat changes on activity and community structure

High-Temperature Aquifer Thermal Energy Storage (HT-ATES) is a sustainable approach for integrating thermal energy from various sources into complex energy systems. Temperatures ≥45°C, which are relevant in impact zones of HT-ATES systems, may dramatically influence the structure and activities of indigenous aquifer microbial communities. Here, we characterized an acetate-mineralizing, sulfate-reducing microbial community derived from an aquifer and adapted to 45°C. Acetate mineralization was strongly inhibited at temperatures ≤25°C and 60°C. Prolonged incubation at 12°C and 25°C resulted in acetate mineralization recovery after 40–80 days whereas acetate was not mineralized at 60°C within 100 days. Cultures pre-grown at 45°C and inhibited for 28 days by incubation at 12°C, 25°C, or 60°C recovered quickly after changing the temperature back to 45°C. Phylotypes affiliated to the order Spirochaetales and to endospore-forming sulfate reducers of the order Clostridiales were highly abundant in microcosms being active at 45°C highlighting their key role. In summary, prolonged incubation at 45°C resulted in active microbial communities mainly consisting of organisms adapted to temperatures between the typical temperature range of mesophiles and thermophiles and being resilient to temporary heat changes.     

Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis

The influence of different sporulation temperatures (30, 37, 44 and 52°C) upon heat resistance of Bacillus subtilis was investigated.
Heat resistance was greater after higher sporulation temperatures. Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested. Heat resistance increased about tenfold in the range of 30–44°C. Sporulation at 52°C did not show any further increase in heat resistance.
This effect was constant over all the range of heating temperatures tested (100–120°C). z value remained constant ( z = 9°C).
Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature. Spores obtained from cells incubated at 32 or 52°C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

Temperature-Dependent Spore Dimorphism in Burenella dimorpha (Microspora: Microsporida)

Burenella dimorpha, a microsporidian parasite of the tropical fire ant, Solenopsis geminata, produces two morphologically distinct types of spores. The binucleate free spores (spores not bound by a pansporoblast membrane) develop normally at temperatures at least as low as 20°C and as high as 32°C. The uninucleate octospores (spores bound in octets by a pansporoblast membrane), however, develop in a restricted range of temperature. Octospores constituted 35.9%± 2.6 of the spores in 25 pupae held at 28°C. Raising the temperature to 30°C reduced octospores to < 1% of the total spore population. Lowering the temperature to 25° or 22°C reduced the octospore population to 8.5%± 6.5 or 0.4 ± 0.5, respectively. Inhibition of octospore development was complete at 20°C. In contrast, the octospores of Vairimorpha necatrix and Vairimorpha plodiae are reported to be abundant at 16°C and 21°C, respectively. The critical event blocked in octospore development may be meiosis, as evidenced by an abundance of binucleate sporonts in the octospore sequence of development, and absence of more advanced sporogonic stages in hosts held at inhibitory temperatures. Free spore size is not affected by temperature although yield may be slightly reduced at elevated temperature.     

Perturbation of the heat resistance of bacterial spores by sporulation temperature and ethanol

Spores ofBacillus megaterium, B. subtilis, andB. stearothermophilus, harvested from cultures grown and sporulated at different temperatures or in the presence of ethanol, had different thermal resistance. There was a direct relationship between the sporulation temperature and the spore-killing temperature. The spores were more temperature-sensitive when formed in ethanol-supplemented media. Temperature and ethanol are known to perturb the degree of order within membranes and to alter membrane functions. Thus, alteration of spore membranes is an additional factor in the multifactorial nature of heat resistance. Another interpretation may be that heat shock proteins, known to be induced by heat, are formed during sporulation and may increase the thermostability of the spores.     

In Vivo and In Vitro Function of the Intracellular Proteolytic Apparatus in Nongrowing Bacillus megaterium Under Heat Stress

In Bacillus megaterium sporulating at 35°C, up to 90% of 10-min pulse-labeled proteins were degraded. Degradation proceeded in two waves. Short-lived proteins, i.e., intrinsically labile proteins and proteins made short-lived because of starvation, were mostly degraded during the reversible sporulation phase. Their amount corresponded to 20% or slightly more during 2 h. The second wave of protein degradation, which followed during the irreversible sporulation phase at 35°C, increased the amount of total degradable pulse-labeled proteins to about 90%. This wave was absent in the isogenic asporogenic mutant 27-36 or in the wild strain, whose sporulation was inhibited by increased temperature. The proportion of degradable proteins was thus reduced to less than 40% in the asporogenic mutant incubated at 35°C and to 46% in the wild strain whose sporulation was suppressed by the temperature of 47°C. Unlike sporulating cells, these cells were thus capable of degrading short-lived and denatured proteins, but were not able to degrade most of other proteins. The in vitro protein degradation was substantially enhanced by increasing the Ca²⁺ concentration, suggesting a role of Ca²⁺-dependent proteinase(s) in the process. Received: 23 July 1998 / Accepted: 19 August 1998     

Activities of Cysteine Synthetase and Cystathionase in Bacilhis subtilis during Sporulation

Cysteine synthetase (O-acetylserine sulfhydrylase) was partially purified from cells of Bacillus subtilis by the use of ammonium sulfate fractionation technique and DEAE-Sephadex A–50 chromatography. The cysteine synthetase preparation was compared with cystathionase (cystathionine β-cleavage enzyme) of the same organism in regard to biochemical properties and to changes in activity during sporulation.

The optimal pH and temperature for the cysteine synthetase were 8.5 and 25°C respectively. The enzyme was relatively stable at temperatures below 50°C and fairly resistant to proteases, in contrast to cystathionase. Production by B. subtilis of cysteine synthetase in sulfur-deficient synthetic medium was repressed by the addition of cysteine and derepressed by djenkolic acid. Activity of the enzyme was inhibited by methionine and increased by acetate. The cysteine synthetase activity was almost constant until the late sporulation stage commenced, but the specific activity of cystathionase (Fraction I) decreased rapidly in the course of sporulation and it could not be detected in the free spores.