The effects of alterations in the suspending medium on low temperature activation of spores of Bacillus stearothermophilus NGB101

The effects of eight different sodium salts on the activation of spores of Bacillus stearothermophilus NGB101 at 30°C were examined. Sodium nitrite was a potent activator spores of NGB101. Complete activation of spore populations was obtained after 6 h or less at 30°C. Activation of spores of NGB101 in solutions of sodium nitrite, like activation in distilled water, was temperature dependent, with optimal activation at 30°C. While a potent activator of spores of NGB101 at 30°C, sodium nitrite was ineffective as an initiator of germination at 65°C. Activation of spores of NGB101 produced marked increases in colony forming spores compared with nonactivated populations. Spore populations activated in solutions of sodium nitrite gave higher plate counts compared with spores activated in distilled water.     

Nutritional requirements of keratinophilic fungi and dermatophytes for conidial germination

Germination of spores of Chrysosporium crassitunicatum, Nannizzia fulva (+), Nannizzia fulva (–) and Trichophyton equinum was studied in the presence of various carbon and nitrogen sources. Effect of different temperatures on spore germination was also determined. Maximum spore germination within 24 hours was recorded when glucose was used as a carbon source for all the test fungi. Except sodium nitrate all the inorganic nitrogen sources enhanced the spore germination at 0.05% concentration. Most of the organic nitrogen sources used were found to be stimulatory for the spore germination of test fungi. Optimum temperature i.e. 28 °C supported maximum spore germination of all the test fungi within 24 hours. C. Crassitunicatum, N. fulva(+), N. fulva(–) could germinate upto 35 °C but beyond that no spore germination was noticed in these fungi. T. equinum could germinate at a higher temperature of 40 °C but the percentage of germination was very low.     

Temporal control of autoactivator synthesis and secretion during spontaneous spore germination in Dictyostelium discoideum

SG mutant and aged wild type spores of the cellular slime mold Dictyostelium discoideum germinate in the absence of an externally applied activation treatment. This type of germination is referred to as autoactivation. During the swelling stage of autoactivation, spores release a factor, the autoactivator, capable of stimulating germination in subsequent spore populations. The autoactivator was not present in the dormant spore, but it or a precursor was produced internally during the first hour of autoactivation. This production was sensitive to moderately high temperatures (+31° C) and was completely destroyed by heat activation (45° C for 30 min). Internal production of the autoactivator was not sensitive to protein synthesis inhibitors. However, the release of the activator from the spore appeared to be regulated by protein synthesis. Internal autoactivator was also produced in the aged wild type strain during the postautoactivation lag phase. The activator could not be directly isolated from within the germinating spore. Its activity on the rest of the spore population was dependent upon its release from the germinating spore. A model is presented integrating the effects of heat, cycloheximide, autoinhibitor and autoactivator on spores of D. discoideum.     

Iron and temperature as sporangiospore germination factors of Pilobolus longipes

Sporangiospores of Pilobclus longipes germinated on a medium containing ascorbate and FeSO₄, but neither ascorbate nor FeSO₄ alone caused spores to germinate. The iron chelates (hemin, coprogen, and ferrichrome) that are known to promote mycelial growth of this and other species of Pilobolus had little or no effect on spore germination, suggesting that under these conditions dormant spores are unable to reduce iron III.Regardless of the medium used, maximum germination required treatment at two temperatures. The early stage of germination, spherical growth, was favored by treatment for several hours at about 38°C while optimum germ tube formation required incubation at lower temperatures (25°C). Under most conditions the requirement for a heat treatment was nearly absolute.When the iron-ascorbate and the heat treatments were separated it was found that they need not be applied simultaneously provided that iron and ascorbate are given first. Spores that were heated first and then given iron and ascorbate at lower temperatures did not germinate. Apparently dormancy of these spores is broken by available iron but a heat treatment is usually required to complete the germination process.     

The effect of sporulation temperature on sporal characteristics ofBacillus subtilis A

Spores ofBacillus subtilis A were produced at different temperatures (23°–49°C) and examined for a number of sporal characteristics. Spore heat resistance increased with sporulation temperature to 45°C, with spores grown at 49°C showing a dramatic reduction in resistance. Spore crops showed biphasic thermal death curves whether enumerated on germination medium with or without calcium dipicolinate. This strain produces both rough and smooth variants. Of the spores produced at 23°C, 99% were rough, had a density of 1.305, and an average core/core + cortex volume ratio of 0.1838. At 49°C, 99% were smooth, had a density of 1.275, and an average volume ratio of 0.3098. Between these temperatures both spore types were produced. There appeared to be no direct correlation with sporulation temperature, heat resistance, and dipicolinate content. There was an increase in both the magnesium and calcium contents to 45°C with a dramatic reduction at 49°C. The 1.305 density spores had higher calcium and dipicolinate contents than the 1.275 spores, although both spore types showed biphasic thermal death curves. The mechanisms involved in determining which spore type (rough/smooth) is produced at a specific growth temperature is unknown.Florida Agricultural Experiment Station Journal Series Number R-00312.     

Thermal properties of NADP:ferredoxin oxidoreductase and ferredoxin isolated from a thermophilic blue-green alga

NADP:ferredoxin oxidoreductase (EC. 1.6.7.1.) isolated from a thermophilic blue-green alga, Synechococcus sp., was stable at temperatures up to 65°C. The diaphorase and cytochrome c reductase activities of the enzyme were low at 25°C but increased with elevated temperature to reach a maximum at about 60°C. The pH-profile of the diaphorase activity showed a peak at pH 9.0 at 55°C, whereas the activity was largely independent of pH at 25°C. High concentrations of NaCl suppressed activity at both high and low temperatures. In the cytochrome c reductase activity catalyzed by the enzyme, ferredoxin served as an electron carrier in a temperature-insensitive manner over a wide range of temperature. The results support the view that the optimum and the upper limiting temperatures for photosynthesis in this alga are related to thermal properties of proteins.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Effect of desiccation level and storage temperature on green spore viability of Osmunda japonica

In order to effectively preserve green spores, which have relatively higher water content and lose viability more quickly than non-green spores, we studied the effect of desiccation level and storage temperature on Osmunda japonica spores. The water content of fresh spores was 11.20%. After 12 h desiccation by silica gel, the water content decreased to 6% but spore viability did not change significantly. As the desiccation continued, the decrease in water content slowed, but spore viability dropped. For almost all storage periods, the effects of storage temperature, desiccation level, and temperature × desiccation level were significantly different. After seven days of storage, spores at any desiccation level stored at 4 °C obtained high germination rates. After more than seven days storage, liquid nitrogen (LN) storage obtained the best results. Storage at −18 °C led to the lowest germination rates. Spores stored at room temperature and −18 °C all died within three months. For storage at 4 °C and in LN, spores desiccated 12 and 36 h obtained better results. Spores without desiccation had the highest germination rates after being stored at room temperature, but suffered the greatest loss after storage at −18 °C. These results suggest that LN storage is the best method of long-term storage of O. japonica spores. The critical water content of O. japonica spores is about 6% and reduction of the water content to this level improves outcome after LN storage greatly. The reason for various responses of O. japonica spores to desiccation and storage temperatures are discussed.     

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.     

Studies on the rust,Maravalia cryptostegiae,a potential biological control agent of rubber-vine weed,Cryptostegia grandiflora (Asclepiadaceae: Periplocoideae), in Australia,II: Infection

The parameters which govern infection of rubber-vine weed by the rustMaravalia cryptostegiae were investigated. The infection process, from appressorial formation to sporulation, is described and illustrated. Uredinioid teliospores have an optimum temperature range for germination at 22–27 °C, both in vitro and in vivo. However, germination on the rubber-vine leaf was more than double (81–92%) that in the absence of the host, and appressoria were formed only in vivo. An optimum temperature of 20–22°C and a dew period of 12 hours or more gave the highest level of infection as measured by sporulation density. The latent period from inoculation to pustule formation decreased with increasing temperature; the shortest period (8–11 days) being recorded at 25–27°C. At the lower temperatures (18°C), this was significantly extended (19–21 days). Four successive inoculations significantly reduced plant height and dry weight, although a compensatory growth flush occurred after the third inoculation. The addition of cryoprotectants had a negative affect on spore viability and subsequent infectivity. Cooling dry spores to –196°C at the rate of 10°C min^–1 gave the best results, with high germination (93–65%) up to 8 days after thawing.     

Effects of Temperature on Activation, Germination, and Outgrowth of Bacillus megaterium Spores

The effects of temperature on the activation, glucose-induced germination, and outgrowth of Bacillus megaterium QM B1551 spores were investigated. There was no evidence for discontinuities in the response of spores to temperature in these processes reflecting reported thermal anomalies in the physical structure of water. Increasing the temperature of heat activation (aqueous suspensions, 5 min) increased the germinability of spores. Activation, as measured by extent of germination, was optimal after heating at 62 to 78 C, and the rate of spore germination was maximal after heat activation at 64 to 68 C. Increasing the temperature of activation above 68 C depressed the germination rate and increased the time lag before this rate was reached. Germination occurred over a wide range of temperatures, but was optimal between 28 and 38 C. The highest rate of germination was at 38 C; at lower incubation temperatures, the maximum attained rate was lower and the lag in attaining this rate was extended. Outgrowth (postgerminative development through the first cell division) of the germinated spores in Brain Heart Infusion (BHI) occurred in at least two phases-a temperature-dependent lag phase followed by a relatively temperature-independent phase of maximum outgrowth rate, during which increase in optical density was a linear function of time. Outgrowth time (time required for doubling of the initial optical density), essentially dependent on the time for completion of the lag phase, was shortest at temperatures between 34 and 40 C. The temperature-dependent lag phase was completed in a rich medium (e.g., BHI) but not in the glucose germination medium, suggesting that the endogenous reserves of the germinated spore were inadequate to support the metabolic synthetic events occurring during this period.     

Activation of Bacillus spores at moderately elevated temperatures (30–33 °C)

The time/temperature profiles experienced by spores on the track from their natural sporulation environment to consumable food products may be highly diverse. Temperature has been documented as an important factor that may activate spores, i.e. potentiates spores to germinate. There is, however, limited knowledge about the relationship between the expected temperature history and the subsequent germination characteristics of bacterial spores. We show here that the germination rate of five different Bacillus spore populations, represented by strains of Bacillus cereus, Bacillus weihenstephanensis, Bacillus pumilus, Bacillus licheniformis and Bacillus subtilis could be increased following 1 week storage at moderately elevated temperatures, 30–33 °C, compared to spores stored at 3–8 °C. The results imply that spores contamination routes to foods, specifically the temperature history, could be highly relevant data in predictive modeling of food spoilage and safety. Activation at these moderately elevated temperatures may be a native form of spore activation in their natural habitats, knowledge that also could be useful in development of decontamination strategies for mildly heated foods.     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

Spore Germination in Two Tropical Mosses: Fissidenssp. and Racopilum sp

Optimum germination of spores of a Fissidens species occurredat a relatively higher temperature (30 °C) than the optimumgermination of those of a Racopilum species (25 °C). Subsequentprotonemal growth of the two mosses also showed the same differentialtemperature optima. The high-temperature requirement for germinationof Fissidens spores happens to coincide with the maturationand dispersal of the spores in the dry season, and apparentlyfavours the establishment of new shoots. Fissidens sp, Racopilum sp, tropical mosses, spore germination, temperature adaptation     

Effects of temperature on microbial utilization of lignocellulosic detritus in a thermally impacted stream

The effects of temperature on rates of mineralization of [¹⁴C]lignocellulose were investigated in water and sediment from a thermally impacted stream and from a nearby unimpacted swamp at the Savannah River Plant, South Carolina. The temperature optimum for lignocellulose mineralization remained near 35°C at the unimpacted site throughout the sampling period from November 1986 to May 1987. The temperature optimum for lignocellulose mineralization in the thermally impacted stream was near 45°C when thermal effluents from a nuclear reactor were released to the stream, and was near 35°C when the reactor was not operating. Microbial populations capable of rapidly degrading lignocellulose at higher temperatures (45–55°C) developed between 9 and 27 days under conditions of thermal stress, indicating that under favorable conditions thermophilic microorganisms became dominant components of the microbiota. Removal of thermal stress for periods of 75 days or less resulted in a collapse of the thermophilic degrading population.     

Factors affecting spore germination in Aspergillus niger

Temperature markedly affected germination and germ tube length of A. niger. More than 90% of the spores were germinated in the range 30°–34 °C and formed maximum length of germ tubes. At temperatures from 38° to 43 °C, the proportion of the spores that germinated as well as the germ tube length were both gradually decreased. However, at 47°C germ tube formation was completely inhibited up to 15 hrs. after inoculation.High relative humidity was found necessary for the spore germination of A. niger. Germination failed to occur at 76% relative humidity. At 78 and 81% relative humidity germination was detected 15 hrs. after inoculation while at the higher humidities germination was started after 6 hrs. only.Conidiospores of A. niger were very sensitive to changes in the hydrogen ion concentration, pH. Complete inhibition of germination was found at pH less than 3.5. The germination and the length of the formed germ tubes increased with pH to reach their maximum rates at pH 4.5.     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus

The influence of sporulation temperature (20, 30 and 37 °C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 °C were the most heat-resistant. However, spores sporulated at 20 °C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 °C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 °C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 °C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 °C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 °C, respectively.     

Temperature-pH effect upon germination of bacterial spores.

Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures. The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C. A possible mechanism involved in this phenomenon is discussed.