Study of heat resistance in a bacterial nucleosidase

The enzyme nucleosidase (EC. 3.2.2.1.) is present in the intact spores, germinated spores as well as vegetative cells of $\text{Bacillus cereus}$ T. In the intact spores the enzymeis resistant to heat and, in fact, has a high temperature optimum. Though the spores themselvesbecome sensitive to heat on germination, the enzyme retains its resistance to heat on germination as well as its high temperature optimum. The vegetative cell enzyme is sensitive to heat. The enzyme in all types of cells &; spores is resistant to octyl alcohol. There is a close correlation between the development of heat resistance in the sporulating cells and that of heat resistance of the enzyme.     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

Cold shock response in sporulating Bacillus subtilis and its effect on spore heat resistance

Cold shock and ethanol and puromycin stress responses in sporulating Bacillus subtilis cells have been investigated. We show that a total of 13 proteins are strongly induced after a short cold shock treatment of sporulating cells. The cold shock pretreatment affected the heat resistance of the spores formed subsequently, with spores heat killed at 85 or 90 degrees C being more heat resistant than the control spores while they were more heat sensitive than controls that were heat treated at 95 or 100 degrees C. However, B. subtilis spores with mutations in the main cold shock proteins, CspB, -C, and -D, did not display decreased heat resistance compared to controls, indicating that these proteins are not directly responsible for the increased heat resistance of the spores. The disappearance of the stress proteins later in sporulation suggests that they cannot be involved in repairing heat damage during spore germination and outgrowth but must alter spore structure in a way which increases or decreases heat resistance. Since heat, ethanol, and puromycin stress produce similar proteins and similar changes in spore heat resistance while cold shock is different in both respects, these alterations appear to be very specific.     

Heat and UV light resistance of vegetative cells and spores of Bacillus subtilis Rec-mutants.

The heat and UV light resistance of spores and vegetative cells of Bacillus subtilis BD170 (rec+) were greater than those of B. subtilis BD224 (recE4). Strain BD170 can repair DNA whereas BD224 is repair deficient due to the presence of the recE4 allele. Spores of a GSY Rec+ strain were more heat resistant than spores of GSY Rec- and Uvr- mutants. The overall level of heat and UV light resistance attained by spores may in part be determined by their ability to repair deoxyribonucleic acid after exposure to these two physical mutagens.     

Effect of intracellular carbohydrates on heat resistance of Dictyostelium discoideum spores.

The effect of intracellular trehalose and glycogen on the survival of spores of Dictyostelium discoideum ATCC 25697 after exposure to supraoptimal temperatures was examined. Cells metabolically perturbed by incubation in glucose and inorganic phosphate have intracellular trehalose and glycogen concentrations fivefold and twofold higher, respectively, than those of the controls. These cells were more resistant to the lethal effects of wet heat (45 degrees to 55 degrees C) than were control cells. The presence of 40 mM trehalose in the buffer during heat stress increased the survival of nonperturbed cells to approximately the level of the perturbed cells. No protection was observed when cells were heated in the presence of exogenous glycogen. Glucose or disaccharides other than trehalose when present during heat stress, had no effect on heat resistance. Nonperturbed cells preincubated in 40 mM trehalose and washed before heat stress were more resistant to killing than were controls. Cells perturbed with inorganic phosphate, which has been shown to increase trehalose concentrations but decrease glycogen concentrations, were also more resistant to the lethal effects of wet heat than were controls. The data suggest that trehalose has an effect on the wet-heat resistance of D. discoideum. Some possible mechanisms are suggested.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

Repair of Radiation Damage to Deoxyribonucleic Acid in Germinating Spores of Bacillus subtilis

The repair of deoxyribonucleic acid (DNA) in germinating spores was studied in comparison with that in vegetative cells. Radiation-induced single-strand breaks in the DNA of spores and of vegetative cells of Bacillus subtilis were rejoined during postirradiation incubation. The molecular weight of single-stranded DNA was restored to the level of nonirradiated cells. The rate of the rejoining of DNA strand breaks in irradiated spores was essentially equal to that in irradiated vegetative cells. The rejoining in spores germinating in nutrient medium occurred in the absence of detectable DNA synthesis. In this state, normal DNA synthesis was not initiated. Very little DNA degradation occurred during the rejoining process. On the other hand, in vegetative cells the rejoining process was accompanied by a relatively large amount of DNA synthesis and DNA degradation in nutrient medium. The rejoining occurred in phosphate buffer in vegetative cells but not in spores in which germination was not induced. Chloramphenicol did not interfere with the rejoining process in either germinating spores or vegetative cells, indicating that the rejoining takes place in the absence of de novo synthesis of repair enzyme. In the radiation-sensitive strain uvs-80, the capacity for rejoining radiation-induced strand breaks was reduced both in spores and in vegetative cells, suggesting that the rejoining mechanism of germinating spores is not specific to the germination process.     

Sensitivity of Saccharomyces cerevisiae vegetative cells and spores to antimicrobial compounds

The sensitivity of Saccharomyces cerevisiae spores and vegetative cells to various antimicrobial compounds was compared. Sulphur dioxide, benzoic acid, potassium sorbate, salicylic acid, nystatin, actidione and pimaricin were tested. Generally, the Saccharomyces spores were more resistant than the corresponding vegetative cells. It was also observed that this greater resistance shown by the spores varied with the antimicrobial compound used. Only potassium sorbate was not selective and killed both vegetative cells and spores at about the same rate.     

Sensitivity of Saccharomyces cerevisiae vegetative cells and spores to antimicrobial compounds

The sensitivity of Saccharomyces cerevisiae spores and vegetative cells to various antimicrobial compounds was compared. Sulphur dioxide, benzoic acid, potassium sorbate, salicylic acid, nystatin, actidione and pimaricin were tested. Generally, the Saccharomyces spores were more resistant than the corresponding vegetative cells. It was also observed that this greater resistance shown by the spores varied with the antimicrobial compound used. Only potassium sorbate was not selective and killed both vegetative cells and spores at about the same rate.     

产乳酸凝结芽胞杆菌N001的抗逆性研究

目的研究前期经初筛的产乳酸凝结芽胞杆菌N001芽胞的抗逆性。方法在模拟饲料制粒条件下和动物消化道内逆境条件下的存活能力,测定N001芽胞的抗热、抗酸、耐胆盐性能和对抗生素的敏感性。结果凝结芽胞杆菌N001芽胞具有很强的耐高温、耐酸、耐胆盐能力;同时N001芽胞对营养体敏感的抗生素也有良好的耐受性。结论凝结芽胞杆菌N001芽胞具有很强的抗逆性,可以作为益生菌制剂的良好菌种。     

Comparative Effects of Osmotic, Sodium Nitrite-Induced, and pH-Induced Stress on Growth and Survival of Clostridium perfringens Type A Isolates Carrying Chromosomal or Plasmid-Borne Enterotoxin Genes

About 1 to 2% of Clostridium perfringens isolates carry the enterotoxin gene (cpe) necessary for causing C. perfringens type A food poisoning. While the cpe gene can be either chromosomal or plasmid borne, food poisoning isolates usually carry a chromosomal cpe gene. Previous studies have linked this association between chromosomal cpe isolates (i.e., C-cpe isolates) and food poisoning, at least in part, to both the spores and vegetative cells of C-cpe isolates being particularly resistant to high and low temperatures. The current study now reveals that the resistance phenotype of C-cpe isolates extends beyond temperature resistance to also include, for both vegetative cells and spores, enhanced resistance to osmotic stress (from NaCl) and nitrites. However, by omitting one outlier isolate, no significant differences in pH sensitivity were detected between the spores or vegetative cells of C-cpe isolates versus isolates carrying a plasmid-borne cpe gene. These results indicate that both vegetative cells and spores of C-cpe isolates are unusually resistant to several food preservation approaches in addition to temperature extremes. The broad-spectrum nature of the C-cpe resistance phenotype suggests these bacteria may employ multiple mechanisms to persist and grow in foods prior to their transmission to humans.     

Photodynamic inactivation of Bacillus spores, mediated by phenothiazinium dyes

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.     

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.     

Action of egg white lysozyme on Clostridium tyrobutyricum.

A 500-U ml-1 portion of egg white lysozyme was able to kill 99% of 5 X 10(5) resting vegetative cells of Clostridium tyrobutyricum within 24 h of incubation at 25 degrees C. Spores were completely resistant to lysozyme. Proliferating vegetative cells were severely inhibited, although lysozyme-resistant cells developed in growing cultures in the presence of lysozyme. Whereas early stages of spore germination (loss of optical refractility and heat resistance) were not inhibited by lysozyme, the overall outgrowth of spore cells into vegetative cells was delayed by 1 day in the presence of 500 U of lysosyme ml-1. This delay was independent of the lysozyme sensitivity or resistance of the mother culture of the used spores. It is suggested that this inhibition by lysozyme of the outgrowth of spore cells into vegetative cells of the lactate-fermenting C. tyrobutyricum is the basis for the observation that lysozyme can substitute for nitrate in preventing the "late gas" defect of Edam- and Gouda-type cheeses.     

Action of egg white lysozyme on Clostridium tyrobutyricum

A 500-U ml-1 portion of egg white lysozyme was able to kill 99% of 5 X 10(5) resting vegetative cells of Clostridium tyrobutyricum within 24 h of incubation at 25 degrees C. Spores were completely resistant to lysozyme. Proliferating vegetative cells were severely inhibited, although lysozyme-resistant cells developed in growing cultures in the presence of lysozyme. Whereas early stages of spore germination (loss of optical refractility and heat resistance) were not inhibited by lysozyme, the overall outgrowth of spore cells into vegetative cells was delayed by 1 day in the presence of 500 U of lysosyme ml-1. This delay was independent of the lysozyme sensitivity or resistance of the mother culture of the used spores. It is suggested that this inhibition by lysozyme of the outgrowth of spore cells into vegetative cells of the lactate-fermenting C. tyrobutyricum is the basis for the observation that lysozyme can substitute for nitrate in preventing the "late gas" defect of Edam- and Gouda-type cheeses.     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores.

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bile salts and glycine as cogerminants for Clostridium difficile spores

Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.     

Growth of Paenibacillus larvae,the causative agent of American foulbrood in honey bees and Bacillus popilliae,the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

TNM-FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild-type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM-FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX-CELL 405 serum-free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology-forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild-type strain of Bacillus popilliae, vegetative cells grew only in TNM-FH medium in the presence of mid-gut BTI-Tn-MG and ovarian (Tn-368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI-Tn-MG and Tn-368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI-Tn-MG cell line in TNM-FH medium produced as many as 12,000 spores/ml after 21 days in culture.     

Resistance of Vegetative Cells and Microcysts of Myxococcus xanthus

The resistance of vegetative cells and of microcysts of Myxococcus xanthus to several destructive agents was compared. Fruiting-body microcysts were 300 times more resistant to 60 C, 5.4 times more resistant to ultraviolet light, and 19.3 times more resistant to sonic vibration than were vegetative cells. Whereas resistance to sonic vibration developed during the conversion of rods to refractile spheres, resistance to heat did not appear until after the conversion was complete. Both vegetative cells and microcysts of the yellow variant of this strain were more resistant to ultraviolet irradiation than was the tan variant.