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.     

Levels of Germination Proteins in Bacillus subtilis Dormant,Superdormant, and Germinating Spores

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.     

Increasing Activity of Germinating Bacillus subtilis Spores to Incorporate Thymidine Triphosphate into Deoxyribonucleic Acid After Detergent Treatment

The incorporation of (3)H-labeled thymidine triphosphate ((3)H-dTTP) into deoxyribonucleic acid (DNA) of germinated and then Brij 58-treated Bacillus subtilis spores was measured to study DNA replication activity of cells. The dTTP incorporation rate was very low in dormant spores, gradually increased as germination proceeded, and reached a level of the vegetative cell activity approximately 4 hr after the start of germination. This is in contrast to the DNA polymerase activity in the cell extract which remained at the same level throughout the germination period. The increase of the dTTP incorporation activity was inhibited by chloramphenicol or phenethyl alcohol. When these inhibitors were added after germination had proceeded, the elevated dTTP incorporation activity gradually decreased. Permeability to dTTP of spores germinated in the presence of chloramphenicol and then treated with Brij 58 was confirmed by (i) (3)H-dTTP incorporation into the treated spores following either electron or ultraviolet irradiation and (ii) release of radioactivity from the treated spores containing radioactively labeled DNA after deoxyribonuclease I treatment.     

Environmental Persistence of Bacillus anthracis and Bacillus subtilis Spores

There is a lack of data for how the viability of biological agents may degrade over time in different environments. In this study, experiments were conducted to determine the persistence of Bacillus anthracis and Bacillus subtilis spores on outdoor materials with and without exposure to simulated sunlight, using ultraviolet (UV)-A/B radiation. Spores were inoculated onto glass, wood, concrete, and topsoil and recovered after periods of 2, 14, 28, and 56 days. Recovery and inactivation kinetics for the two species were assessed for each surface material and UV exposure condition. Results suggest that with exposure to UV, decay of spore viability for both Bacillus species occurs in two phases, with an initial rapid decay, followed by a slower inactivation period. The exception was with topsoil, in which there was minimal loss of spore viability in soil over 56 days, with or without UV exposure. The greatest loss in viable spore recovery occurred on glass with UV exposure, with nearly a four log₁₀ reduction after just two days. In most cases, B. subtilis had a slower rate of decay than B. anthracis, although less B. subtilis was recovered initially.     

Completed Chromosomes in Thymine-Requiring Bacillus subtilis Spores

Origin:terminus genetic marker ratios (both purA: metB and purA:ilvA) were measured in extracts of spores of Bacillus subtilis strains W23 thy his and 168 thy. For strain W23 thy his, normalized to W23 spore deoxyribonucleic acid, both ratios were equal to unity and were consistent with the presence of only completed chromosomes in the spores. The same ratios in extracts of spores of 168 thy, normalized to strain 168 or the prototroph SB19, were abnormal, i.e., 2.26 +/- 0.10 and 0.71 +/- 0.06 for purA:metB and purA:ilvA, respectively. These values were unaffected by the extent of extraction of the spore deoxyribonucleic acid, the richness of the medium on which they are formed, and the thymine phenotype. The high ratio for purA:metB is in agreement with the results of earlier workers but, because of the low purA:ilvA ratio, cannot be explained simply by the presence of partially replicated chromosomes in spores of strain 168 thy. Furthermore, purA:leuA in such extracts is 1.01 +/- 0.06, consistent with the presence of only completed chromosomes. It is concluded that the abnormal origin:terminus marker ratios are only apparent and result from non-isogenicity between strains 168 thy and 168 in the metB thyB ilvA chromosome region introduced during construction of 168 thy by transformation of strain 168 with W23 thy deoxyribonucleic acid. It is concluded further that the chromosomes of strain 168 thy spores are in a completed form.     

Viability of Bacillus subtilis Spores in Rocket Propellants

The sporicidal activity of components used in liquid and solid rocket propellants was tested by use of spores of Bacillus subtilis dried on powdered glass. Liquid propellant ingredients tested were N₂O₄, monomethylhydrazine and 1,1-dimethylhydrazine. N₂O₄ was immediately sporicidal; the hydrazines were effective within several days. Solid propellants consisted of ammonium perchlorate in combination with epoxy resin (EPON 828), tris-1-(2-methyl) aziridinyl phosphine oxide, bis-1-(2-methyl) aziridinyl phenylphosphine oxide, and three modified polybutadiene polymers. There was no indication of appreciable sporicidal activity of these components.     

Resistance and Structure of Spores of Bacillus subtilis

Spores of Bacillus subtilis NCDO 2130 were produced on five different media and their structure and resistance to chemicals, to heat and to ultraviolet (uv) irradiation compared. Resistance to one chemical was not necessarily related to resistance to another, to uv irradiation or to heat. Spores with the smallest protoplasts and largest cortices were most resistant to heat, while the least resistant were those in which the protoplasts became electron dense after staining and which had the lowest concentrations of calcium and dipicolinic acid.     

Initiation of Deoxyribonucleic Acid Replication in Germinating Spores of Bacillus subtilis 168 Carrying the dnaB(Ts)134 Mutation

The nature of the deoxyribonucleic acid synthesis reported by others to occur at 45 degrees C in germinating spores of the temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of B. subtilis 168, one carrying and the other without the dnaB (Ts)134 mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 45 degrees C. At a slightly higher temperature, 49 degrees C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts(+) strain. Thus, it is concluded that, after the germination of a spore, the action of the dnaB134 gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 45 degrees C is presumably due to incomplete inactivation of the dnaB134 gene product under such conditions.     

Peptide Synthesis by Extracts from Bacillus subtilis Spores

Cell-free peptide synthesis by extracts from vegetative cells and spores of Bacillus subtilis was analyzed and compared. The initial rate of phenylalanine incorporation in a polyuridylate-directed system was found to be in a similar range for the two extracts. However, spore extracts frequently incorporated less total phenylalanine as did the vegetative cell system. Optimal conditions for amino acid incorporation by spore extracts were found to be similar to those of vegetative cell extracts. Polyphenylalanine synthesis was stimulated by preincubation of both extracts prior to the addition of polyuridylic acid (poly U) and labeled phenylalanine. Both systems showed a dependence on an energy-generating system and were inhibited by chloramphenicol and puromycin. Ribonuclease, but not deoxyribonuclease, inhibited the reaction significantly. The presence of methionine transfer ribonucleic acid (tRNA(F)) and methionyl-tRNA(F) transformylase was demonstrated in spore extracts. An analysis of several aminoacyl-tRNAs in spores revealed that the relative amounts of these tRNAs were similar to those found in vegetative cells. Only lysine tRNA was found to be present in relatively greater amounts in spores. These results indicate that dormant spores of B. subtilis contain the machinery for the translation of genetic information.     

Effect of Monosodium n-Alkylsalicylates on Spores of Bacillus subtilis

The effects of several monosodium n-alkylsalicylates on washed spores of Bacillus subtilis have been examined. None of the compounds was sporicidal, but in general they prevented dormancy induced by incubating the spores in water. This effect was related to the position in the ring and size of the alkyl groups substituted on the salicylic acid nucleus.     

Messenger Ribonucleic Acid of Dormant Spores of Bacillus subtilis

Evidence of the presence of messenger ribonucleic acid (mRNA) in dormant spores of Bacillus subtilis has been obtained. The bulk RNA from spores was isolated and labeled in vitro with tritiated dimethyl sulfate. The spore RNA hybridized to 2.4 to 3.2% of the B. subtilis genome. The RNA hybridized to both the complementary heavy and light fractions of deoxyribonucleic acid (DNA). Bulk RNA from log-phase cells competed with virtually all the spore RNA for the heavy DNA fraction and with part of the spore RNA for the light DNA fraction. Bulk RNA from stage IV cells in sporulation also competed with all of the spore RNA for the heavy DNA fraction and with essentially all the spore RNA for the light DNA fraction. These results indicate that dormant spores contain mRNA species present in both log-phase cells and stage IV cells of sporulation. The RNA polymerase in the developing forespore must be able to recognize promotor sites for both log-phase and sporulation genes.     

The X-ray Diffraction Pattern of Spores of Bacillus subtilis

ORD and CD of d(+) pantothenic acid, d(+) pantothenyl alcohol and d(?) pantolactone were studied. The acid and the alcohol gave positive Cotton effects with a peak at 227 and 225 mμ, respectively, and the lactone gave a negative Cotton effect with trough at 233 mμ. They gave CD maxima at 214, 213 and 219 mµ corresponding to the inflection points of their ORD curves.

The concentrations were found to be linear to the rotation angles and the possibility of the application to quantitative analysis of the ORD was cited. The ORD showed the quantitative formation of the lactone by acid treatment of the vitamins without any racemization and hence the determination via lactone was suggested.     

Heat Injury of Bacillus subtilis Spores at Ultrahigh Temperatures

The following three criteria indicated that Bacillus subtilis A spores were injured, but not completely inactivated, by ultrahigh temperature treatment. (i) Significant reductions in survivors were observed when spores were enumerated with a standard medium but not when the medium contained added CaCl(2) and sodium dipicolinate. (ii) After a damaging heat treatment, more survivors were enumerated with the standard medium after incubation at 32 C than at 45 C, which was opposite to the result with untreated or slightly heated spores. (iii) Apparent numbers of survivors increased during the initial period of 3 C storage when enumerated with the standard medium at 45 C. No injury was evident when survivors were enumerated at either incubation temperature with the medium containing added CaCl(2) and sodium dipicolinate. Heat activation of the spores did not significantly influence the appearance of heat injury. The data suggested that the heat injury occurred in a germination system which was required in the absence of CaCl(2) and sodium dipicolinate.     

Bacillus subtilis Spores Germinate in the Chicken Gastrointestinal Tract

A number of poultry probiotics contain bacterial spores. In this study, orally administered spores of Bacillus subtilis germinated in the gastrointestinal (GI) tracts of chicks. Furthermore, 20 h after spores were administered, vegetative cells outnumbered spores throughout the GI tract. This demonstrates that spore-based probiotics may function in this host through metabolically active mechanisms.     

Sensitivities of Germinating Spores and Carvacrol-Adapted Vegetative Cells and Spores of Bacillus cereus to Nisin and Pulsed-Electric-Field Treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

Cold shock response in Bacillus subtilis

Following a rapid decrease in temperature, the physiology of Bacillus subtilis cells changes profoundly. Cold shock adaptation has been monitored at the level of membrane composition, adjustment in DNA topology, and change in cytosolic protein synthesis/composition. Some major players in these processes (cold-stress induced proteins and cold acclimatization proteins, CIPs and CAPs) have been identified and mechanisms in cold shock acclimatization begin to emerge; however, important questions regarding their cellular function still need to be answered.     

Thermal Inactivation Characteristics of Bacillus subtilis Spores at Ultrahigh Temperatures

The thermal inactivation characteristics of Bacillus subtilis A spores suspended in skim milk with the use of large-scale ultrahigh temperature (UHT) processing equipment were investigated in terms of survival as measured with two plating media. Data on survival immediately after UHT treatments were recorded in temperature-survivor curves, time-survivor curves, and decimal reduction time (DRT) curves. The temperature-survivor curves emphasized that inactivation is accelerated more by increases in the treatment temperature than by increases in the exposure time. Time-survivor curves and DRT curves were not linear. Generally, exceedingly concave time-survivor curves were observed with the standard plating medium; however, only slightly concave curves were observed when CaCl(2) and sodium dipicolinate were added to the medium. For a given UHT sample, larger D values were obtained by use of the medium with the added CaCl(2) and sodium dipicolinate. The DRT curves of all data were concave and appeared to have two discrete slopes (z(D) values). The z(D) values observed in the upper UHT range (above 260 F; 127 C) were twice those observed at lower test temperatures.