Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Relationship between Bacillus sphaericus spore heat resistance and sporulation temperature

Bacillus sphaericus 9602 was grown in batch culture at various temperatures. At 10°C and 12°C the maximum sporulation yield was <10%, while at 15°C, 20°C and 30°C, a sporulation yield of >95% was achieved. However at 40°C B. sphaericus grew only vegetatively. The heat resistances (D values at 90°C) of spores grown at 15°C and 20°C were significantly higher than those grown at 30°C.     

Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation

The first ～10% of spores released from sporangia (early spores) during Bacillus subtilis sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ～24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca(2+) but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.     

Role of DNA repair in Bacillus subtilis spore resistance.

Wet-heat or hydrogen peroxide treatment of wild-type Bacillus subtilis spores did not result in induction of lacZ fusions to three DNA repair-related genes (dinR, recA, and uvrC) during spore outgrowth. However, these genes were induced during outgrowth of wild-type spores treated with dry heat or UV. Wet-heat, desiccation, dry-heat, or UV treatment of spores lacking major DNA-binding proteins (termed alpha-beta- spores) also resulted in induction of the three DNA repair genes during spore outgrowth. Hydrogen peroxide treatment of alpha-beta-spores did not result in induction of dinR- and rerA-lacZ but did cause induction of uvrC-lacZ during spore outgrowth. Spores of a recA mutant were approximately twofold more UV sensitive and approximately ninefold more sensitive to dry heat than were wild-type spores but were no more sensitive to wet heat and hydrogen peroxide. In contrast, alpha-beta- recA spores were significantly more sensitive than were alpha-beta- spores to all four treatments, as well as to desiccation. Surprisingly, RecA levels were quite low in dormant spores, but RecA was synthesized during spore outgrowth. Taken together, these data (i) are consistent with previous suggestions that some treatments (dry heat and UV with wild-type spores; desiccation, dry and wet heat, hydrogen peroxide, and UV with alpha-beta- spores) that kill spores do so in large part by causing DNA damage and (ii) indicate that repair of DNA damage during spore outgrowth is an important component of spore resistance to a number of treatments, as has been shown previously for UV.     

Effects of phosphorelay perturbations on architecture, sporulation, and spore resistance in biofilms of Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is able to form highly organized multicellular communities called biofilms. This coordinated bacterial behavior is often lost in domesticated or laboratory strains as a result of planktonic growth in rich media for many generations. However, we show here that the laboratory strain B. subtilis 168 is still capable of forming spatially organized multicellular communities on minimal medium agar plates, exemplified by colonies with vein-like structures formed by elevated bundles of cells. In line with the current model for biofilm formation, we demonstrate that overproduction of the phosphorelay components KinA and Spo0A stimulates bundle formation, while overproduction of the transition state regulators AbrB and SinR leads to repression of formation of elevated bundles. Time-lapse fluorescence microscopy studies of B. subtilis green fluorescent protein reporter strains show that bundles are preferential sites for spore formation and that flat structures surrounding the bundles contain vegetative cells. The elevated bundle structures are formed prior to sporulation, in agreement with a genetic developmental program in which these processes are sequentially activated. Perturbations of the phosphorelay by disruption and overexpression of genes that lead to an increased tendency to sporulate result in the segregation of sporulation mutations and decreased heat resistance of spores in biofilms. These results stress the importance of a balanced control of the phosphorelay for biofilm and spore development.     

Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.     

Binding of small, acid-soluble spore proteins to DNA plays a significant role in the resistance of Bacillus subtilis spores to hydrogen peroxide.

Dormant spores of Bacillus subtilis which lack the majority of the alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha- beta- spores) that coat the DNA in wild-type spores are significantly more sensitive to hydrogen peroxide than are wild-type spores. Hydrogen peroxide treatment of alpha- beta- spores causes DNA strand breaks more readily than does comparable treatment of wild-type spores, and alpha- beta- spores, but not wild-type spores, which survive hydrogen peroxide treatment have acquired a significant number of mutations. The hydrogen peroxide resistance of wild-type spores appears to be acquired in at least two incremental steps during sporulation. The first increment is acquired at about the time of alpha/beta-type SASP synthesis, and the second increment is acquired approximately 2 h later, at about the time of dipicolinic acid accumulation. During sporulation of the alpha- beta- strain, only the second increment of hydrogen peroxide resistance is acquired. In contrast, sporulation mutants which accumulate alpha/beta-type SASP but progress no further in sporulation acquire only the first increment of hydrogen peroxide resistance. These findings strongly suggest that binding of alpha/beta-type SASP to DNA provides one increment of spore hydrogen peroxide resistance. Indeed, binding of alpha/beta-type SASP to DNA in vitro provides strong protection against cleavage of DNA by hydrogen peroxide.     

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.     

spoVIC, a new sporulation locus in Bacillus subtilis affecting spore coats, germination and the rate of sporulation

A mutation near cysB on the Bacillus subtilis chromosome marks a new sporulation locus, spoVIC. It causes spores to germinate more slowly than those of the wild-type under all conditions and, from indirect evidence, it does not appear to alter the affinity for the germinant L-alanine. The mutant spores have some deficiency of coat proteins (particularly the alkalisoluble coat protein, Mr = 12 000) and the spore coat layers are disorganized. The mutant strain grows normally and sporulates normally until stage II, after which its sporulation is delayed by about 2 h compared to that of the wild-type. This delay results in the prolonged synthesis of some coat proteins and the late synthesis of others. The abnormal coat may be the cause of the germination deficiency. A double mutant strain carrying the spoVIC610 mutation together with gerE36 sporulates slowly. Its spores have very little coat protein, are sensitive to heat, lysozyme and organic solvents, but germinate as well as the strain carrying the spoVIC mutation alone. The role of the spore coat in germination is discussed in the light of these findings.     

Emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168

Abstract The emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168 was examined. Resistance to an organic solvent (toluene), heat and lysozyme were included for comparison. A sequential development of resistance was observed, with toluene resistance occuring early on in sporulation (stages III and IV), thermal resistance at early stage V, lysozyme resistance at middle stage V and glutaraldehyde resistance arising late in stage V. Studies with sporulation mutants also indicate that glutaraldehyde resistance is acquired even later than lysozyme resistance and may therefore possibly be considered as a very late marker event for sporulation, characterizing late stages of B. subtilis 168 spore formation.     

Development of resistance to biocides during sporulation of Bacillus subtilis

From synchronized sporulation and spore mutant studies, the order of development of resistance to biocides during sporulation of Bacillus subtilis strain 168 was toluene, formaldehyde, sodium lauryl sulphate, phenol, phenylmercuric nitrate, m -cresol, chlorocresol, chlorhexidine gluconate, cetylpyridinium chloride, moist heat, sodium dichlorisocyanurate, sodium hypochlorite, lysozyme and glutaraldehyde. These resistances could be assigned to different stages in spore development.     

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.     

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

The influence of different sporulation temperatures (30, 37, 44 and 52 degrees 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 degrees C. Sporulation at 52 degrees C did not show any further increase in heat resistance. This effect was constant over all the range of heating temperatures tested (100-120 degrees C). z value remained constant (z = 9 degrees 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 degrees C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

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.     

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Summary Specitinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc1–11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1–11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.     

Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde

G orman , S.P. S cott , E.M. H utchinson , E.P. 1984. Interaction of the Bacillus subtilis spore protoplast, cortex, ion-exchange and coatless forms with glutaraldehyde. Journal of Applied Bacteriology 56 , 95–102.
Bacillus subtilis spores with altered ionic content were tested for their susceptibility to lysis with lysozyme or sodium nitrite following treatment with glutaraldehyde. The Ca-form was more sensitive to glutaraldehyde (pH 4.0.and pH 7.9) than the untreated or H-form. Removal of spore coat dramatically increased sensitivity of the spore to glutaraldehyde. Pretreatment of spores, the coats of which had been extensively removed, with glutaraldehyde (pH 7.9) reduced the rate of lysis by lysozyme and by sodium nitrite, whereas glutaraldehyde at pH 4.0.had little effect. Glutaraldehyde pretreatment (pH 4.0 and pH 7.9) reduced the amount of hexosamine released by lysozyme but not by nitrite from isolated cortical fragments. Spore protoplasts were more susceptible to 0.01% (w/v) glutaraldehyde at pH 4.0 and isolated spore coats adsorbed alkaline glutaraldehyde more rapidly. These results are discussed in terms of a possible mode of action of glutaraldehyde on the bacterial spore.     

Role of spore coat proteins in the resistance of Bacillus subtilis spores to Caenorhabditis elegans predation

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.