Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins.

Spores of a Bacillus subtilis strain with an insertion mutation in the dacB gene, which codes for an enzyme involved in spore cortex biosynthesis, have a higher core water content than wild-type spores. Spores lacking the two major alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha-beta- spores) have the same core water content as do wild-type spores, but alpha-beta- dacB spores had more core water than did dacB spores. The resistance of alpha-beta-, alpha-beta- dacB, dacB, and wild-type spores to dry and moist heat, hydrogen peroxide, and UV radiation has been determined, as has the role of DNA damage in spore killing by moist heat and hydrogen peroxide. These data (i) suggest that core water content has little if any role in spore UV resistance and are consistent with binding of alpha/beta-type SASP to DNA being the major mechanism providing protection to spores from UV radiation; (ii) suggest that binding of alpha/beta-type SASP to DNA is the major mechanism unique to spores providing protection from dry heat; (iii) suggest that spore resistance to moist heat and hydrogen peroxide is affected to a large degree by the core water content, as increased core water resulted in large decreases in spore resistance to these agents; and (iv) indicate that since this decreased resistance (i.e., in dacB spores) is not associated with increased spore killing by DNA damage, spore DNA must normally be extremely well protected against such damage, presumably by the saturation of spore DNA by alpha/beta-type SASP.     

Role of the Nfo (YqfS) and ExoA apurinic/apyrimidinic endonucleases in protecting Bacillus subtilis spores from DNA damage

The Bacillus subtilis enzymes ExoA and Nfo (originally termed YqfS) are endonucleases that can repair apurinic/apyrimidinic (AP) sites and strand breaks in DNA. We have analyzed how the lack of ExoA and Nfo affects the resistance of growing cells and dormant spores of B. subtilis to a variety of treatments, some of which generate AP sites and DNA strand breaks. The lack of ExoA and Nfo sensitized spores (termed alpha-beta-) lacking the majority of their DNA-protective alpha/beta-type small, acid-soluble spore proteins (SASP) to wet heat. However, the lack of these enzymes had no effect on the wet-heat resistance of spores that retained alpha/beta-type SASP. The lack of either ExoA or Nfo sensitized wild-type spores to dry heat, but loss of both proteins was necessary to sensitize alpha-beta- spores to dry heat. The lack of ExoA and Nfo also sensitized alpha-beta-, but not wild-type, spores to desiccation. In contrast, loss of ExoA and Nfo did not sensitize growing cells or wild-type or alpha-beta- spores to hydrogen peroxide or t-butylhydroperoxide. Loss of ExoA and Nfo also did not increase the spontaneous mutation frequency of growing cells. exoA expression took place not only in growing cells, but also in the forespore compartment of the sporulating cell. These results, together with those from previous work, suggest that ExoA and Nfo are additional factors that protect B. subtilis spores from DNA damage accumulated during spore dormancy.     

Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid

Treatment of wild-type spores of Bacillus subtilis with glutaraldehyde or an iodine-based disinfectant (Betadine) did not cause detectable mutagenesis, and spores (termed alpha-beta-) lacking the major DNA-protective alpha/beta-type, small, acid-soluble proteins (SASP) exhibited similar sensitivity to these agents. A recA mutation did not sensitize wild-type or alpha-beta- spores to Betadine or glutaraldehyde, nor did spore treatment with these agents result in significant expression of a recA-lacZ fusion when the treated spores germinated. Spore glutaraldehyde sensitivity was increased dramatically by removal of much spore coat protein, but this treatment had no effect on Betadine sensitivity. In contrast, nitrous acid treatment of wild-type and alpha-beta- spores caused significant mutagenesis, with alpha-beta- spores being much more sensitive to this agent. A recA mutation further sensitized both wild-type and alpha-beta- spores to nitrous acid, and there was significant expression of a recA-lacZ fusion when nitrous acid-treated spores germinated. These results indicate that: (a) nitrous acid kills B. subtilis spores at least in part by DNA damage, and alpha/beta-type SASP protect against this DNA damage; (b) killing of spores by glutaraldehyde or Betadine is not due to DNA damage; and (c) the spore coat protects spores against killing by glutaraldehyde but not Betadine. Further analysis also demonstrated that spores treated with nitrous acid still germinated normally, while those treated with glutaraldehyde or Betadine did not.     

Role of dipicolinic acid in resistance and stability of spores of Bacillus subtilis with or without DNA-protective alpha/beta-type small acid-soluble proteins

Dipicolinic acid (DPA) comprises approximately 10% of the dry weight of spores of Bacillus species. Although DPA has long been implicated in spore resistance to wet heat and spore stability, definitive evidence on the role of this abundant molecule in spore properties has generally been lacking. Bacillus subtilis strain FB122 (sleB spoVF) produced very stable spores that lacked DPA, and sporulation of this strain with DPA yielded spores with nearly normal DPA levels. DPA-replete and DPA-less FB122 spores had similar levels of the DNA protective alpha/beta-type small acid-soluble spore proteins (SASP), but the DPA-less spores lacked SASP-gamma. The DPA-less FB122 spores exhibited similar UV resistance to the DPA-replete spores but had lower resistance to wet heat, dry heat, hydrogen peroxide, and desiccation. Neither wet heat nor hydrogen peroxide killed the DPA-less spores by DNA damage, but desiccation did. The inability to synthesize both DPA and most alpha/beta-type SASP in strain PS3664 (sspA sspB sleB spoVF) resulted in spores that lost viability during sporulation, at least in part due to DNA damage. DPA-less PS3664 spores were more sensitive to wet heat than either DPA-less FB122 spores or DPA-replete PS3664 spores, and the latter also retained viability during sporulation. These and previous results indicate that, in addition to alpha/beta-type SASP, DPA also is extremely important in spore resistance and stability and, further, that DPA has some specific role(s) in protecting spore DNA from damage. Specific roles for DPA in protecting spore DNA against damage may well have been a major driving force for the spore's accumulation of the high levels of this small molecule.     

Sensitization of Bacillus subtilis spores to dry heat and desiccation by pretreatment with oxidizing agents

Aims: To determine if pretreatment with oxidizing agents sensitizes Bacillus subtilis spores to dry heat or desiccation. Methods: Bacillus subtilis spores were killed approx. 90% by oxidizing agents, and the sensitivity of treated and untreated spores to dry heat and desiccation was determined. The effects of pyruvate on spore recovery after oxidizing agent pretreatment and then dry heat or desiccation were also determined. Conclusions: Spores pretreated with Oxone? or hypochlorite were not sensitized to dry heat or freeze‐drying. However, hydrogen peroxide or t‐butylhydroperoxide pretreatment sensitized spores to dry heat or desiccation, and the desiccation caused mutagenesis in the survivors. Pyruvate increased recovery of spores treated with hydrogen peroxide alone or plus dry heat or desiccation, and with t‐butylhydroperoxide and desiccation, but not with t‐butylhydroperoxide alone or plus dry heat. Significance and Impact of the Study: Pretreatment with peroxides sensitizes bacterial spores to subsequent stress. This finding may suggest improved regimens for spore inactivation.     

Role of DNA repair by nonhomologous-end joining in Bacillus subtilis spore resistance to extreme dryness, mono- and polychromatic UV, and ionizing radiation

The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation, and ultrahigh vacuum was studied in wild-type and DNA repair mutants (recA, splB, ykoU, ykoV, and ykoU ykoV mutants) of Bacillus subtilis. NHEJ-defective spores with mutations in ykoU, ykoV, and ykoU ykoV were significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.     

Base-change mutations induced by various treatments of Bacillus subtilis spores with and without DNA protective small,acid-soluble spore proteins

Previous work has shown that spores of wild-type Bacillus subtilis are more resistant to killing by dry and wet heat, low vacuum lyophilization and hydrogen peroxide than are spores lacking the majority of their DNA protective alpha/beta-type small, acid-soluble spore proteins (SASP) (termed alpha(-)beta(-) spores). These four treatments kill alpha(-)beta(-) spores in large part by DNA damage with accompanying mutagenesis, but only dry heat kills wild-type spores by DNA damage and mutagenesis. DNA sequence analysis of nalidixic acid-resistant (nal(r)) mutants generated by these treatments has now shown that the nal(r) mutations are base changes in the gyrA gene that encodes one subunit of DNA gyrase. Analysis of the DNA sequence of the gyrA gene in a large number of nal(r) mutants also indicates that: (1) base changes induced by hydrogen peroxide and wet heat in alpha(-)beta(-) spores are similar to those in spontaneous nal(r) mutants with only a few notable differences; (2) base changes induced by dry heat in wild-type spores and low vacuum lyophilization of alpha(-)beta(-) spores are similar, and include a high level of a tandem base change seen previously only in spores treated with very high vacuum and (3) base changes induced by lyophilization and dry heat are very different from those in spontaneous mutants in wild-type and alpha(-)beta(-) spores, which exhibit only one significant difference. While the initial DNA damage generated in spores by dry heat, lyophilization or high vacuum is almost certainly different than that generated by hydrogen peroxide or wet heat, the precise nature of the DNA damage remains to be determined.     

Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone. METHODS AND RESULTS: Killing of B. subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity. Spores (termed alpha-beta-) lacking the two major DNA protective alpha/beta-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and alpha-beta- and alpha-beta-recA spores exhibited identical ozone sensitivity. Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat. Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores. However, ozone-killed spores did not germinate with either nutrients or Ca(2+)-DPA and could not be recovered by lysozyme treatment. CONCLUSIONS: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat. Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of spore killing by and resistance to ozone.     

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.     

Effects of inactivation or overexpression of the sspF gene on properties of Bacillus subtilis spores.

Inactivation of the Bacillus subtilis sspF gene had no effect on sporulation, spore resistance, or germination in a wild-type strain or one lacking DNA protective alpha/beta-type small, acid-soluble proteins (SASP). Overexpression of SspF in wild-type spores or in spores lacking major alpha/beta-type SASP (alpha- beta- spores) had no effect on sporulation but slowed spore outgrowth and restored a small amount of UV and heat resistance to alpha- beta- spores. In vitro analyses showed that SspF is a DNA binding protein and is cleaved by the SASP-specific protease (GPR) at a site similar to that cleaved in alpha/beta-type SASP. SspF was also degraded during spore germination and outgrowth, and this degradation was initiated by GPR.     

Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents.

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.     

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.     

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.     

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 (H₂O₂) 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% H₂O₂, 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.     

Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from killing by dry heat.

Dry Bacillus subtilis spores lacking their two major DNA-binding proteins (small, acid-soluble proteins [SASP] alpha and beta) were much more sensitive to dry heat than were wild-type spores. Survivors of dry heat treatment of both wild-type and mutant spores exhibited a high frequency of mutations, and the DNA from the heated spores had increased numbers of single-strand breaks. These data indicate that SASP alpha and beta provide significant protection to spore DNA against the damaging effects of dry heat. This DNA damage may be in part depurination, and a purified alpha/beta-type SASP gave significant protection against dry heat-induced DNA depurination in vitro.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

Heat inactivation of Bacillus subtilis spores lacking small, acid-soluble spore proteins is accompanied by generation of abasic sites in spore DNA.

Previous work has shown that lethal heat treatment of Bacillus subtilis spores lacking the major DNA-binding proteins SASP-alpha and -beta (alpha-beta- spores) causes significant DNA damage, including many single-strand breaks. In this work we have used a reagent specific for aldehydes present in abasic sites in DNA to show that DNA from wild-type spores killed by heat treatment to levels of < 0.05% survival had at most two aldehydes (i.e., abasic sites) per 10(4) nucleotides, while DNA from alpha(-)beta- spores killed to similar levels had 7 to 20 times as many abasic sites per 10(4) nucleotides. These data were generally consistent with the level of single-strand breaks in DNA from these heated spores and strongly suggest that a major mechanism responsible for the heat killing of alpha(-)beta- (but not wild-type) spores is DNA depurination followed by strand breakage at the resultant abasic site. In contrast, hydrogen peroxide killing of alpha(-)beta - spores was not accompanied by generation of a high level of DNA aldehydes.     

Bacillus subtilis RecA and its accessory factors,RecF, RecO,RecR and RecX,are required for spore resistance to DNA double-strand break

Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions generates a replication stress to germinating spores, and the RecA·ssDNA filament might act by preventing potentially dangerous forms of DNA repair occurring during replication. RecA might stabilize a stalled fork or prevent or promote dissolution of reversed forks rather than its cleavage that should require end-processing.     

Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light.

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.     

Comparative Study of Pressure-Induced Germination of Bacillus subtilis Spores at Low and High Pressures

We have studied pressure-induced germination of Bacillus subtilis spores at moderate (100 MPa) and high (500 to 600 MPa) pressures. Although we found comparable germination efficiencies under both conditions by using heat sensitivity as a criterion for germination, the sensitivity of pressure-germinated spores to some other agents was found to depend on the pressure used. Spores germinated at 100 MPa were more sensitive to pressure (>200 MPa), UV light, and hydrogen peroxide than were those germinated at 600 MPa. Since small, acid-soluble proteins (SASPs) and dipicolinic acid (DPA) are known to be involved in spore resistance to UV light and hydrogen peroxide, we studied the fate of these compounds during pressure germination. DPA was released upon both low- and high-pressure germination, but SASP degradation, which normally accompanies nutrient-induced germination, occurred upon low-pressure germination but not upon high-pressure germination. These results adequately explain the UV and hydrogen peroxide resistance of spores germinated at 600 MPa. The resistance to pressure inactivation of 600-MPa-germinated spores could also, at least partly, be attributed to α/β-type SASPs, since mutants deficient in α/β-type SASPs were more sensitive to inactivation at 600 MPa. Further, germination at 100 MPa resulted in rapid ATP generation, as is the case in nutrient-induced germination, but no ATP was formed during germination at 600 MPa. These results suggest that spore germination can be initiated by low- and high-pressure treatments but is arrested at an early stage in the latter case. The implications for the use of high pressure as a preservation treatment are discussed.