Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to the general biological decontamination agents, Decon and Oxone. METHODS AND RESULTS: Spores of B. subtilis treated with Decon or Oxone did not accumulate DNA damage and were not mutagenized. Spore killing by these agents was increased if spores were decoated. Spores prepared at higher temperatures were more resistant to these agents, consistent with a major role for spore coats in this resistance. Neither Decon nor Oxone released the spore core's depot of dipicolinic acid (DPA), but Decon- and Oxone-treated spores more readily released DPA upon a subsequent normally sublethal heat treatment. Decon- and Oxone-killed spores initiated germination with dodecylamine more rapidly than untreated spores, but could not complete germination triggered by nutrients or Ca(2+)-DPA and did not degrade their peptidoglycan cortex. However, lysozyme treatment did not recover these spores. CONCLUSIONS: Decon and Oxone do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents is the spore coat. Spore killing by both agents renders spores defective in germination, possibly because of damage to the inner membrane of spore. SIGNIFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of the killing of bacterial spores by Decon and Oxone.     

Studies on the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide

AIMS: To determine the mechanism of killing of Bacillus subtilis spores by hydrogen peroxide. METHODS AND RESULTS: Killing of spores of B. subtilis with hydrogen peroxide caused no release of dipicolinic acid (DPA) and hydrogen peroxide-killed spores were not appreciably sensitized for DPA release upon a subsequent heat treatment. Hydrogen peroxide-killed spores appeared to initiate germination normally, released DPA and hydrolysed significant amounts of their cortex. However, the germinated killed spores did not swell, did not accumulate ATP or reduced flavin mononucleotide and the cores of these germinated spores were not accessible to nucleic acid stains. CONCLUSIONS: These data indicate that treatment with hydrogen peroxide results in spores in which the core cannot swell properly during spore germination. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide further information on the mechanism of killing of spores of Bacillus species by hydrogen peroxide.     

Mechanisms of Bacillus subtilis spore killing by and resistance to an acidic Fe-EDTA-iodide-ethanol formulation

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to an acidic solution containing Fe(3+), EDTA, KI and ethanol termed the KMT reagent. METHODS AND RESULTS: Wild-type B. subtilis spores were not mutagenized by the KMT reagent but the wild-type and recA spores were killed at the same rate. Spores (alpha(-)beta(-)) lacking most DNA-protective alpha/beta-type small, acid-soluble spore proteins were less resistant to the KMT reagent than wild-type spores but were also not mutagenized, and alpha(-)beta(-) and alpha(-)beta(-)recA spores exhibited nearly identical resistance. Spore resistance to the KMT reagent was greatly decreased if spores had defective coats. However, the level of unsaturated fatty acids in the inner membrane did not determine spore sensitivity to the KMT reagent. Survivors in spore populations killed by the KMT reagent were sensitized to killing by wet heat or nitrous acid and to high salt in plating medium. KMT reagent-killed spores had not released their dipicolinic acid (DPA), although these killed spores released their DPA more readily when germinated with dodecylamine than did untreated spores. However, KMT reagent-killed spores did not germinate with nutrients or Ca(2+)-DPA and were recovered only poorly by lysozyme treatment in a hypertonic medium. CONCLUSIONS: The KMT reagent does not kill spores by DNA damage and a major factor in spore resistance to this reagent is the spore coat. KMT reagent treatment damages the spore's ability to germinate, perhaps by damaging the spore's inner membrane. However, this damage is not oxidation of unsaturated fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanism of spore resistance to and killing by the KMT reagent developed for killing Bacillus spores.     

Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them. METHODS AND RESULTS: Spores of B. subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective alpha/beta-type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway. Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins. Spores prepared at a higher temperature were also much more resistant to these agents. Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite- and chlorine dioxide-treated spores much more readily released DPA upon a subsequent normally sub-lethal heat treatment than did untreated spores. Hypochlorite-killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+-DPA, and these spores could not be recovered by lysozyme treatment. Chlorine dioxide-treated spores also did not germinate with Ca2+-DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients. However, while germinated chlorine dioxide-killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties. CONCLUSIONS: Hypochlorite and chlorine dioxide do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat. Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane. While chlorine dioxide-killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide.     

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.     

Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane

AIMS: To determine the mechanisms of Bacillus subtilis spore resistance to and killing by a novel sporicide, dimethyldioxirane (DMDO) that was generated in situ from acetone and potassium peroxymonosulfate at neutral pH. METHODS AND RESULTS: Spores of B. subtilis were effectively killed by DMDO. Rates of killing by DMDO of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha- beta- spores) or the major DNA repair protein, RecA, were very similar to that of wild-type spore killing. Survivors of wild-type and alpha- beta- spores treated with DMDO also exhibited no increase in mutations. Spores lacking much coat protein due either to mutation or chemical decoating were much more sensitive to DMDO than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with DMDO were sensitized to wet heat. The DMDO-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by very high pressures and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after their germination, and none of these treatments were able to revive the DMDO-killed spores. CONCLUSIONS: DMDO is an effective reagent for killing B. subtilis spores. The spore coat is a major factor in spore resistance to DMDO, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent appears to kill spores by damaging the spore's inner membrane in some fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that DMDO is an effective decontaminant for spores of Bacillus species that can work under mild conditions, and the killed spores cannot be revived. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent.     

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.     

Effect of mechanical abrasion on the viability, disruption and germination of spores of Bacillus subtilis

AIMS: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. METHODS AND RESULTS: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. CONCLUSIONS: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.     

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

AIMS: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. METHODS AND RESULTS: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. CONCLUSIONS: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.     

Killing of spores of Bacillus subtilis by tert-butyl hydroperoxide plus a TAML activator

AIMS: To determine the effectiveness of tert-butyl hydroperoxide (tBHP) plus the cationic surfactant cetyltrimethyl ammonium bromide (CTAB) and a tetra-amido macrocyclic ligand (TAML) activator in killing spores of Bacillus subtilis and the mechanisms of spore resistance to and killing by this reagent. METHODS AND RESULTS: Killing of spores of B. subtilis by tBHP was greatly stimulated by the optimum ratio of concentrations of a TAML activator (1.7 micromol l(-1)) to tBHP (4.4%, vol/vol) plus a low level (270 mg l(-1)) of CTAB. Rates of killing of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha(-)beta(-) spores) or the major DNA repair protein, RecA, by tBHP plus CTAB and a TAML activator were essentially identical to that of wild-type spore killing. Survivors of wild-type and alpha(-)beta(-) spores treated with tBHP plus CTAB and a TAML activator also exhibited no increase in mutations. Spores lacking much coat protein either because of mutation or chemical decoating were much more sensitive to this reagent than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with this reagent were sensitized to wet heat. The tBHP plus CTAB and TAML activator-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by application of 150 and 500 megaPascals of pressure for 15 min and by lysozyme treatment in hypertonic medium, but these spores lysed shortly after their germination. CONCLUSIONS: The combination of tBHP plus CTAB and a TAML activator is effective in killing B. subtilis spores. The spore coat is a major factor in spore resistance to this reagent system, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent system appears to kill spores by damaging the spore's inner membrane in some fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that tBHP plus CTAB and a TAML activator is an effective and mild decontaminant for spores of Bacillus species. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent system.     

Characterization of spores of Bacillus subtilis which lack dipicolinic acid

Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Deltager3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Deltager3 spores. The Deltager3 spoVF spores lack DPA and have higher levels of core water than Deltager3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to Deltager3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Deltager3 spoVF spores also contain minimal levels of the processed active form (termed P(41)) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P(41) formed in Deltager3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-gamma. Analysis of the resistance of wild-type, Deltager3, and Deltager3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Deltager3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.     

Effect of microwave radiation on Bacillus subtilis spores

AIMS: To compare the killing efficacy and the effects exerted by microwaves and conventional heating on structural and molecular components of Bacillus subtilis spores. METHODS AND RESULTS: A microwave waveguide applicator was developed to generate a uniform and measurable distribution of the microwave electric-field amplitude. The applicator enabled the killing efficacy exerted by microwaves on B. subtilis spores to be evaluated in comparison with conventional heating at the same temperature value. The two treatments produced a similar kinetics of spore survival, while remarkably different effects on spore structures were seen. The cortex layer of the spores subjected to conductive heating was 10 times wider than that of the untreated spores; in contrast, the cortex of irradiated spores did not change. In addition, the heated spores were found to release appreciable amounts of dipicolinic acid (DPA) upon treatment, while extracellular DPA was completely undetectable in supernatants of the irradiated spores. These observations suggest that microwave radiation may promote the formation of stable complexes between DPA and other spore components (i.e. calcium ions); thus, making any release of DPA from irradiated spores undetectable. Indeed, while a decrease in measurable DPA concentrations was not produced by microwave radiation on pure DPA solutions, a significant lowering in DPA concentration was detected when this molecule was exposed to microwaves in the presence of either calcium ions or spore suspensions. CONCLUSIONS: Microwaves are as effective as conductive heating in killing B. subtilis spores, but the microwave E-field induces changes in the structural and/or molecular components of spores that differ from those attributable only to heat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the effect of microwaves on B. subtilis spore components.     

Killing of Bacillus subtilis spores by a modified Fenton reagent containing CuCl2 and ascorbic acid

Bacillus subtilis spores were killed by CuCl(2)-ascorbic acid, chloride ions were essential for killing of spores, and spores with defective coats were killed more rapidly. CuCl(2)-ascorbic acid did not damage spore DNA, and spores killed by this reagent initiated germination. However, spores killed by CuCl(2)-ascorbic acid may have damage to their inner membrane.     

A Bacillus subtilis mutant requiring dipicolinic acid for the development of heat-resistant spores.

A Bacillus subtilis mutant is described which forms heat-resistant spores only in the presence of external dipicolinic acid (DPA). The mutation, dpa-1, is localized in a new sporulation locus, linked to pyrA. The dpa-1 strain is unable to synthesize DPA but can incorporate external DPA. The amount of DPA incorporated, the frequency of heat-resistant spores and their degree of resistance are all dependent on the concentration of external DPA. Spores of dpa- 1 strains exhibit normal resistance to most chemicals, including octanol and chloroform, but not to ethanol, pyridine, phenol and trichloroacetic acid. Complete resistance to the latter group depends on DPA. DPA incorporation is slow and apparently requires an energy supply but not protein synthesis. Direct involvement of DPA in the heat-resistance of the spores is suggested. Thin sections of DPA-less spores exhibit clearly visible cytoplasmic membranes and ribosomes. These structures are absent or less visible in the core of spores obtained with added DPA.     

Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of approximately 9 and a temperature optimum of 60 degrees C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl(2). Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca(2+) almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca(2+) and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca(2+)-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca(2+)-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.     

Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.     

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.     

Studies of the release of small molecules during pressure germination of spores of Bacillus subtilis

AIMS: To measure rates of release of small molecules during pressure germination of Bacillus subtilis spores, and the role of SpoVA proteins in dipicolinic acid (DPA) release. METHODS AND RESULTS: Rates of DPA release during B. subtilis spore germination with pressures of 150 or 500 megaPascals were much higher in spores with elevated levels of SpoVA proteins, and spores with a temperature-sensitive mutation in the spoVA operon were temperature-sensitive in DPA release during pressure germination. Spores also released arginine and glutamic acid, but not AMP, during pressure germination. CONCLUSIONS: Pressure germination of B. subtilis spores causes release of many small molecules including DPA. SpoVA proteins are involved in the release of DPA, perhaps because SpoVA proteins are a component of a DPA channel in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of pressure germination of spores of Bacillus species, a process that has significant potential for usage in the food industry.