A Note on the Effect of Hydrogen Chloride on the Morphology of Bacillus subtilis Spores

Gaseous hydrogen chloride, in the presence of a minute amount of water vapour, rapidly inactivated bacterial spores. Scanning electron microscopy showed that the treatment caused spores of Bacillus subtilis to collapse. Modern theories of spore structure and resistance suggest that it is likely that hydrogen chloride inactivates and causes collapse of spores by breaking disulphide bonds in coat protein and neutralizing, by protonation, peptidoglycan carboxyl groups in the underlying cortex.     

Luminometric and differential scanning calorimetry (DSC) studies on heat- and radiation inactivation of Bacillus subtilis luxAB spores

A bioluminescent derivative of Bacillus subtilis containing a plasmid encoding a luxAB fusion under control of a vegetative promoter and gives bioluminescence upon addition of an exogenous long-chain aldehyde has been used as test organism. Its spore populations have been produced and their heat- and radiation survival curves established. Heat-sensitization effect of pre-irradiation of spores was proven not only by colony counting but also with differential scanning calorimetry. Under a linearly programmed temperature increase, the heat destruction of spores surviving 2.5 kGy gamma irradiation resulted in at a few centigrade lower temperature than that of untreated spores. Heat denaturation endotherms in the DSC-thermogram of irradiated spores were shifted to lower temperatures as well. Comparative turbidimetric, luminometric and phase-contrast microscopic studies of untreated, heat-treated and irradiated spore populations showed that the kinetics of germination and the light emission during germination of radiation-inactivated spores were the same as those of untreated spores, revealing that the pre-formed luciferase enzyme packaged into the spores during sporulation remained intact after an irradiation dose causing 90% decrease in number of colony forming spores. Therefore, in contrast to heat-treated spores, the initial bioluminescence reading upon germination of irradiated spores does not reflect the viable count of their population.     

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.     

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.     

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.     

A quantitative histochemical study of sulphydryl and disulphide content during normal epidermal keratinization

Summary A quantitative histochemical study was carried out on the distribution of protein thiol and disulphide groups in normal human plantar epidermal tissue. Histochemical demonstration of reactive groups was achieved by addition ofN-(4-aminophenyl) maleimide, subsequent diazotization and final coupling with a Nitro Red or chromotropic acid label as first described by Sippel. The quantitative reliability of the method was tested by absorption cytophotometry, and evaluated on the basis of the internal consistency of the results reported.Our histological observations and histophotometric data support accepted views on epidermal keratinization. A limited, though reproducible, amount of disulphide bonds was observed near the basement membrane. The free thiol concentration in basal and prickle cells was low and almost constant, but was higher in the granular cells, where deposition of sulphur-containing proteins on cell membranes is initiated. In Malpighian layers, disulphide cross-links only occurred just beneath the transition zone in thickened cell membranes. The staining pattern of the inner stratum corneum resembled a mosaic and was characterized by a sharp rise of the disulphide content, which exceeded the decrease in free thiol groups. The free thiol concentration decreased further throughout the cornified layers whilst the disulphide content remained fairly constant. Staining of thiol and disulphide groups together corresponded, within the limits of the standard error, to the sum of the thiol and disulphide concentrations when they were assayed separately in living and horny cells. These results confirm that living cells are the main site of free thiol groups, while horny cells are the most prominent site of disulphide cross-links.     

Effect of trichloroacetic acid treatment on certain properties of spores of Bacillus cereus T.

Spores of Bacillus cereus T treated with trichloroacetic acid (6.1--61.2 mM) were compared with untreated spores, and as the concentration of the chemical increased, the following alterations in spore properties were found: (1) the extent of germination decreased irrespective of the germination medium used; (2) the spores became sensitive to sodium hydroxide (1 N) and hydrochloric acid (0.27 N), but not to lysozyme (200 micrograms/ml); (3) loss of dipicolinate increased on subsequent heating; and (4) the spores became more sensitive to heat. However, trichloroacetic acid-treated spores were still viable and there was no significant change in spore components. The mechanism of action of trichloroacetic acid is discussed.     

Efficacy of iodine-treated biocidal filter media against bacterial spore aerosols

Aims: To assess the effectiveness of iodine-treated biocidal filter media against bacterial spore aerosols. Methods and Results: Bacillus subtilis spores were aerosolized and introduced into a filtration system. Both treated and untreated filters exhibited high viable removal efficiency (>99·996%) with negligible variation in pressure drop during the entire experiment. The viability of collected spores on the filter was investigated by enumeration of spores extracted from the filter by vortexing. At room temperature and low relative humidity (RH), the survival fraction of the treated filter was significantly lower than that of the untreated filter (P-value < 0·05). Meanwhile, at room temperature and high RH and at high temperature and high RH, the survival fractions on the treated medium were statistically the same as the untreated control at room temperature and low RH. Conclusions: Both treated and untreated filters achieved excellent viable removal efficiency for spores. The pressure drop of the treated filter was not affected by the iodine treatment. The viability of collected bacterial spores was decreased because of the exertion of iodine disinfectant. Significance and Impact of the Study: The evaluation demonstrates that the iodine-treated filter is a viable medium for respiratory protection against infectious spore aerosols. The results warrant further evaluation of smaller biological agents, which exhibit higher penetration.     

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.     

Water Distribution in Bacterial Spores: A Key Factor in Heat Resistance

The role of water, its distribution and its implication in the heat resistance of dried spores was investigated using DSC (Differential Scanning Calorimetry). Bacillus subtilis spores equilibrated at different water activity levels were heat treated under strictly controlled conditions. The temperature was increased linearly in pans with different resistances to pressure. Data from the heat-related transitions occurring in the spores were recorded and spore viability was assessed at different stages during DSC. The thermodynamic transitions observed were related to the water status in the spores and spore survival. The results demonstrated that water still remained in the spore core when water activity was as low as 0.13. The first transition occurred at around 150 °C and was assumed to be related to a mobile fraction of water from the outer layers of the spore. The second occurred at around 200 °C, which could correspond to a fraction of water embedded in the spore core. Moreover, the results showed that spore destruction during heating was favored by the amount of water remaining in the spore. The changes in their structure were also evaluated by FTIR (Fourier Transform Infrared Spectroscopy). This work offers new understanding about the distribution of water in spores and presents new elements on the heat resistance of spores in relation to their water content.     

Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals

A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents. Spore resistance to wet heat is determined largely by the water content of spore core, which is much lower than that in the growing cell protoplast. A lower core water content generally gives more wet heat-resistant spores. The level and type of spore core mineral ions and the intrinsic stability of total spore proteins also play a role in spore wet heat resistance, and the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP) protects DNA against wet heat damage. However, how wet heat kills spores is not clear, although it is not through DNA damage. The alpha/beta-type SASP are also important in spore resistance to dry heat, as is DNA repair in spore outgrowth, as Bacillus subtilis spores are killed by dry heat via DNA damage. Both UV and gamma-radiation also kill spores via DNA damage. The mechanism of spore resistance to gamma-radiation is not well understood, although the alpha/beta-type SASP are not involved. In contrast, spore UV resistance is due largely to an alteration in spore DNA photochemistry caused by the binding of alpha/beta-type SASP to the DNA, and to a lesser extent to the photosensitizing action of the spore core's large pool of dipicolinic acid. UV irradiation of spores at 254 nm does not generate the cyclobutane dimers (CPDs) and (6-4)-photoproducts (64PPs) formed between adjacent pyrimidines in growing cells, but rather a thymidyl-thymidine adduct termed spore photoproduct (SP). While SP is formed in spores with approximately the same quantum efficiency as that for generation of CPDs and 64PPs in growing cells, SP is repaired rapidly and efficiently in spore outgrowth by a number of repair systems, at least one of which is specific for SP. Some chemicals (e.g. nitrous acid, formaldehyde) again kill spores by DNA damage, while others, in particular oxidizing agents, appear to damage the spore's inner membrane so that this membrane ruptures upon spore germination and outgrowth. There are also other agents such as glutaraldehyde for which the mechanism of spore killing is unclear. Factors important in spore chemical resistance vary with the chemical, but include: (i) the spore coat proteins that likely react with and detoxify chemical agents; (ii) the relative impermeability of the spore's inner membrane that restricts access of exogenous chemicals to the spore core; (iii) the protection of spore DNA by its saturation with alpha/beta-type SASP; and (iv) DNA repair for agents that kill spores via DNA damage. Given the importance of the killing of spores of Bacillus species in the food and medical products industry, a deeper understanding of the mechanisms of spore resistance and killing may lead to improved methods for spore destruction.     

Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

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.     

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.     

Survival of Clostridium botulinum Spores

Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a “tail” comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The “tail” was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation “inactivated” spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth.     

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

AIMS: To determine the mechanisms of killing of Bacillus subtilis spores by ethanol or strong acid or alkali. METHODS AND RESULTS: Killing of B. subtilis spores by ethanol or strong acid or alkali was not through DNA damage and the spore coats did not protect spores against these agents. Spores treated with ethanol or acid released their dipicolinic acid (DPA) in parallel with spore killing and the core wet density of ethanol- or acid-killed spores fell to a value close to that for untreated spores lacking DPA. The core regions of spores killed by these two agents were stained by nucleic acid stains that do not penetrate into the core of untreated spores and acid-killed spores appeared to have ruptured. Spores killed by these two agents also did not germinate in nutrient and non-nutrient germinants and were not recovered by lysozyme treatment. Spores killed by alkali did not lose their DPA, did not exhibit a decrease in their core wet density and their cores were not stained by nucleic acid stains. Alkali-killed spores released their DPA upon initiation of spore germination, but did not initiate metabolism and degraded their cortex very poorly. However, spores apparently killed by alkali were recovered by lysozyme treatment. CONCLUSIONS: The data suggest that spore killing by ethanol and strong acid involves the disruption of a spore permeability barrier, while spore killing by strong alkali is due to the inactivation of spore cortex lytic enzymes.SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide further information on the mechanisms of spore killing by various chemicals.     

Accessibilities and reactivities of cysteine thiols during refolding of reduced bovine pancreatic trypsin inhibitor

The experimental basis of the pathway of refolding of reduced bovine pancreatic trypsin inhibitor that accompanies disulphide bond formation is explained in the light of a recent suggestion that the inability of certain Cys residues to form disulphide bonds could be explained simply by their thiol groups being inaccessible to disulphide reagents. This explanation is not valid, because part of the experimental evidence for inability to form disulphides is that the Cys residues accumulate as mixed-disulphides with the reagent. That these thiol groups are observed to react normally with the reagent, and with iodoacetic acid, is direct positive proof that they were not inaccessible or otherwise unreactive. The experimentally determined refolding pathway accurately reflects the energetics of the protein folding transitions and is consistent with all general observations of the folding transitions of other small proteins, whether or not disulphide bond formation is involved.     

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.     

Properties of spores of Bacillus subtilis blocked at an intermediate stage in spore germination

Germination of mutant spores of Bacillus subtilis unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.     

Analysis of damage due to moist heat treatment of spores of Bacillus subtilis

Aims:  To determine conditions for generation and recovery of Bacillus subtilis spore populations heavily damaged by moist heat treatment.
Methods and Results:  Bacillus subtilis spores were treated with moist heat and spore viability was assessed on different media. A rich medium and several minimal media gave similar spore recoveries after moist heat treatment, but lack of glucose in minimal media greatly decreased spore recovery. High NaCl levels also greatly decreased the recovery of moist heat-treated spores on minimal media, and addition of good osmoprotectants reversed this effect. Moist heat treatment did not decrease spore recovery on minimal media with high salt through DNA damage or by eliminating spore germination, but by affecting spore outgrowth.
Conclusions:  Conditions for generating B. subtilis spore populations with high levels of conditional moist heat damage have been determined. The major conditional damage appears to be in spore outgrowth, perhaps because of damage to one or more important metabolic enzymes.
Significance and Impact of the Study:  This work has provided new insight into the mechanism of B. subtilis spore killing by moist heat.