Mode of action of a germination-specific cortex-lytic enzyme, SleC, of Clostridium perfringens S40

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.     

The Identification of in Vitro Production of Lectin from Callus Cultures of Korean Mistletoe (Viscum album L. var. coloratum)

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.     

The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization

Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL (yaaH), which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N-acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL-encoded N-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.     

Interactions between Clostridium perfringens spores and Raw 264.7 macrophages

Clostridium perfringens is the causative agent of a variety of histotoxic infections in humans and animals. Studies on the early events of C. perfringens infections have been largely focused on the interactions between their vegetative cells and macrophages. Consequently, in the current study we have examined the interactions between C. perfringens spores and Raw 264.7 macrophages. Raw 264.7 cells were able to interact and phagocytose Clostridium perfringens spores of a food poisoning isolate, strain SM101, and a non-food borne isolate, strain F4969, albeit to different extents. Phagocytosis and to a lesser extent, association, of C. perfringens spores by Raw 2647 macrophages was completely inhibited in presence of cytochalasin D. Complement increased association and phagocytosis of C. perfringens spores by Raw 264.7 macrophages. Survival of C. perfringens spores during macrophage infection seems to depend on the ability of spore germination during infection as: (i) F4969 spores germinated during infection with Raw 264.7 macrophages and subsequently killed by macrophages; and (ii) SM101 spores remained dormant inside Raw 264.7 macrophages and thus survived up to 24 h of infection. The in vitro spore-resistance factors, α/β-type SASP, SpmA/B proteins and spore's core water content, seems to play no role in mediating SM101 spore-resistance to macrophages. Collectively, these results might well have implications in understanding the initial stages of infections by C. perfringens spores.     

Mechanisms of induction of germination of Bacillus subtilis spores by high pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

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.     

Clostridium perfringens spore germination: characterization of germinants and their receptors

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.     

Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells

Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host's intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p?相似文献   

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.     

Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity

A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.     

Identification of a Novel Lipoprotein Regulator of Clostridium difficile Spore Germination

Clostridium difficile is a Gram-positive spore-forming pathogen and a leading cause of nosocomial diarrhea. C. difficile infections are transmitted when ingested spores germinate in the gastrointestinal tract and transform into vegetative cells. Germination begins when the germinant receptor CspC detects bile salts in the gut. CspC is a subtilisin-like serine pseudoprotease that activates the related CspB serine protease through an unknown mechanism. Activated CspB cleaves the pro-SleC zymogen, which allows the activated SleC cortex hydrolase to degrade the protective cortex layer. While these regulators are essential for C. difficile spores to outgrow and form toxin-secreting vegetative cells, the mechanisms controlling their function have only been partially characterized. In this study, we identify the lipoprotein GerS as a novel regulator of C. difficile spore germination using targeted mutagenesis. A gerS mutant has a severe germination defect and fails to degrade cortex even though it processes SleC at wildtype levels. Using complementation analyses, we demonstrate that GerS secretion, but not lipidation, is necessary for GerS to activate SleC. Importantly, loss of GerS attenuates the virulence of C. difficile in a hamster model of infection. Since GerS appears to be conserved exclusively in related Peptostreptococcaeace family members, our results contribute to a growing body of work indicating that C. difficile has evolved distinct mechanisms for controlling the exit from dormancy relative to B. subtilis and other spore-forming organisms.     

Release of small molecules during germination of spores of Bacillus Species

Free amino acids, dipicolinic acid, and unidentified small molecules were released early in Bacillus spore germination before hydrolysis of the peptidoglycan cortex, but adenine nucleotides and 3-phosphoglycerate were not. These results indicate that early in germination there is a major selective change in the permeability of the spore's inner membrane.     

Kinetics of germination of wet-heat-treated individual spores of Bacillus species, monitored by Raman spectroscopy and differential interference contrast microscopy

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores of Bacillus cereus and Bacillus megaterium, as well as of several isogenic Bacillus subtilis strains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca2+ and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time, T(lag), at which spores began release of the great majority of their CaDPA during the germination of B. subtilis spores with different nutrient germinants and also increased the variability of T(lag) values. (iii) The time period, ΔT(release), between T(lag) and the time, T(release), at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values of I(release), the intensity of a spore's DIC image at T(release), than did untreated spores and had much longer time periods, ΔT(lys), for the reduction in I(release) intensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases in T(lag) and ΔT(release) were also observed when wet-heat-treated B. subtilis spores were germinated with the nonnutrient dodecylamine, while the change in I(release) was less significant. (vi) The effects of wet-heat treatment on nutrient germination of B. cereus and B. megaterium spores were generally similar to those on B. subtilis spores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.     

Spore germination

The germination of dormant spores of Bacillus species is the first crucial step in the return of spores to vegetative growth, and is induced by nutrients and a variety of non-nutrient agents. Nutrient germinants bind to receptors in the spore's inner membrane and this interaction triggers the release of the spore core's huge depot of dipicolinic acid and cations, and replacement of these components by water. These latter events trigger the hydrolysis of the spore's peptidoglycan cortex by either of two redundant enzymes in B. subtilis, and completion of cortex hydrolysis and subsequent germ cell wall expansion allows full spore core hydration and resumption of spore metabolism and macromolecular synthesis.     

Inactivation of the gene (cpe ) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.     

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores'' return to life within minutes in the process of germination. This process requires a number of spore-specific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores'' IM through which the spore core''s huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. (i) How do nutrient germinants penetrate through spores'' outer layers to access GRs in the IM? (ii) What happens during the highly variable and often long lag period between the exposure of spores to nutrient germinants and the commitment of spores to germinate? (iii) What do GRs and GerD do, and how do these proteins interact? (iv) What is the structure of the SpoVA channel in spores'' IM, and how is this channel gated? (v) What is the precise state of the spore IM, which has a number of novel properties even though its lipid composition is very similar to that of growing cells? (vi) How is CLE activity regulated such that these enzymes act only when germination has been initiated? (vii) And finally, how does the germination of spores of clostridia compare with that of spores of bacilli?     

Production of small, acid-soluble spore proteins in Clostridium perfringens nonfoodborne gastrointestinal disease isolates

The molecular basis for the differences in heat resistance between spores of Clostridium perfringens food-borne versus nonfoodborne isolates remains unknown. Since a recent study demonstrated the role of small, acid-soluble spore proteins (SASPs) in heat resistance of spores of food-borne isolates, in the current study, we evaluated the expression of SASP-encoding genes (ssp) and the production of SASPs in nonfoodborne isolates. Our results demonstrated the presence of all three ssp genes in five surveyed nonfoodborne isolates. A beta-glucuronidase assay showed that these ssp genes are expressed specifically during sporulation. Furthermore, nonfoodborne isolate F4969 produced SASPs at a level similar to that of food-borne isolate SM101. Collectively, these results suggest that the difference in the levels of heat resistance between spores of food-borne and the nonfoodborne isolates is not the result of impaired expression of ssp genes and (or) decreased production of SASPs in nonfoodborne isolates.     

Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.     

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.