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. Dormant spores of Bacillus species initiate germination in response to a variety of nutrients, with the precise nature of the nutrient dependent on the species and strain (14, 21). These nutrients, termed germinants, bind to one or more receptors in the spore, and this binding somehow triggers both permeability changes in the spore's inner membrane and activation of enzymes that initiate hydrolysis of the spore's peptidoglycan cortex (14, 21). The permeability changes in the spore's inner membrane allow excretion of the spore core's large depots of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and divalent cations and uptake of a significant amount of water into the spore core (14, 21). More core water is then taken up as the spore core expands once the restraining cortex has been acted upon by cortex-lytic enzymes (CLEs) (14, 21). However, spore cortex hydrolysis does not appear to be essential for at least the initial permeability changes in response to interaction of germinants with their receptor (10,22,27).It appears likely that spores in which germination has been initiated but cortex degradation has not taken place represent an intermediate stage in spore germination, since cortex hydrolysis is normally significantly slower than DPA excretion and initial water uptake (21). However, this intermediate stage is impossible to study during wild-type spore germination because of the rapidity of cortex hydrolysis and the asynchrony of germination in spore populations. Therefore, we have analyzed the properties of mutant spores in which germination has been initiated but cortex hydrolysis cannot take place in order to characterize this intermediate stage in spore germination that we propose to call stage I.Bacillus subtilis was used for this work, and all strains were isogenic, except as noted otherwise, and were PS832 (wild type), PS2307 (cwlD and also carrying a chloramphenicol resistance marker) (22), and FB113 (cwlJ sleB and also carrying spectinomycin and tetracycline resistance markers) (19); all three strains are derivatives of strain 168. In strain PS2307, the cwlD mutation blocks the formation of muramic acid lactam in the spore cortex (22). Since muramic acid lactam is necessary for the action of CLEs on the spore cortex, there is no cortex degradation during the germination of spores of strain PS2307, although DPA is released, albeit slightly mo...
Aims: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition. Methods and Results: Bacillus subtilis spores prepared at temperatures from 22 to 48°C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures. As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures. Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox. However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures. In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased. However, this latter change was not due to a reduction in cotA gene expression at higher temperatures. Conclusions: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition. Significance and Impact of the Study: The precise conditions for the formation of B. subtilis spores have a large effect on many spore properties.
Aims: To determine the mechanism whereby the new disinfectant Sterilox Ò kills spores of Bacillus subtilis. Methods and Results: Bacillus subtilis spores were readily killed by Sterilox and spore resistance to this agent was due in large part to the spore coats. Spore killing by Sterilox was not through DNA damage, released essentially no spore dipicolinic acid and Sterilox-killed spores underwent the early steps in spore germination, including dipicolinic acid release, cortex degradation and initiation of metabolism. However, these germinated spores never swelled and many had altered permeability properties. Conclusions: We suggest that Sterilox treatment kills dormant spores by oxidatively modifying the inner membrane of the spores such that this membrane becomes non-functional in the germinated spore leading to spore death. Signi®cance and Impact of the Study: This work provides information on the mechanism of spore resistance to and spore killing by a new disinfectant.
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.
During an infection of a higher eukaryote, dormant spores of a Bacillus species have been previously shown to be present in cells that can generate the toxic agent peroxynitrite (PON). Dormant spores of Bacillus subtilis were much more resistant to killing by PON than were growing cells, and spore-coat alteration or removal greatly decreased PON resistance. Spores were not killed by PON through DNA damage and lost no dipicolinic acid (DPA) during PON treatment. However, PON-killed spores lost DPA during subsequent heat treatments that caused much less DPA release from untreated spores. Although dead, the PONkilled spores germinated and initiated metabolism but never went through outgrowth ; the great majority of germinated PON-killed spores also took up propidium iodide, indicating that they had suffered significant membrane damage and were dead. Together these data suggest that spore killing by PON is through some type of damage to the spore's inner membrane.
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