Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures.

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 degrees 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.

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.

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the alpha/beta-type protect spores against this DNA damage.

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed alpha-beta-) lacking the two major alpha/beta-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and alpha-beta- spores to formaldehyde treatment, which caused significant expression of a recA-lacZ fusion when the treated spores germinated. Formaldehyde also caused protein-DNA cross-linking in both wild-type and alpha-beta- spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) alpha/beta-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.