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 α−β−) lacking the two major DNA protective α/β-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and α−β− and α−β−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 Ca2+-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.