Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid.

Treatment of wild-type spores of Bacillus subtilis with glutaraldehyde or an iodine-based disinfectant (Betadine) did not cause detectable mutagenesis, and spores (termed α–β–) lacking the major DNA-protective α/β-type, small, acid-soluble proteins (SASP) exhibited similar sensitivity to these agents. A recA mutation did not sensitize wild-type or α–β– spores to Betadine or glutaraldehyde, nor did spore treatment with these agents result in significant expression of a recA-lacZ fusion when the treated spores germinated. Spore glutaraldehyde sensitivity was increased dramatically by removal of much spore coat protein, but this treatment had no effect on Betadine sensitivity. In contrast, nitrous acid treatment of wild-type and α–β– spores caused significant mutagenesis, with α–β– spores being much more sensitive to this agent. A recA mutation further sensitized both wild-type and α–β– spores to nitrous acid, and there was significant expression of a recA-lacZ fusion when nitrous acid-treated spores germinated. These results indicate that: (a) nitrous acid kills B. subtilis spores at least in part by DNA damage, and α/β-type SASP protect against this DNA damage; (b) killing of spores by glutaraldehyde or Betadine is not due to DNA damage; and (c) the spore coat protects spores against killing by glutaraldehyde but not Betadine. Further analysis also demonstrated that spores treated with nitrous acid still germinated normally, while those treated with glutaraldehyde or Betadine did not.