Expression of the 2Duf protein in wild-type Bacillus subtilis spores stabilizes inner membrane proteins and increases spore resistance to wet heat and hydrogen peroxide

Aims: This work aimed to characterize spore inner membrane (IM) properties and the mechanism of spore killing by wet heat and H₂O₂ with spores overexpressing the 2Duf protein, which is naturally encoded from a transposon found only in some Bacillus strains with much higher spore resistance than wild-type spores.
Methods and Results: Killing of Bacillus subtilis spores by wet heat or hydrogen peroxide (H₂O₂) was slower when 2Duf was present, and Ca-dipicolinic acid release was slower than killing. Viabilities on rich plates of wet heat- or H₂O₂ -treated spores +/- 2Duf were lower when NaCl was added, but higher with glucose. Addition of glucose but not Casamino acids addition increased treated spores' viability on minimal medium plates. Spores with 2Duf required higher heat activation for germination, and their germination was more wet-heat resistant than that of wild-type spores, processes that involve IM proteins. IM permeability and lipid mobility were lower in spores with 2Duf, although IM phospholipid composition was similar in spores +/- 2Duf.
Conclusions: These results and previous work suggests that wet heat and H₂O₂ kill spores by damaging an IM enzyme or enzymes involved in oxidative phosphorylation.