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

J Appl Microbiol. 2023 Mar 1;134(3):lxad040. doi: 10.1093/jambio/lxad040.


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.

Keywords: Bacillus; antimicrobials; bacterial spores; disinfection; metabolism.

MeSH terms

  • Bacillus subtilis / metabolism
  • Glucose / metabolism
  • Hot Temperature*
  • Hydrogen Peroxide* / metabolism
  • Hydrogen Peroxide* / pharmacology
  • Membrane Proteins / metabolism
  • Picolinic Acids / metabolism
  • Spores, Bacterial / metabolism


  • Hydrogen Peroxide
  • Membrane Proteins
  • Glucose
  • Picolinic Acids