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 B. 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 suggest that wet heat and H2O2 kill spores by damaging an IM enzyme or enzymes involved in oxidative phosphorylation.

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is to perform different acquisitions of the same object to determine the precision of the reconstruction (i.e. fluctuations or changes under different realizations of the noise). To show the repeatability of the reconstructed images we used consecutive acquisitions of fluorescent microscopy images of F-actin filaments and microtubules over the same region after refocusing. The differences between images due to both the different noise realization and the randomness of the genetic algorithm are quantified by introducing a metric computation of the distance between solutions. In a second study, the accuracy of the method under different noise levels is discussed by applying a similar metric for the comparison of the retrieved solution with the ground truth of synthetic images. Two photon microscopy images and also the improvement of STED images of the nuclear pores complex will also be presented to discuss the accuracy of the reconstruction.

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