New insights into peroxide toxicology: sporulenes help Bacillus subtilis endospores from hydrogen peroxide

Aim The purpose of the present study was to understand the possible events involved in the toxicity of hydrogen peroxide (H2O2) to wild and sporulene-deficient spores of Bacillus subtilis, as H2O2 was previously shown to have deleterious effects.Methods and results The investigation utilized two strains of B. subtilis, namely the wild-type PY79 (WT) and the sporulene-deficient TB10 (Delta sqhC mutant). Following treatment with 0.05% H2O2 (v/v), spore viability was assessed using a plate count assay, which revealed a significant decrease in cultivability of 80% for the Delta sqhC mutant spores. Possible reasons for the loss of spore viability were investigated with microscopic analysis, dipicholinic acid (DPA) quantification and propidium iodide (PI) staining. Microscopic examinations revealed the presence of withered and deflated morphologies in spores of Delta sqhC mutants treated with H2O2, indicating a compromised membrane permeability. This was further substantiated by the absence of DPA and a high frequency (50%-75%) of PI infiltration. The results of fatty acid methyl ester analysis and protein profiling indicated that the potentiation of H2O2-induced cellular responses was manifested in the form of altered spore composition in Delta sqhC B. subtilis. The slowed growth rates of the Delta sqhC mutant and the heightened sporulene biosynthesis pathways in the WT strain, both upon exposure to H2O2, suggested a protective function for sporulenes in vegetative cells.Conclusions Sporulenes serve as a protective layer for the inner membrane of spores, thus assuming a significant role in mitigating the adverse effects of H2O2 in WT B. subtilis. The toxic effects of H2O2 were even more pronounced in the spores of the Delta sqhC mutant, which lacks this protective barrier of sporulenes.