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bioRxiv · 10.1101/2025.06.24.660303

High-salinity sporulation in Bacillus subtilis results in coat dependant enhanced resistance to both wet heat and hydrogen peroxide.

Freire, V.·Orera, I.·Condon, S.
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Abstract

In natural niches such as soil, spore-formers encounter fluctuating conditions, leading to sporulation in non-optimal environmental conditions that can affect spore germination and resistance properties. However, while the influence of sporulation temperature on spore behaviour has been widely studied, the data concerning the influence of other factors such as water activity (aw) is scarce. We reported that reducing the awof the sporulation medium from the optimum (aw 0.99; Scontrol spores) to 0.98, using sodium chloride (Ssalt spores) increased their wet heat resistance, yet by unknown means. The present work aimed to examine the mechanism behind the increased wet heat resistance observed in the spores produced in the presence of high salinity. Crust morphogenetic protein CotY was required for the increased wet heat resistance observed in spores of Bacillus subtilis formed in the presence of salt. Interestingly, this protein was also related to increased H2O2 resistance. Proteomic analysis of coat extracts from Ssalt and Scontrol spores revealed markedly different protein profiles. Specifically, Ssalt spores displayed significant increase in abundance of proteins involved in redox homeostasis, structure of the coat and also, others involved in favouring or establish cross-linking. Increased cross-linking, such as disulfide bond formation in the coat and the cysteine-rich crust is known to influence resistance to both agents. Furthermore, deletion of CotY increased DPH lipid probe mobility regardless of sporulation condition, indicating that it plays an active role in stabilizing the IM. Therefore, all the presented data suggested that increased coat cross-link directly or indirectly related to CotY in Ssalt grants enhanced wet heat resistance by protecting the IM, which was supported by DPA release data under lethal heat stress. This work advances our understanding of how the coat modulates resistance in bacterial spores, helping develop effective control strategies against problematic spore populations due to soil salinization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/660303v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@16140bborg.highwire.dtl.DTLVardef@1e0e3dborg.highwire.dtl.DTLVardef@18f66ceorg.highwire.dtl.DTLVardef@1a6937f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Freire, V., Orera, I., Condon, S.. 2025-06-24. High-salinity sporulation in Bacillus subtilis results in coat dependant enhanced resistance to both wet heat and hydrogen peroxide.. https://doi.org/10.1101/2025.06.24.660303

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