Abstract
New effectors of spore properties have been identified in two Bacillus subtilis strains-bacillithiol, the major low-molecular-weight thiol in B. subtilis, and sporulene, a multi-ring compound synthesized from curcumene in spores' outer layers. The absence of either bacillithiol or sporulene caused faster germination of spores of one wild-type B. subtilis strain, PS832, with two germinant receptors (GR)-dependent germinants, L-valine for the GerA GR, and the AGFK mixture for the cooperative action of the GerB and GerK GRs, as well as the GR-independent germinant dodecylamine. However, these effects on PS832 spore germination were due to the presence of the antibiotic marker used to replace the genes for bacillithiol or sporulene synthesis. The absence of bacillithiol also caused reduced spore resistance to wet heat but not to hydrogen peroxide or UV radiation, while sporulenes' absence had no effect on spore wet heat resistance, but reduced spore resistance to hydrogen peroxide as found previously (T. Bosak, R. M. Losick, and A. Pearson, Proc Natl Acad Sci USA 105:6725-6729, 2008, https://doi.org/10.1073/pnas.0800199105) and to UV radiation, but the presence of the antibiotic markers in these mutant strains was not responsible for these effects. Notably, spores lacking sporulene were much whiter than wild-type (wt) spores, suggesting that sporulene and its precursor curcumene contribute to spore pigmentation that can absorb UV, thus reducing spores' UV resistance. Analyses of reasons for the effects of bacillithiol's or sporulene's absence on spore resistance, specifically core water content and/or altered spore inner membrane (IM) permeability, found no major differences from values in wt spores. However, other analyses showed that sporulene's absence led to slight changes in spore IM fluidity. IMPORTANCE: The work in this paper has identified two new factors, bacillithiol and sporulene, as modulators of the resistance and germination of spores of two Bacillus subtilis strains, and by extension, probably spores of other Bacillota. Since spores of some species can give rise to cells that can cause food spoilage and/or disease, new knowledge about spore resistance and germination could have applied utility.