Sporulation at reduced water activity impairs germination kinetics of Bacillus subtilis spores

While the effect of sporulation temperature on spore germination has been extensively studied, the effect of other relevant environmental factors such as water activity (a_w) has been overlooked, despite the fact that sporulation niches with lower humidity than ideal culture media, such as soil, are common in nature. In this work, we characterized the germination kinetics of B. subtilis 168 spores produced at reduced a_w (0.98) using either glycerol (S_gly spores) or NaCl (S_salt spores) in comparison to spores prepared under optimal conditions (S_control spores, a_w ~0.99) in a variety of nutrient and chemical stimuli, along with the effect of thermal activation. Spores produced at reduced a_w showed impaired germination to varying degrees depending on the nutrient and solute used to depress a_w. While S_gly spores exhibited germination defects in all the nutrients tested (a rich growth medium, L-alanine, L-valine, and the AGFK mixture) compared to S_control spores, S_salt populations showed an impaired response to L-alanine and L-valine. These nutrient germination defects of the S_gly and S_salt spores could not be reversed by heat activation. In addition, both populations displayed impaired germination in Ca-DPA, but increased germination rate in dodecylamine. The phenotypes of spores produced at reduced a_w suggested plausible alterations in their coat properties. Using mutant spores with coat morphogenesis defects and a 4 kDa FITC-dextran probe permeability test, we could infer that the impaired germination of S_salt spores in nutrients and Ca-DPA may involve alterations in the crust and/or outer coat, leading to reduced permeability.IMPORTANCEBacterial spores are causative agents of relevant zoonoses and foodborne diseases and are involved in food spoilage. Natural sporulation niches, such as soil, are exposed to a variety of changing environmental conditions, such as temperature and water activity (a_w), which strongly influence the dynamics of spore germination. This work provides the first data on the effect of lowering the a_w of the sporulation medium on spore germination kinetics, which, together with previous data on the effect of other environmental sporulation conditions and inter- and intraspecific variability, will help accurately predict germination and thus prevent the negative impacts of pathogenic and food spoiling spores. Furthermore, we inferred that alterations related to the coat are associated with impaired nutrient germination of spores produced at reduced a_w with the addition of NaCl. These findings may help develop novel and efficient strategies to control germination or eradicate spores.