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) similar to 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.