Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation

• Protein digestion efficiency is an estimate of the extent of protein cross-linking.
• Coat and crust layers in maturing Bacillus subtilis spores show a decrease in protein digestion.
• Endospore maturation is coupled to protein cross-linking and enhanced thermal resistance.
• Spore maturation lengthens the average germination time of unstressed spores.

Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically 15N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.