Observation and control of sporulation failures in Bacillus subtilis

Spore formation in Bacilli is a complex developmental process in which vegetative cells form dormant endospores. However, not all produced spores are necessarily viable, yet high purity of spore preparations is crucial for many spore-based applications.

Here, we characterized how such sporulation failures arise by tuning sporulation medium conditions in liquid batch culture in small-scale bioreactors. Supplementation of a chemically defined sporulation medium with L-alanine improved the sporulation outcome of Bacillus subtilis. We attribute this to alanine catabolism by alanine dehydrogenase, which prevents premature germination triggered by the major germinant receptor GerA. Pyruvate biosynthesis from alanine catabolism in turn supports spore formation via central metabolism.

By using automated time-lapse microscopy, we further investigated spore morphogenesis during successful and failed sporulation events. We found that sporulation failures were associated with premature mother cell lysis and linked to an additional, understudied late step in the sporulation program: a centering motion of the forespore body within the mother cell.

Our observations suggest a tight link between central metabolism and the outcome of the late stages of spore development. Furthermore, our new methods and insights offer new opportunities for improving spore production of Bacilli in applied contexts.