Modulation of the Sporulation Dynamics in the Plant-Probiotic Bacillus velezensis 83 via Carbon and Quorum-Sensing Metabolites

Spore-forming Bacilli, such as the plant-associated Bacillus velezensis strains, are widely used as probiotics, known for their safety and substantial health benefits for both animal and plant species. Through differentiation pathways mediated by quorum-sensing metabolites (QSMs), these bacteria develop multiple isogenic subpopulations with distinct phenotypes and ecological functions, including motile cells, matrix-producing/cannibalistic cells, competent cells, spores, and others. However, the heterogeneity in Bacillus populations is a significant limitation for the development of spore-based probiotics, as nutrients supplied during fermentation are consumed through non-target pathways. One of these pathways is the generation of overflow metabolites (OMs), including acetoin and 2,3-butanediol. This study elucidates, using a 2³ full factorial experimental design, the individual effects of OMs, QSMs, and their interactions on the sporulation dynamics and subpopulation distribution of B. velezensis 83. The results showed that OMs play a relevant role as external reserves of carbon and energy during in vitro nutrient limitation scenarios, significantly affecting sporulation dynamics. OMs improve sporulation efficiency and reduce cell autolysis, but they also decrease cellular synchronization and extend the period of spore formation. Although QSMs significantly increase sporulation synchronization, the desynchronization caused by OMs cannot be mitigated even with the addition of autoinducer QSM pro-sporulation molecules, including competence and sporulation stimulating factor "CSF" and cyclic lipopeptides. Indeed, the interaction effect between OMs and QSMs displays antagonism on sporulation efficiency. Modulating the levels of OMs and QSMs is a potential strategy for regulating the distribution of subpopulations within Bacillus cultures.