Cannibalism Shapes Biofilm Structure and Composition in Bacillus subtilis

Bacillus subtilis can form robust colony biofilms in which different phenotypic subpopulations co-exist in highly structured and complex multicellular communities with tissue-like properties. These subpopulations provide the colony with an overall competitive fitness and the ability to respond to everchanging environmental conditions. But biofilms also provide a structural framework for both colony expansion and long-term survival in the form of highly resilient endospores. In this context, cannibalism is thought to delay sporulation by enabling one subpopulation of cells, the cannibals, to produce the sporulation delay protein SDP, the sporulation killing factor SKF and the epipeptide EPE. These cannibalism toxins lyse susceptible non-producers, thereby releasing nutrients to prevent premature sporulation. Here, we comprehensively characterized a collection of mutants defective in either toxin production or the corresponding autoimmunity by combining luminescence reporters, colony biopsy, multi-parameter flow cytometry and MALDI-mass spectrometry imaging to resolve cannibalism function and distribution. Our findings demonstrate that EPE and SDP are critical in delaying sporulation, while SKF does not significantly influence spore abundance. The three toxins are produced in distinct, only partially overlapping areas of the colony and interdepend in their spatial distribution. The absence of all three toxins (ΔΔΔ mutant) led to hyper-sporulation and small colonies characterized by excessive wrinkle formation, indicating that cannibalism-driven cell death is essential for maintaining biofilm structure and lateral expansion. Furthermore, the loss of autoimmunity against EPE and SDP resulted in severe morphological changes and stress-induced occurrence of suppressor mutants, thereby underscoring the importance of toxin resistance mechanisms. Our results highlight the complex interplay between the three cannibalism toxins, which together influence the spatiotemporal organisation, morphology and subpopulation dynamics within B. subtilis biofilms. By integrating different methodologies, we provide the first evidence of the complex interactions that shape biofilm architecture through bacterial programmed cell death mediated by localized toxin production and spatial distribution.