In Bacillus subtilis colony biofilms, phenotypic diversification confers tissue-like properties and enhanced competitive fitness within a structural framework that allows both colony expansion and long-term survival via endospore formation. Cannibalism is a sporulation delay strategy, in which one subpopulation produces the sporulation delay protein SDP, the sporulation killing factor SKF, and the epipeptide EPE. These toxins are thought to lyse susceptible nonproducers, thereby releasing nutrients to prevent premature sporulation. However, the molecular mechanisms orchestrating this bacterial type of programmed cell death during biofilm development are poorly understood. Here, we comprehensively characterized mutants defective in either toxin production or the corresponding autoimmunity by a multiscale approach, combining luminescence reporters, colony biopsy, multi-parameter flow cytometry, and MALDI-mass spectrometry imaging to resolve cannibalism function and distribution. The toxins are produced in distinct, only partially overlapping areas of the colony, and are interdependent in their spatial distribution. Both EPE and SDP, but not SKF, are crucial for delaying sporulation. Loss of EPE or SDP autoimmunity resulted in severe morphological changes and stress-induced occurrence of suppressor mutants. The absence of all three toxins led to small, hyper-sporulating colonies with excessive wrinkle formation, indicating that cannibalism is essential for maintaining biofilm structure and lateral expansion. Our results provide the first evidence for the complex interactions between the three cannibalism toxins that shape biofilm architecture through bacterial programmed cell death. Localized toxin production and its spatial distribution affect the spatiotemporal organization, morphology, and subpopulation dynamics within B. subtilis biofilms.

IMPORTANCE: Programmed cell death (PCD) is a ubiquitous and crucial mechanism to structure eukaryotic multicellular tissues. PCD-like processes have also been described in bacteria, but their contribution to multicellular development is poorly understood. Cannibalism in Bacillus subtilis has been described as a sporulation delay strategy, in which one subpopulation produces antimicrobial peptides that kill susceptible nonproducing siblings. Their lysis is thought to release nutrients that delay the sporulation in the producing subpopulation. This study comprehensively analyses the role of the three cannibalism toxins in shaping colony biofilms. By combining MALDI-mass spectrometry imaging, colony biopsy, flow cytometry, and luminescence reporters, we demonstrate that cannibalism toxins are crucial for biofilm structure. They show a discrete and interdependent localization within the colonies. While cannibalism inhibits sporulation and causes severe envelope stress within biofilms, our data challenge the established role of cannibalism-dependent killing as the mechanism behind this sporulation delay.