Cannibalism in Bacillus subtilis colonies: Role of programmed cell death in shaping and functionalizing differentiated multicellular populations

同类相食是一种独特的细菌程序性细胞死亡（PCD）形式，将枯草芽孢杆菌的多细胞分化与孢子内形成联系起来。在固体表面，未驯化的菌株形成结构复杂的菌落，其中不同表型的细胞类型在时空上协调形成强大的微生物组织和子实体。这些结构提供了一种多细胞形式，用于控制种群扩张，平衡生长与生存，并最终形成休眠内生孢子。同类相食是基于至少三种毒素的产生，即产孢杀死因子SKF、产孢延迟蛋白SDP和EPE。同类相食似乎代表了组织形成的一个中心检查点，因为它(i)提供了延迟或最终促进孢子形成的资源，（ii）构建和功能化菌落，并可能最终服务于（iii）平衡结构多细胞群体中不同细胞类型的比例。在这个联合项目中，我们旨在揭示由同类相食毒素和其他次生代谢物介导的PCD在分化枯草芽孢杆菌菌落结构和功能中的作用。我们的目标是在单细胞分辨率下的2D微菌落和3D分化大菌落（Mascher）中机械地理解食人毒素的作用。菌落自相残杀毒素的产生也将作为应用和开发微生物MALDI质谱成像（MSI）的生物模型系统，这是该优先项目的核心技术。这项强大的技术将被开发用于在单细胞分辨率（µm范围）下在时间和3D空间上研究细菌菌落（cm范围）的复杂化学。将MSI与进一步的成像方式相结合，包括生命细胞成像、多参数流式细胞术和单细胞转录组学，将以前所未有的分辨率获得微生物组织动力学和结构的全面时空图。为了开发MALDI-MSI (Dreisewerd)，将优化目前用于二维俯视图和横截面分析的设置，并扩大检测化合物的范围。通过在微米分辨率下分析菌落的垂直薄切片，将建立一个整合传输几何形状的协议。结合2D俯视图，这将允许分析细菌菌落在3D高分辨率到单细胞水平。此外，已建立的MALDI-MSI程序应应用于枯草芽孢杆菌之外，例如研究大肠杆菌中的PCD和分析铜绿假单胞菌生物膜中的化学通讯。

Cannibalism is a unique bacterial form of programmed cell death (PCD) that links multicellular differentiation to endospore formation in Bacillus subtilis. On solid surfaces, undomesticated strains form intricately structured colonies, in which phenotypically different cell types are spatiotemporally coordinated to form robust microbial tissues and fruiting bodies. These structures provide a multicellular form for controlling population expansion, balancing growth with survival, and ultimately culminating in the formation of dormant endospores. Cannibalism is based on the production of at least three toxins, the sporulation killing factor SKF, the sporulation delay protein SDP and the epipeptide EPE. Cannibalism seems to represent a central checkpoint in tissue formation, since it (i) provides resources to either delay or ultimately fuel sporulation, (ii) structures and functionalizes the colonies, and might ultimately serve to (iii) balance the ratio of different cell types in structured multicellular populations. In this joint project, we aim at unravelling the role of PCD, mediated by cannibalism toxins and other secondary metabolites, for the structure and function of differentiated B. subtilis colonies. We aim at mechanistically understanding cannibalism toxin action both in 2D microcolonies at single-cell resolution, and in 3D differentiated macrocolonies (Mascher).Cannibalism toxin production in colonies will also serve as a biological model system to apply and develop microbial MALDI mass spectrometry imaging (MSI), a core technology of this priority program. This powerful technique will be developed to enable studying the complex chemistry of bacterial colonies (cm range) in time and 3D space at single cell resolution (µm range). Combining MSI with further imaging modalities, including life cell imaging, multi-parameter flow cytometry and single cell transcriptomics will allow obtaining a comprehensive spatiotemporal map of microbial tissue dynamics and structure at unprecedented resolution. For developing MALDI-MSI (Dreisewerd), the current set-up for 2D topview and cross-section analysis will be optimized and the range of compounds detected will be expanded. A protocol for incorporating transmission geometries will be established by analyzing vertical thin-sections of colonies at µm resolution. Combined with 2D topview, this will allow analyzing bacterial colonies in 3D at high resolution down to single-cell level. Moreover, the established MALDI-MSI procedures shall be applied beyond B. subtilis, e.g. for studying PCD in E. coli and analyzing chemical communication in P. aeruginosa biofilms.