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

食人是枯草芽孢杆菌中一种独特的细菌形式的程序性细胞死亡(PCD)，它将多细胞分化与内孢子形成联系起来。在固体表面，未驯化的菌株形成结构复杂的菌落，在这些菌落中，不同的细胞类型在时空上进行协调，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来控制种群扩张，平衡生长和生存，并最终形成休眠的内孢子。食人是基于至少三种毒素的产生，即孢子形成杀伤因子SKF、孢子形成延迟蛋白SDP和多肽EPE。食人现象似乎代表着组织形成中的一个中心检查点，因为它(I)提供了延迟或最终促进孢子形成的资源，(Ii)结构和使菌落功能化，并最终可能起到(Iii)平衡结构化多细胞群体中不同类型细胞的比例的作用。在这个联合项目中，我们的目标是揭示PCD在同类毒素和其他次级代谢产物介导下对已分化的枯草杆菌菌落结构和功能的作用。我们的目标是在单细胞分辨率下从机制上理解食人族毒素在2D微菌落和3D分化大菌落(Mascher)中的作用。菌落中的食人菌毒素生产也将作为应用和开发微生物MALDI质谱学成像(MSI)的生物模型系统，这是这一优先计划的核心技术。这项强大的技术将被开发成能够在单个单元分辨率(微米范围)下在时间和3D空间上研究细菌菌落(厘米范围)的复杂化学。将MSI与进一步的成像手段相结合，包括生命细胞成像、多参数流式细胞术和单细胞转录，将能够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)，目前的2D俯视和截面分析设置将得到优化，检测化合物的范围将扩大。将通过分析微米分辨率的菌落垂直薄片来制定合并传输几何形状的协议。与2D Topview相结合，这将使我们能够以高分辨率分析3D细菌菌落，精确到单细胞水平。此外，已建立的MALDI-MSI程序应应用于枯草杆菌以外的领域，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.