Deciphering the activity of individual microbes within complex communities and environments remains a challenge. Here we describe the development of microbiome single-cell transcriptomics using droplet-based single-cell RNA sequencing and pangenome-based computational analysis to characterize the functional heterogeneity of the rumen microbiome. We generated a microbial genome database (the Bovine Gastro Microbial Genome Map) as a functional reference map for the construction of a single-cell transcriptomic atlas of the rumen microbiome. The atlas includes 174,531 microbial cells and 2,534 species, of which 172 are core active species grouped into 12 functional clusters. We detected single-cell-level functional roles, including a key role for Basfia succiniciproducens in the carbohydrate metabolic niche of the rumen microbiome. Furthermore, we explored functional heterogeneity and reveal metabolic niche trajectories driven by biofilm formation pathway genes within B. succiniciproducens. Our results provide a resource for studying the rumen microbiome and illustrate the diverse functions of individual microbial cells that drive their ecological niche stability or adaptation within the ecosystem.

破译复杂群落和环境中单个微生物的活动仍然是一个挑战。在这里，我们描述了微生物组单细胞转录组学的发展，使用基于液滴的单细胞 RNA 测序和基于泛基因组的计算分析来表征瘤胃微生物组的功能异质性。我们生成了微生物基因组数据库（牛胃肠微生物基因组图谱）作为构建瘤胃微生物组单细胞转录组图谱的功能参考图谱。该图谱包括 174,531 个微生物细胞和 2,534 个物种，其中 172 个是核心活性物种，分为 12 个功能簇。我们检测了单细胞水平的功能作用，包括Basfia succiniciproductns在瘤胃微生物组碳水化合物代谢生态位中的关键作用。此外，我们探索了功能异质性并揭示了由B. succinici Producens内生物膜形成途径基因驱动的代谢生态位轨迹。我们的结果为研究瘤胃微生物组提供了资源，并说明了单个微生物细胞的多种功能，这些功能驱动其生态位稳定性或在生态系统内的适应。