Single-cell RNA-sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes but current methods are incompatible with bacteria. Here, we introduce microSPLiT, a high-throughput scRNA-seq method for gram-negative and gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction, and also identified novel and unexpected gene expression states including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities otherwise not amenable to single-cell analysis such as natural microbiota.
