Probe-based bacterial single-cell RNA sequencing predicts toxin regulation

ProBac-seq is a method that uses libraries of DNA probes and commercial microfluidics for single-cell RNA-seq, leveraged here to show heterogeneous gene expression in clonal bacterial cultures including variable toxin expression in an agricultural strain of Clostridium. Clonal bacterial populations rely on transcriptional variation across individual cells to produce specialized states that increase fitness. Understanding all cell states requires studying isogenic bacterial populations at the single-cell level. Here we developed probe-based bacterial sequencing (ProBac-seq), a method that uses libraries of DNA probes and an existing commercial microfluidic platform to conduct bacterial single-cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. Applied to Bacillus subtilis and Escherichia coli, ProBac-seq correctly identifies known cell states and uncovers previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, application of the approach to Clostridium perfringens reveals heterogeneous expression of toxin by a subpopulation that can be controlled by acetate, a short-chain fatty acid highly prevalent in the gut. Overall, ProBac-seq can be used to uncover heterogeneity in isogenic microbial populations and identify perturbations that affect pathogenicity.

Automated summary

ProBac-seq is a method using DNA probes and microfluidics for single-cell RNA-seq, demonstrating heterogeneous gene expression in clonal bacterial cultures and identifying variable toxin expression in an agricultural strain of Clostridium. It allows the study of transcriptional variation and specialized states in bacterial populations at the single-cell level, correctly identifying known cell states and uncovering previously unreported transcriptional heterogeneity. The application of ProBac-seq to Clostridium perfringens reveals heterogeneity in toxin expression controlled by acetate, a prevalent fatty acid in the gut, highlighting its potential for understanding bacterial pathogenesis.