Massively-parallel Microbial mRNA Sequencing (M3-Seq) reveals heterogeneous behaviors in bacteria at single-cell resolution

Abstract

Bacterial populations are highly adaptive, enabling them to respond to and surviving in shifting environments or stresses. Yet, how these single-cell organisms vary and organize their behavior to tolerate stressors is poorly understood. This is because many bacterial subpopulations are rare and cannot be readily discovered by existing single-cell sequencing methods due to limitations in cell number and sequencing depth. Here we develop Massively-parallel Microbial mRNA sequencing (M3-Seq), which addresses these challenges by using combinatorially-indexed cells to overload droplets in combination with RNA amplification and post-hoc rRNA depletion. In a single M3-Seq experiment, we profile hundreds of thousands of bacterial cells from multiple species under a wide range of conditions. In addition to validating our approach and findings, we exploit the scale of M3-Seq to make several unexpected discoveries, including new insights into bet hedging strategies in stress responses, bacterial responses to antibiotics, and host responses to phage infection.

Targeted rRNA depletion enables efficient mRNA sequencing in diverse bacterial species and complex co-cultures

“…In addition, the use of blocking primers to deplete rRNA can potentially also be scaled down to single cells as we previously showed that EMBR-seq efficiently depletes rRNA from as little as 0.02 ng input total RNA ( 25 ). This corresponds to a pool of ~200 single bacteria (assuming ~0.1 pg of RNA per bacterial cell) and is in line with the sensitivity requirements of the existing methods that employ combinatorial barcoding strategies ( 49 , 51 , 56 , 57 ). Therefore, we anticipate that in the future EMBR-seq+ could potentially be applied to single cells, thereby adding to the growing toolkit for profiling individual bacterial cells.…”

Transcription-replication interactions reveal principles of bacterial genome regulation

“…This work represents only an initial effort in this direction, but provides a foundational framework for genome-wide exploration of novel bacterial regulatory phenomena. As bacterial scRNA-seq methods evolve in scale, capture efficiency, and cost [61][62][63][64] , we predict that these methods, in combination with microscopy and molecular genetics approaches that allow mechanistic dissection of these phenomena, will illuminate a diverse ecosystem of dynamic transcriptional processes.…”