Single-Cell RNA Sequencing Elucidates the Structure and Organization of Microbial Communities.

Clonal bacterial populations exhibit various forms of heterogeneity, including co-occurrence of cells with different morphological traits, biochemical properties, and gene expression profiles. This heterogeneity is prevalent in a variety of environments. For example, the productivity of large-scale industrial fermentations and virulence of infectious diseases are shaped by cell population heterogeneity and have a direct impact on human life. Due to the need and importance to better understand this heterogeneity, multiple methods of examining single-cell heterogeneity have been developed. Traditionally, fluorescent reporters or probes are used to examine a specific gene of interest, providing a useful but inherently biased approach. In contrast, single-cell RNA sequencing (scRNA-seq) is an agnostic approach to examine heterogeneity and has been successfully applied to eukaryotic cells. Unfortunately, current extensively utilized methods of eukaryotic scRNA-seq present difficulties when applied to bacteria. Specifically, bacteria have a cell wall which makes eukaryotic lysis methods incompatible, bacterial mRNA has a shorter half-life and lower copy numbers, and isolating an individual bacterial species from a mixed community is difficult. Recent work has demonstrated that these technical hurdles can be overcome, providing valuable insight into factors influencing microbial heterogeneity. This perspective describes the emerging microbial scRNA-seq toolkit. We outline the benefit of these new tools in elucidating numerous scientific questions in microbiological studies and offer insight about the possible rules that govern the segregation of traits in individual microbial cells.