Ushering in a new era of single-cell transcriptomics in bacteria

Abstract

Transcriptome analysis of individual cells by single-cell RNA-seq (scRNA-seq) has become routine for eukaryotic tissues, even being applied to whole multicellular organisms. In contrast, developing methods to read the transcriptome of single bacterial cells has proven more challenging, despite a general perception of bacteria as much simpler than eukaryotes. Bacterial cells are harder to lyse, their RNA content is about two orders of magnitude lower than that of eukaryotic cells, and bacterial mRNAs are less stable than their eukaryotic counterparts. Most importantly, bacterial transcripts lack functional poly(A) tails, precluding simple adaptation of popular standard eukaryotic scRNA-seq protocols that come with the double advantage of specific mRNA amplification and concomitant depletion of rRNA. However, thanks to very recent breakthroughs in methodology, bacterial scRNA-seq is now feasible. This short review will discuss recently published bacterial scRNA-seq approaches (MATQ-seq, microSPLiT, and PETRI-seq) and a spatial transcriptomics approach based on multiplexed in situ hybridization (par-seqFISH). Together, these novel approaches will not only enable a new understanding of cell-to-cell variation in bacterial gene expression, they also promise a new microbiology by enabling high-resolution profiling of gene activity in complex microbial consortia such as the microbiome or pathogens as they invade, replicate, and persist in host tissue.

Keywords: MATQ-seq; PETRI-seq; heterogeneity; microSPLiT; par-seqFISH; single-cell RNA-seq.

Figure 1.

Challenges in bacterial single-cell transcriptomics. Bacterial single-cell transcriptomic studies come along with various challenges related to the specific features of bacterial mRNA, cell wall composition, and cell scale. Thus, method development requires consideration of technical methods for resolution of these challenges. Created with Biorender.com.

Figure 2.

Bacterial scRNA-seq workflows. Generic bacterial scRNA-seq workflows: (A) MATQ-seq is a multiple annealing and tailing-based workflow, which enables targeting of low abundant transcripts in single bacteria. After RT, poly-C tailing, second strand synthesis, and PCR amplification, cDNA suitable for library preparation and sequencing is generated. (B) microSPLiT as well as PETRI-seq are based on split-pool barcoding system. After permeabilization and fixation of cells, three rounds of barcoding using RT and ligation process are performed. Thereby, each cDNA within a cell receives a unique barcode combination, which is used as cell identity. Barcoding is followed by cell lysis, library preparation, and sequencing. (C) Spatial transcriptomics of single bacteria: par-seqFISH is based on sequential FISH technology using sets of primary and secondary probes specifically targeting a selection of genes for high-throughput microscopy. Rounds of hybridization and stripping of three probes per cycle allow detection of gene expression profiles gene by gene. An overlay of the generated images results in a merged transcriptome map providing gene expression profiles of individual bacteria including spatial information.