Single-cell analyses to dissect morphological heterogeneity in the gut commensal Bacteroides thetaiotaomicron

Genetically identical bacteria within the same micro-environment may not behave alike. This is exemplified by the abundant gut microbiota member Bacteroides thetaiotaomicron, whose name derives from the three morphotypes (dubbed "Theta", "Iota" and "Omicron") that these bacteria can adopt. However, the molecular basis of this morphological heterogeneity and its functional consequences are unknown. Here, we map the transcriptome of Bacteroides morphotypes using sensitive "mini-bulk" RNA-seq. We observed that across four biological replicates, the different morphotypes upregulate specific sets of marker genes, encoding proteins involved in primary metabolism and membrane-associated processes. A subset of morphotype-specific markers are validated using single-molecule fluorescence in-situ hybridization and by single-bacterium RNA-seq. Lastly, reanalysis of existing high-throughput imaging data derived from an ordered transposon insertion library of B.thetaiotaomicron in combination with confocal microscopy of clean deletion mutants, reveals the influence of individual genes on bacterial cell shape. Together, our work lays the ground to dissect the extent and impact of cell-to-cell variability in this dominant bacterial species of the human microbiota.