Fig. 4: M3-seq characterizes independent activation of prophages in B. subtilis.
a, UMAP of B. subtilis transcriptomes from ciprofloxacin- and nalidixic acid-treated cells in exponential phase (OD = 0.3). Colours indicate treatment conditions (90 min). b, Same as a but with colours indicating clusters of transcriptionally similar cells. c, Pseudobulk gene expression of the two prophages in the DNA-damaging antibiotic-treated conditions (yellow) compared to exponential phase (grey). d, Same as a but with colour gradient indicating percentage of PBSX prophage UMIs within each cell. Percentages were calculated by dividing the total number of PBSX UMIs by the total number of UMIs in each cell. e, Same as a but with colour gradient indicating percentage of SPβ prophage UMIs within each cell. f, Schematic of B. subtilis genome with location of PBSX and SPβ prophages indicated. g, Zero-centred and normalized expression of marker genes for each of 7 clusters identified in b. Marker genes were defined as detailed in Methods, where a maximum of 5 genes were included per cluster. h, Classification of cells with induced prophages. Green indicates cells with relative expression of PBSX genes >8.4% per cell, which is >10th percentile of PBSX prophage gene expression in cluster 5 from b. Red indicates cells with relative expression of SPβ genes >15.0% per cell, which is >10th percentile of SPβ prophage gene expression in cluster 6 from b. Brown indicates cells above both thresholds. i, Schematic of prophage classification results. The expected independent co-induction probability (calculated from observed PBSX and SPβ percentages) is 2.5%. j, Dual-colour smFISH of B. subtilis with no-drug treatment (left), or B. subtilis treated with ciprofloxacin for 90 min (right). Probes hybridizing to PBSX genes were labelled with a green fluorophore. Probes hybridizing to SPβ genes were labelled with a red fluorophore. Scale bar, 5 μm. k, Fluorescent reporter fusions of B. subtilis PL-gfp (PBSX promoter) and PyonO-mKate2 (SPβ promoter) treated with no drug (left), or treated with ciprofloxacin (right) for 150 min to allow for maturation of the fluorescent protein. Percentages of induction were calculated from a single set of acquired images (N = 1,394 cells). Scale bar, 5 μm.
