Fig. 2: M3-seq reveals an acid-tolerant, bet-hedging subpopulation of E. coli in early stationary phase.

a, Uniform Manifold Approximation and Projection (UMAP) of E. coli MG1655 transcriptomes from cells at early stationary phase (OD = 2.8). Colours indicate clusters of transcriptionally similar cells. b, GO-term enrichment of select biological processes calculated with marker genes identified for cluster 2 in a. Marker gene identification and GO-term analyses were performed as described in Methods. c, Same as a but with colour gradient indicating expression of gadABC genes (in normalized UMI counts). d, Zero-centred and normalized expression of marker genes for each cluster identified in a. Marker genes were determined as described in Methods. e, Same as a but with colour gradient indicating number of UMIs captured in each cell. f, Boxplot of normalized cluster percentage for each BC1 barcode in each cluster. The normalized cluster percentage and boxplot limits were determined as described in Methods. g, Normalized fluorescence distribution of early stationary phase E. coli transformed with P_gadB-gfp. Inset is a representative composite image with phase and GFP channels overlaid. The gad+ percentage was determined as described in Methods (N = 3 biological replicates, 5,034, 1,219 and 2,171 cells analysed, respectively). Scale bar, 5 μm. h, Schematic of acid-stress recovery assay. Tubes are adapted from BioRender.com. i, Representative composite images of E. coli expressing P_gadB-gfp during recovery phase of acid-stress recovery assay depicted in h. Arrows indicate cells that divided during the recovery period. Scale bar, 5 μm. j, Distributions of fluorescence intensity of E. coli expressing P_gadB-gfp before and after acid-stress recovery assay. Orange depicts measurements from cells before acid treatment (1,833 cells) and green depicts measurements from cells at t = 0 that ultimately divided over the course of recovery (that is, survived acid treatment, N = 38 cells). Inset is a representative composite overlay of the cells 180 min after the start of recovery from the same experiment as in i. Arrows indicate cells that divided during the recovery period. Scale bar, 5 μm. P = 2.99 × 10⁻¹⁵⁶ from independent, two-sided t-test. k, Growth curves of E. coli MG1655 transformed with gfp or gadBC transgene (overexpression plasmids) and grown with or without 10 μM of IPTG (dashed curves) for 1,000 min. Curves indicate mean values and the shaded regions the 95% confidence interval of 3 technical replicates. l, Representative composite images of a mixed population of E. coli MG1655 transformed with gfp or gadBC transgene grown on an LB-agarose pad with 100 μM of IPTG. m, Single-cell growth rates of E. coli MG1655 transformed with gfp or gadBC transgene after transgene induction. Growth rates were computed as described in Methods from 539 and 112 observations, respectively, within a single set of videos (N = 1). P = 1.06 × 10⁻²³⁰ from independent, two-sided t-test. Boxplot limits are as defined in Methods.