Fig. 4: Extracellular metabolite measurements reveal spatiotemporal cross-feeding of pyruvate.

a, To measure extracellular metabolites, cells were removed from the swarming plate before extracting a small piece of agar from up to 3 positions indicated by stars in different colours (orange position: p = 0 mm, pink position: p = 10 mm, blue position: p = 20 mm). Positions p were measured as radial distances from the point of inoculation. Similar samples were acquired for swarms that were grown for different times to measure extracellular metabolites at 3 spatial locations over time. Metabolites were extracted from the agar and measured using mass spectrometry. Created using BioRender.com. b, In comparison to the RNA-seq measurements (grey tiles), sampling points for extracellular metabolites (coloured stars) were sparser in space but were acquired for a longer period of time. c,d, Extracellular malate and succinate concentrations in the agar decreased to zero with increasing time, indicating that these compounds were consumed by the cells and eventually depleted. Colours indicate the 3 different sampling positions as described in a. e, The concentration of pyruvate increased over time before decreasing again, indicating that this metabolite was deposited by cells growing in malate- and succinate-rich medium, and later consumed by daughter cells as well as cells that have migrated to this position. Pyruvate is therefore cross-fed in space and time. In c–e, dots represent acquired samples, lines and shaded regions correspond to the mean ± s.d. calculated using n = 4 biologically independent samples in all cases except for the last timepoint, where n = 3 biologically independent samples were used.