Fig. 4: Increased metabolite export activity in auxotrophic cells promotes drug tolerance.
a, SeMeCos cultures were incubated with DiOC5(3), and flow cytometry was used to assess fluorescent populations as described. Prototrophs, single and double auxotrophs (red gradient) and triple-auxotrophic (yellow) populations in SeMeCos were gated by TagRFP657 fluorescence. Gaussian curve fitting and cluster assignment via mclust88 identified subpopulations carrying different levels of auxotrophy. Prototrophic populations proportionally retain, on average, greater dye fluorescence, indicating a slower export of DiOC5(3). n = 20,000 cells from a single culture, of which 6,330 live cells were taken for downstream analysis. H, L and M indicate auxotrophy for histidine, leucine and methionine, respectively; PRO, prototrophic subpopulation. b, SeMeCos cultures were incubated with DiOC5(3), fixed and analyzed via fluorescence microscopy. Prototrophs and auxotrophs were identified by fluorescence, with two populations corresponding to low and high dye retention identified. Boxplots represent median (50% quantile (middle line), lower (25%) and upper (75%) quantiles, respectively); n = total number of cells counted per population; med, median fluorescence intensity per cell. Data are from one independent experiment. c, Intracellular concentration of uniconazole in auxotrophic and prototrophic subpopulations as sorted from singly auxotrophic SeMeCos, and measured by LC–MS/MS. Mean ± s.d. from a single injection from n = 3 biological replicates. Statistical significance was determined using a two-sided Student’s t-test, ****P < 0.00005. Exact P values are available in Source Data 4. d, Plating of auxotrophic and prototrophic subpopulations from sorted single auxotrophic SeMeCos onto SM or SM-supplemented medium with the complementary amino acids (+H/L/U/M) permitted titration of A:P ratios. Cropped DDA denoted by red-bounded region. e, DDA for the four sorted SeMeCos strains exposed to uniconazole plated onto SM or SM + H/L/U/M. DDAs were generated from a single-sort experiment, plated and exposed to uniconazole. a.u., Arbitrary units.
