Fig. 2: Auxotrophs promote a rich metabolic environment that increases drug tolerance in prototrophs.
a, Left: genome-scale metabolic modeling in SeMeCos composed of auxotrophic and prototrophic subpopulations (n = 4, H/L/U/M community models). Significant increase (change >10%) in the number of metabolic fluxes (second from left, P = 7 × 10–4, metabolite exchange (second from right, P = 0.02) and exchange of amino acids (right, P = 0.002) in auxotrophs compared to prototrophs, shown as boxplots. Significance was calculated by two-sided Student’s t-test. b, Prototrophic community generated by genomic repair of HIS3, LEU2, URA3 and MET15 (WT), as opposed to SeMeCos containing auxotrophs due to the stochastic segregation of plasmids containing the four auxotrophic markers. c, Left, middle: quantification of intra- and extracellular metabolites by mass spectroscopy39 in exponentially growing SeMeCos compared to WT cultures in SM medium. Metabolite concentrations were normalized to biomass, as assessed by optical density at OD600. Grouped metabolite comparison (box plots) significance was determined using a one-sided Kruskal–Wallis rank-sum test. Mean ± s.e.m. of n = 8 independent cultures per strain from two independent experiments. Individual metabolite comparison (bar plots) significance was determined using an unpaired two-sided Wilcoxon rank-sum test: *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005; exact P values are given in Source data 2. Right: proportion of auxotrophs and prototrophs in SeMeCos analyzed above, calculated by spotting colonies onto selective medium. Mean ± s.e.m. of n = 6 independent cultures from two independent experiments. d, Drug resistance (diameter of inhibition halo) and tolerance (growth within halo) measured by DDA22 in WT colonies in minimal (SM) or SM + HLUM-supplemented medium treated with uniconazole or miconazole, respectively. DDAs generated from WT cultures plated onto SM or SM + HLUM and/or azoles. One DDA per drug is illustrated. e, Growth of WT yeast cultures, assessed by OD600 and plotted as AUC, under increasing concentrations of uniconazole/miconazole and following increasing HLUM supplementation. Boxplots represent median (50% quantile (middle line)), lower (25%) and upper (75%) quantiles, respectively, of change in metabolic flux and amino acid and metabolite exchanges in auxotrophs compared to prototrophs in a, and pooled metabolite FC levels compared to WT in c.
