Fig. 4: FACS and proteomics link producer/consumer status to an antimicrobial resistance phenotype.

a, Experimental scheme used to test for phenotypic differences between producer and consumer subpopulations. A ¹³C-labelled, heterogeneous population of cells from a colony is resuspended and challenged with the fungicidal drug amphotericin B. Live and dead populations are then separated physically using FACS and their producer/consumer status determined using targeted proteomics as before. This method can directly link a metabolic phenotype to one with a fluorescent readout. b, Lysine producer cells of colonies were found to be significantly more likely to survive amphotericin B treatment (mean fraction of producer cells in live population 26.6% versus 12.1% in the dead population; P = 0.0004, paired two-sided t-test, n = 4 biological replicates (biol. rep.)). Error bars indicate s.d. Lines indicate peptide pairs across colour-coded biological replicates. Following convention, box-plot elements are defined as follows: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. c, This difference in susceptibility is not due to lysine production alone. No clear difference in susceptibility was observed when whole colonies, which were either producers (no supplement) or consumers (400 μM lysine), were challenged with varying concentrations of amphotericin B. d, Difference in susceptibility is linked to the position of cells in the colony. Physically separated top and bottom cells from non-supplemented colonies were challenged with various concentrations of amphotericin B. Bottom-layer cells were significantly more susceptible (at 5 μg ml^–1: mean fraction of dead cells in bottom layer 32% versus 22% in the top layer; P = 0.0005, paired two-sided t-test (see inset)). c,d, Lines and shaded areas represent mean and s.d., respectively. **P < 0.005, ***P < 0.0005.