Extended Data Fig. 10: Auxotrophy correlates with DIOC5(3) export and azole tolerance independent of cell size in SeMeCos.
(a) Construction of the 3-plasmid, TagRFP657 fluorescent SeMeCo strain. Auxotrophy was repaired in the parental BY4741 strain via plasmid complementation with pH, pL and pM (all encoding TagRFP657) and genomic knock-in of URA3. (b) Scatter plot of DIOC5(3) against TagRFP657 fluorescence intensity in 20,000 cells. Correlation was tested using a two-sided, Spearman’s Rank Correlation Coefficient (P value < 2.2e-16) with a R-value of 0.53 indicating a positive correlation. (c) Total ion chromatograms (TIC) and extracted ion chromatograms (XIC) corresponding to uniconazole in standard and cell pellet extracts from sorted cells. Peaks at <1 and ~4 min retention time correspond to highly hydrophilic and hydrophobic metabolites respectively. XIC at m/z of 292.121 was used to calculate concentration of uniconazole in extracts from a standard curve generated from the analytical standard (Sigma, 37044). Fragments at m/z 70.030 and 43.010 correspond to protonated triazole and loss of CNH thereof respectively. (d) DDA for all four sorted single-plasmid-CFP SeMeCos exposed to miconazole plated onto SM or SM + H/L/U/M. DDAs were generated from a single sort experiment and exposed to miconazole. (e) Summary of the changes in cell size against SeMeCo composition change as measured in the drug screen. Cell size was defined as the pixel area covered by a cell capture via high-throughput microscopy. DMSO (mean) indicates the global mean value from all DMSO treated wells. Drugs (Azoles) indicate azoles within the 900-FDA compounds collection, dotted lines indicate range of cell size changes for azole treated cells. Error bars indicate standard deviation of prototroph percentage or cell size from 3 biological replicates. Data are presented as mean values + /- SD.
