Fig. 2: Genetic alteration in cellular metabolism changes mutational buffering.

a Schematic for activity assay for glycine-doublet substitution library of Gm-R. Gm-R activity is inferred from competitive fitness of the mutants under gentamicin selection and deep-sequencing. b Schematic for activity assay of GFP-mutant library. An arabinose inducible promoter drives the bicistronic construct of GFP and mCherry. GFP is mutated using random mutagenesis. GFP mutants are sorted into population of compromised mutants (Pc) and active mutants (Pa) based on GFP Fluorescence. c Gene Ontology (GO) classes upregulated in WG350 transcriptome with respect to CSH4 shown as fold enrichment on the left-axis and Benjamini-Hochberg FDR corrected p-values obtained using DAVID⁶³ on the right axis from two biological replicates. d Comparison of metabolites in WG350 and CSH4 using untargeted metabolomics. Colored circles represent significantly different metabolites between CSH4 and WG350 obtained using paired 2-sided Student’s t-test (p-value < 0.05, five biological replicates for each sample). e Mean of normalized read counts of Gm-R GG-mutant library at comparative selection pressure of gentamicin (Gm) from two biological replicates. Pink shaded area marks 99% confidence interval. Mutants marked in red show lower read counts in WG350 than in CSH4. f Conservation score (calculated using Consurf) for each residues that are mutated in the clones studied. g Fractional ASA (calculated using VADAR) of the mutated residues against the temperature factor of the residues obtained from PDB file 1BO4 (left panel). h Distance of the residues mutated from the ligand plotted against the predicted alteration in protein stability. Error bar represents standard deviation from three replicates (calculated using FoldX). i Heatmap representing growth of Gm-R mutants in WG350 and in CSH4 along with Wt Gm-R in increasing gentamicin (Gm) concentration (left panel). MIC of each mutant normalized with respect to Wt Gm-R in the respective strains (right panel). Mean is plotted with standard deviations as error bars from four biological replicates. Significance is calculated using 2-sided Students’ t-test with respect to CSH4 (*p-value M1:0.03, M3:0.03; ***p-value M2:0.0001, M4:0.0001). Also see Supplementary Fig. 1 (Source data are provided as source data file Fig. 2).