Fig. 8: Wild-type cells evolve altered buffering capacity with evolution of a different metabolome. | Nature Communications

Fig. 8: Wild-type cells evolve altered buffering capacity with evolution of a different metabolome.

From: Distinct metabolic states of a cell guide alternate fates of mutational buffering through altered proteostasis

Fig. 8

a Schematic of strategy for Laboratory Adaptive Evolution of osmotolerant strains of E. coli starting from BW (wild-type E. coli K-12, BW25113). b Growth curve of unevolved BW and evolved osmotolerant strains E5, F12, and H8 in 500 mM of NaCl added in excess to LB medium while growing at 37 °C, 200 rpm. c Genetic interaction network map of evolved strains E5 and H8 based on mutations obtained in genome sequencing. d Metabolite features of representative evolved osmotolerant strains (E5, F12, and H8) and its comparison with BW. The colored circled represent metabolites that are significantly altered in the evolved strains. e Histogram for GFP/mCherry fluorescence of Wt, C1, and C6 GFP in the BW and evolved strains (E5, F12, and H8). f Comparative quantification of chaperone proteins DnaK and GroEL in BW and evolved strains (E5, F12, and H8) done by immunoblotting with specific antibodies. Quantification of the chaperone levels are shown as bar graphs representing mean values (2-sided Student’s t-test p-value > 0.05). g. Chloramphenicol based chase for checking protein degradation rates were performed in BW and evolved strains (E5, F12, and H8) as discussed earlier. Mean of C1 GFP levels at different time points are shown in different strains. h Heatmap for growth-based activity of Gm-R mutants and Wt Gm-R in unevolved strain (BW) and evolved strains (E5, F12, and H8) in increasing concentrations of gentamicin. i Bar graph representing percentage MIC of a selected set of Gm-R mutants w.r.t Wt Gm-R in the respective strains. Mean of four biological replicates is plotted with standard deviation as error bars (unpaired 2-sided Student’s t-test *p-value for M1(H8:0.04, F12:0); M2(H8:0.002, F12:0.0002); M3(H8, F12:0); M4(H8:0.002); GG1(F8, F12, E5:0); GG2(H8, E5:0); GG3(H8:0.01, E5:0.002); GG4(H8, F12:0). For b, f, and g: Standard deviations are plotted as error bars from 3 biological replicates. Also see Supplementary Fig. 6 (Source data are provided as source data file Fig. 8).

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