Figure 3. Overexpression of endogenous DHFR triggers a major metabolic shift.
(A) Heat-map of the intracellular levels of various metabolites detected in bacterial cells over-expressing endogenous ADK and DHFR proteins from a pBAD plasmid. While ADK over-expression does not alter the metabolite levels, DHFR over-expression results in a pronounced up-regulation of AICAR and dUMP levels, and down-regulation of purines and pyrimidine nucleotides (dTMP, dTTP, AMP, IMP) (See Materials and methods). In all experiments, sample size was three biological replicates. For statistical significance, levels in DHFR over-expression were compared to ADK over-expression, where * denotes p-value<0.05 and ** denotes p-value<0.001. (B) Metabolic complementation by addition of purine and pyrimidine nucleotides along with several amino acids, like serine and tryptophan, results in a partial rescue of fitness. 1 mM of each of the metabolites was added to supplemented M9 medium at 37°C. Growth rates under different conditions were normalized by the growth rate obtained with empty plasmid without addition of any metabolite, and was referred to as ‘relative growth rate’. For all experiments the arabinose concentration used was 0.05%, which corresponds to ~850 fold over-expression of DHFR and ~5000 fold increase of ADK.
Figure 3—figure supplement 1. Overlay of growth curves of E.coli over-expressing DHFR and those in the presence of various metabolites.
