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. 2016 Dec 10;5:e20309. doi: 10.7554/eLife.20309

Figure 4. Orthologous DHFR proteins do not cause metabolic imbalance and dosage-related toxicity.

Fitness is shown as a function of (A) intracellular DHFR abundance and (B) (abundance * kcat/KM) for overexpression of E. coli DHFR as well as those from five mesophilic bacteria Listeria innocua (DHFR 6), Bordetella avium (DHFR 11), Leuconostoc mesenteroides (DHFR 12), Aeromonas hydrophila (DHFR 13), Clostridium cellulolyticum (DHFR 16) and Streptococcus dysgalactiae (DHFR 17) expressed from the same plasmid under pBAD-promoter using an inducer concentration of 0.05%. Fitness of cells over-expressing orthologous DHFRs was significantly different from those expressing E. coli DHFR at equivalent concentrations (in all cases, p-value<0.001) (also see Materials and methods) and only endogenous EcDHFR was highly toxic to E. coli. The intracellular abundance of orthologous DHFR was measured by Western Blot with anti-His antibodies (See Materials and methods). Overexpression of orthologous DHFR proteins lacking the His-tag produced identical results (Figure 4—figure supplement 2). (C) Heat-map of the intracellular levels of various metabolites detected in cells overexpressing endogenous ADK and E. coli DHFR as well as two orthologous DHFRs (6 and 11). An empty pBAD plasmid is used as a control. Metabolite levels in orthologous DHFR over-expression were compared to EcDHFR over-expression for statistical significance, where * denotes p-value<0.05 and ** denotes p-value<0.001. Hence when compared to E. coli DHFR, orthologous DHFRs do not cause any major perturbation in the intracellular purine and pyrimidine levels. In all experiments, sample size was three biological replicates. (D) Growth rate of E. coli over-expressing ADK, E. coli DHFR, and orthologous DHFRs 6, 11 and 16 in the presence of 0.05% arabinose is shown as a function of the intracellular dTTP levels. All data were normalized by growth rate of E. coli transformed with the empty plasmid. The data shows that fitness is tightly correlated to cellular metabolite levels (Pearson r = 0.99, p=0.0002).

DOI: http://dx.doi.org/10.7554/eLife.20309.008

Figure 4—source data 1. Raw and normalized data for metabolomics.
DOI: http://dx.doi.org/10.7554/eLife.20309.009
elife-20309-fig4-data1.xlsx (14.1KB, xlsx)
DOI: 10.7554/eLife.20309.009
Figure 4—source data 2. Molecular properties of orthologous DHFR proteins.
DOI: http://dx.doi.org/10.7554/eLife.20309.010
elife-20309-fig4-data2.xlsx (15KB, xlsx)
DOI: 10.7554/eLife.20309.010

Figure 4.

Figure 4—figure supplement 1. Activity (vmax) of over-expressed DHFR from whole cell lysate.

Figure 4—figure supplement 1.

The initial rate of conversion of 50 µM Dihydrofolate (DHF) to THF was measured in the presence of 100 µM NADPH in 50 mM MES buffer for E. coli over-expressing different DHFRs. As expected from their in vitro activity parameters, over-expressed orthologous DHFR proteins were more active than E. coli DHFR even in the lysate.
DOI: http://dx.doi.org/10.7554/eLife.20309.011
Figure 4—figure supplement 2. The hexa-histidine tag on DHFR is neutral to fitness.

Figure 4—figure supplement 2.

E. coli DHFR, DHFR 6 and 11 were cloned in pBAD plasmid with and without a C-terminal his-tag and growth rate of BW27783 cells transformed with the plasmids were measured using an inducer concentration of 0.05%.
DOI: http://dx.doi.org/10.7554/eLife.20309.012