Figure 1. Over-expression of endogenous DHFR is detrimental to bacterial growth.
(A) The effect of variation in DHFR dosage on fitness of E. coli. Change in the intracellular DHFR abundance (in log scale) is plotted against growth rate. DHFR abundance and bacterial growth are normalized against the parameters observed for wild-type strain. In case of over-expression, the growth rates were normalized with values obtained with no inducer (arabinose) (also see Figure 1—source data 1). Controlled drop (over 100 fold) in DHFR abundance (blue circles) was induced by IPTG titration of the chromosomal folA gene of E. coli MG1655 strain at 30°C which was modified to contain LacI binding sites (see Materials and methods and [Bershtein et al., 2013]) and shows a Michaelis-Menten type dependence on fitness described in (Bershtein et al., 2013). A controlled increase (~850 fold) in DHFR abundance (red circles) was achieved by arabinose titration of E. coli BW27783 strain transformed with a plasmid at 37°C carrying the endogenous folA gene under the control pBAD promoter. DHFR over-expression shows a strong dose-dependent drop in fitness (Spearman r = −1, p=0.0167). The obtained abundance vs fitness function shows that the basal endogenous DHFR levels approach a physiological optimum with respect to E. coli growth rate. Overexpression of C-terminal His-tagged EcDHFR generated identical fitness drop (Figure 4—figure supplement 2). (B) Fitness as a function of protein abundance shown for EcDHFR, E. coli Adenylate Kinase (ADK) and a eukaryotic non-endogenous protein H-ras p21 (Ras). Only the expression of DHFR shows a dose-dependent toxicity and, therefore, it is not a generic effect of an over-expression burden. The intracellular DHFR, Ras, and ADK abundances were measured by Western Blot with custom raised antibodies (also see Materials and methods).
DOI: http://dx.doi.org/10.7554/eLife.20309.002
Figure 1—figure supplement 1. Growth rate (relative to untransformed cells) of E.coli cells transformed with an empty pBAD plasmid over a range of arabinose concentrations.
