Escherichia coli response to uranyl exposure at low pH and associated protein regulations

Abstract

Better understanding of uranyl toxicity in bacteria is necessary to optimize strains for bioremediation purposes or for using bacteria as biodetectors for bioavailable uranyl. In this study, after different steps of optimization, Escherichia coli cells were exposed to uranyl at low pH to minimize uranyl precipitation and to increase its bioavailability. Bacteria were adapted to mid acidic pH before exposure to 50 or 80 µM uranyl acetate for two hours at pH≈3. To evaluate the impact of uranium, growth in these conditions were compared and the same rates of cells survival were observed in control and uranyl exposed cultures. Additionally, this impact was analyzed by two-dimensional differential gel electrophoresis proteomics to discover protein actors specifically present or accumulated in contact with uranium.Exposure to uranium resulted in differential accumulation of proteins associated with oxidative stress and in the accumulation of the NADH/quinone oxidoreductase WrbA. This FMN dependent protein performs obligate two-electron reduction of quinones, and may be involved in cells response to oxidative stress. Interestingly, this WrbA protein presents similarities with the chromate reductase from E. coli, which was shown to reduce uranyl in vitro.

Figure 1. Schematic view of E. coli cells exposure to uranium and details of the optimized analysis protocol.

Figure 2. Uranium speciation in the exposure medium.

Uranium L_III-edge XAS spectra (A), normalized to equal intensity at 17.176 keV; and k³-weighted EXAFS curves (B) of uranyl acetate 50 µM prepared in LB-glucose medium at pH 2.7 (U-LBG pH 2,7) compared to reference spectra.

Figure 3. Cell survival after exposure to uranyl at acidic pH.

Numbering of Colony Forming Units from the E. coli culture in LB-Mes pH 5.5, and in the exposure LB 1/10 Glucose medium at pH 2.7 before (T0) and after exposure for 2 hours to 50 µM sodium acetate, 50 µM of uranyl acetate, and 80 µM of uranyl acetate.

Figure 4. Protein regulations from 2D-gel analysis.

A: Principal component analysis performed on the proteomes of control cells and cells exposed to 50 and 80 µM of uranyl acetate. Each colored cross represents one independent replicate. The numbers in grey represent the protein spots which are impacted by the uranium stress in a statistically pertinent manner. B: Two-dimensional gel-electrophoresis obtained with a control sample (2 hours sodium acetate), showing the position of up-regulated proteins (red circles) and down-regulated proteins (blue circles) in uranyl exposed cells (protein loading: 100 µg; silver nitrate staining).