Fructose-1,6-bisphosphate aldolase (class II) is the primary site of nickel toxicity in Escherichia coli

Abstract

Nickel is toxic to all forms of life, but the mechanisms of cell damage are unknown. Indeed, environmentally relevant nickel levels (8 µM) inhibit wild-type Escherichia coli growth on glucose minimal medium. The same concentration of nickel also inhibits growth on fructose, but not succinate, lactate or glycerol; these results suggest that fructose-1,6-bisphosphate aldolase (FbaA) is a target of nickel toxicity. Cells stressed by 8 µM Ni(II) for 20 min lost 75% of their FbaA activity, demonstrating that FbaA is inactivated during nickel stress. Furthermore, overexpression of fbaA restored growth of an rcnA mutant in glucose minimal medium supplemented with 4 µM Ni(II), thus confirming that FbaA is a primary target of nickel toxicity. This class II aldolase has an active site zinc and a non-catalytic zinc nearby. Purified FbaA lost 80 % of its activity within 2 min when challenged with 8 µM Ni(II). Nickel-challenged FbaA lost 0.8 zinc and gained 0.8 nickel per inactivated monomer. FbaA mutants (D144A and E174A) affecting the non-catalytic zinc were resistant to nickel inhibition. These results define the primary site of nickel toxicity in E. coli as the class II aldolase FbaA through binding to the non-catalytic zinc site.

Fig. 1

Nickel is toxic at micromolar levels and sensitivity is dependent on the carbon source. Cells were grown aerobically at 37 °C. (A) Wild-type cells (MG1655) and (B) rcnA mutant cells (LEM201) were grown on M9 glucose medium and challenged with 0 µM (□), 2 µM (●), 4 µM (▲), or 8 µM Ni(II) (♦). (C) Wild-type cells (MG1655) were grown in M9 medium containing fructose (open symbols) or glycerol (closed symbols) and challenged with 0 µM (squares) or 8 µM (diamonds) Ni(II). (D) Wild-type cells (MG1655, open symbols) and gnd mutant cells (LEM202, closed symbols) were grown in M9 gluconate medium containing 0 µM (squares) or 16 µM (crucibles) Ni(II). The data are representative of three independent experiments.

Fig. 2

Nickel toxicity occurs by inhibition of fructose-1,6-bisphosphate aldolase. Cells were grown aerobically in M9 glucose medium at 37 °C. (A) At OD₆₀₀ ~0.1, wild-type cells (MG1655) were challenged with 0 µM (□) or 8 µM (♦) Ni(II) and FbaA activity was measured in cell-free extracts after selected time points. (B) rcnA mutant cells containing the vector (pWKS30; LEM233; open symbols) or pFbaA (pLEM1; LEM234; closed symbols) were challenged with 0 µM (squares) or 4 µM (diamonds) Ni(II) and further growth was monitored. The data are representative of three independent experiments.

Fig. 3

RcnA is induced concomitantly with nickel-dependent FbaA damage. LEM273 (rcnA lacZ P_rcnA-lacZ) were grown in M9 glucose medium to OD₆₀₀ of 0.1. Cultures were split and 0 µM NiCl₂ (gray bars) or 1 µM NiCl₂ (black bars) was added. Cultures were incubated aerobically at 37 °C for 2 hours. White bars represent enzyme activity prior to the culture being split. Error bars represent the standard deviation of three independent experiments.

Fig. 4

Nickel-damaged FbaA is reactivated in vitro. Wild-type MG1655 cells were grown aerobically in M9 glucose medium at 37 °C. At OD₆₀₀ ~0.1, the cells were challenged with 16 µM Ni(II). After an hour exposure, chloramphenicol (150 µg ml⁻¹) was added to inhibit protein synthesis. Cells were washed with M9/glucose/chloramphenicol medium to remove nickel, resuspended in the same medium, incubated aerobically at 37 °C, and assayed for FbaA activity at the times indicated. Error bars represent the standard deviation of three independent experiments.

Fig. 5

FbaA is inhibited by nickel, whereas zinc-loaded enzyme is resistant to nickel damage. Purified FbaA was challenged with nickel aerobically at room temperature (25 °C). (A) FbaA (50 nM) was incubated with 0 µM (□) or 8 µM (♦) Ni(II) and enzyme activity was assessed at the indicated times. (B) FbaA as purified (90 nM) (□) or Zn-loaded (30 nM) (■) was incubated with the indicated concentrations of Ni(II) for 5 min and assayed. (C) The zinc contents of FbaA were determined by ICP-OES for FbaA as purified and after Zn-loading, including samples challenged with 0 µM (white) or 256 µM Ni(II) (grey) for 10 min. (D) The nickel content of FbaA was determined by ICP-OES for FbaA as purified (black) and after Zn-loading (white), both challenged with 256 µM Ni(II) for 10 min. (C & D) In each case, unbound metal was removed by gel filtration. Error bars represent the standard deviation of three independent experiments.

Fig. 6

Nickel inactivates FbaA at the secondary zinc binding site. As purified His-tagged FbaA (400 nM; 0.4 U/mg; □) and its corresponding D144A (200 nM; 0.6 U/mg; ■), E174A (300 nM; 0.1 U/mg; ●), C177A (25 nM; 0.4 U/mg; ♦), and E181A (1000 nM; 0.08 U/mg; ▲) variants were challenged with the indicated nickel concentrations for 5 min aerobically at room temperature (25 °C) and tested for remaining activity. Error bars represent the standard deviation of three independent experiments.

Fig. 7

Cellular metabolites affect nickel-dependent damage to FbaA. (A) As purified FbaA (20 nM) was challenged with 0 µM (open symbols) or 16 µM (closed symbols) Ni(II) in the absence (squares) or presence of 100 µM His (circles) or 1 mM reduced glutathione (GSH; triangles) aerobically at 25 °C, and the enzyme was assayed at the indicated times. The error bars represent the standard deviation of three independent experiments. (B) Mutant E. coli cells LEM201 (rcnA, squares) and LEM267 (gshA rcnA, circles) were grown aerobically at 37 °C in M9 glucose medium and challenged with 0 µM (open symbols) or 1 µM (closed symbols) Ni(II). The data are representative of three independent experiments.