Construction and characterization of an Escherichia coli strain genetically engineered for Ni(II) bioaccumulation

Abstract

An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase-pea metallothionein fusion protein. The resulting strain accumulated 15 micromol of Ni(II) per g (dry weight) from a 10 microM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.

FIG. 1

Ni(II) bioaccumulation by E. coli cells carrying different plasmids. Induced JM109 cells containing the indicated plasmids were resuspended in phosphate buffer containing 30 μg of chloramphenicol per ml and 10 μM Ni(II) and shaken at 37°C for 1 h.

FIG. 2

(A) Time course of Ni(II) bioaccumulation. Induced cells containing pKNA and pGPMT were resuspended in phosphate buffer containing 10 μM Ni(II) and were shaken at 37°C for the indicated amounts of time. (B) Ni(II) bioaccumulation isotherms. Induced cells expressing both a nickel transporter and PMT-GST fusion protein (pKNA/pGPMT) and control cells expressing neither (pK187/pGEX-2T) were resuspended in phosphate buffer at various levels of Ni(II). Cells were shaken at 37°C and harvested after 1 h.

FIG. 3

(A) Effect of ionic strength on Ni(II) bioaccumulation. Induced cells containing pKNA and pGPMT were resuspended in phosphate buffer containing 10 μM Ni(II) and the indicated concentrations of NaCl. Cells were shaken at 37°C and harvested after 1 h. (B) Effect of pH on Ni(II) bioaccumulation. Induced cells containing pKNA and pGPMT were resuspended in phosphate buffer adjusted to the indicated pH and containing 10 μM Ni(II). Cells were shaken at 37°C and harvested after 1 h.