Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury

Abstract

Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)(n)Gly, which were shown to bind heavy metals more effectively than metallothioneins. However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides. In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg(2+) transport system with (Glu-Cys)(20)Gly (EC20) or by directly expressing EC20 on the cell surface. Both approaches were equally effective in increasing the bioaccumulation of Hg(2+). Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.

FIG. 1

Expression of EC fusion proteins. [³⁵S]cysteine was added to the cultures at an OD₆₀₀ of 0.3. The cultures were further grown for 15 h. Total cell proteins were separated on SDS–12.5% polyacrylamide gel electrophoresis. The gel was dried and autoradiographed. Expression from induced cultures harboring pLO20 (lane 1), pMC20/pCLTP (lane 2), pMC20 (lane 3), and pMAL-c2x (lane 4), respectively, is shown. The desired fusion proteins are marked with arrows. Molecular mass is shown in kilodaltons on right.

FIG. 2

The Hg²⁺-to-MBP-EC20 stoichiometry expressed as the plot of initial Hg²⁺ concentration against the complexed Hg²⁺-to-peptide ratio. Five nanomoles of purified MBP-EC20 was incubated with 1 to 1,200 nmol of Hg²⁺ in 5 mM dithiothreitol for 1 h. The portion of bound and unbound Hg²⁺was determined by a mercury analyzer.

FIG. 3

Bioaccumulation of Hg²⁺ by resuspended cultures harboring various plasmids from LB medium containing 5 μM Hg²⁺. Data were obtained from three independent experiments.

FIG. 4

(A) Time course of mercury uptake by resting cultures harboring various plasmids. Resting cultures were resuspended in LB medium containing 5 μM Hg²⁺ and were incubated for 20 min. Cells were harvested at various times, and the supernatant was removed by centrifugation for 30 s. (B) Hg²⁺ bioaccumulation capacity of JM109 cells (0.265 mg [dry weight]) harboring either pLO20 or pCLTP/pMC20. Bioaccumulation of Hg²⁺ was measured at various concentrations after 1 h of incubation. (C) Effect of cadmium on bioaccumulation of Hg²⁺. JM109 cells harboring either pLO20 or pCLTP/pMC20 were incubated with 5 nmol of Hg²⁺ and various concentrations of Cd²⁺. The amount of Hg²⁺ accumulated by cells after 1 h was determined.