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Mercury is one of the most toxic heavy metals in the environment. The principal sources of contamination in wastewater are chloralkali plants, battery facilities, mercury switches, and medical wastes (12). In an aqueous environment, Hg²⁺ in sediment is subject to methylation, forming more toxic methylmercury (4). Bioaccumulation of methylmercury through the food chains is a potential risk to consumers of contaminated fish or shellfish (7). One of the most severe cases of mercury poisoning occurred in Minamata Bay, Japan, in which hundreds of people died and thousands were affected by consuming contaminated fish.

Common treatments to remove Hg²⁺ from contaminated sources are based on adsorption with ion-exchange resins (14). These technologies, however, are inadequate to reduce Hg²⁺ concentrations to acceptable regulatory standards. Another emerging technology that is receiving more attention is the use of biosorbents. The first commercial biosorbents developed (MRA and Algasorb) were based on sequestration of toxic metals by cell-surface moieties (8). These biosorbents, however, generally lack the required affinity and specificity.

The availability of genetic engineering technology provides the possibility of specially tailoring microbial biosorbents with the required selectivity and affinity for Hg²⁺. One emerging strategy that is receiving more attention is the use of metal-binding peptides. Naturally occurring metal-binding peptides, such as metallothioneins (MTs) and phytochelatins (17), are the main metal-sequestering molecules used by cells to immobilize metal ions, offering selective, high-affinity binding sites. However, the de novo design of metal-binding peptides is an attractive alternative to MTs, as they offer the potential of enhanced affinity and selectivity for heavy metals. Recently, a new class of metal-binding peptides known as synthetic phytochelatins (ECs) with the repetitive metal-binding motif (Glu-Cys)_nGly were shown to have improved Cd²⁺ binding capability over that of MTs (1).

Overexpression of metal-binding proteins such as MTs in bacterial cells resulted in enhanced Hg²⁺ accumulation and thus offers a promising strategy for the development of microbe-based biosorbents (13, 15) for the removal and recovery of Hg²⁺ from contaminated water or soil. However, Hg²⁺ removal by intracellular accumulation has been problematic because of the limited metal uptake (2). This uptake limitation could be potentially overcome either by coexpressing an Hg²⁺ transport system (3) or by anchoring the metal-binding proteins directly on the cell surface (1). In this paper, we describe the characterization of recombinant Escherichia coli strains with EC20 either anchored on the cell surface or coexpressed intracellularly with the mercury transport proteins MerP and MerT (2, 10). The ability of these strains to accumulate Hg²⁺ was investigated.

Expression of ECs. Synthetic genes coding for EC20 were synthesized as described previously (1). To express EC20 intracellularly, plasmid pM20 (1) was digested with BamHI and HindIII and the DNA fragment coding for EC20 was inserted into pMAL-c2x (New England BioLabs), resulting in pMC20. This construct allows the cytoplasmic expression of EC20 as a fusion to the maltose-binding protein (MBP).

View larger version (63K): [in this window] [in a new window]   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.

Hg²⁺ binding to EC20. To investigate the Hg²⁺ binding stoichiometry of EC20, MBP-EC20 fusion proteins were purified from cultures of JM109/pM20 using an amylose resin affinity column (New England BioLabs). The purity of the protein was confirmed through SDS-12.5% polyacrylamide gel electrophoresis. Five nanomoles of the purified fusion protein was resuspended in 50 mM Tris-Cl buffer (pH 7.4) supplemented with 5 mM dithiothreitol and incubated with 1 to 1,200 nmol of Hg²⁺ for 2 h. Hg(II)-glutathione complexes were used instead of HgCl₂ in order to prevent precipitation as reported previously (9). The protein-Hg²⁺ complex was recovered using a Microcon centrifugal filter membrane (Millipore), and the amount of bound Hg²⁺ was measured by cold-vapor atomic absorption spectroscopy (Coleman Model 5B Mercury Analyzer System). The Hg²⁺-to-MBP-EC20 stoichiometry was determined by plotting the initial Hg²⁺ concentration against the molar ratio of bound Hg²⁺ to MBP-EC20 (Fig. 2). A saturating ratio of 20 Hg²⁺ per MBP-EC20 was obtained, a value much higher than the typical ratio of 7 reported for MTs (11). A similar binding experiment was conducted with purified MBP, with no significant binding of Hg²⁺ observed.

View larger version (11K): [in this window] [in a new window]   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.

Bioaccumulation of Hg²⁺ To investigate the effect of uptake on bioaccumulation of Hg²⁺, the binding capabilities of various E. coli strains were compared. Overnight cultures grown in LB medium at 37°C were harvested, washed with distilled water twice, and resuspended to a final optical density at 600 nm (OD₆₀₀) of 1.0 in LB medium containing 5 µM Hg²⁺. The Hg²⁺ contents were determined after 1 h. As shown in Fig. 3, E. coli strain JM109/pUC18 accumulated a very low level of Hg²⁺. The intracellular accumulation of Hg²⁺ increased by sixfold for cells overexpressing MBP-EC20 (JM109/pMC20). By elimination of Hg²⁺ uptake, cells with EC20 anchored on the cell surface (JM109/pLO20) accumulated about threefold more Hg²⁺ than did cells with EC20 expressed in the cytoplasm. This threefold improvement is in good agreement with our earlier observation of Cd²⁺ accumulation using cells with surface-expressed EC20 (1). In the presence of the Hg²⁺ transporters (JM109/pCLTP/pMC20), intracellular accumulation of Hg²⁺ also increased significantly. The level of Hg²⁺ accumulation was similar to that for cells expressing EC20 on the surface. In both cases, 100% of the added Hg²⁺ was removed after 1 h. These results indicate that uptake is indeed the rate-limiting step for the intracellular accumulation of Hg²⁺.

View larger version (38K): [in this window] [in a new window]   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.

View larger version (21K): [in this window] [in a new window]   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.

Evaluation of bioaccumulation. The maximum bioaccumulation capacity of two best cell lines overexpressing EC20 (JM109/pLO20 and JM109/pMC20/pCLTP) was determined over a range of Hg²⁺ concentrations (Fig. 4B). At the lower levels (<20 nmol), 100% of the added Hg²⁺ was removed within 1 h. Although the level of EC20 expressed on the surface is approximately fivefold lower than the expression of MBP-EC20 from JM109/pCLTP/pMC20, the highest level of accumulation was around 230 nmol/mg (dry weight) for both cell lines. It has been reported previously that the bioaccumulation of Cd²⁺ by cells expressing MT on the surface exceeds by at least 1 order of magnitude the theoretical amount contributed by the surface-exposed MT moiety (16). It appears that the surface-exposed MT helps to increase the local metal concentration around the cells and facilitates interactions of the metal ions with other cell wall components. A similar situation may have occurred here for cells with surface-exposed EC20. It should be noted that the maximum bioaccumulation observed in this study is twofold higher than that reported for cells overexpressing MT and the mercury transporters (3) and falls within the higher range reported for other microorganisms (15 to 290 µmol/g of cells [dry weight]). This increase in capacity may also reflect the improved Hg²⁺ binding stoichiometry offered by EC20 over that of MT.

Conclusions. Two different strategies were used to enhance the uptake and bioaccumulation of Hg²⁺ by cells overexpressing EC20. Our results indicate that the expression of EC20 on the cell surface is as efficient as the coexpression of Hg²⁺ transporters in alleviating the uptake limitation, resulting in rapid, selective, and high-level bioaccumulation of Hg²⁺. Since specific transporters have been identified only for a few heavy metals such as mercury and nickel (5, 10), surface expression of metal-binding peptides may be useful as a common strategy to bypass the uptake of any heavy metal of interest, a highly desirable property not associated with the metal transporter systems.