Expression of the Neurospora crassa metallothionein gene in Escherichia coli and its effect on heavy-metal uptake

Abstract

The gene coding for the Neurospora crassa metallothionein protein was chemically synthesized and cloned into the fusion expression vectors pMal-c and pMal-p. Cell-fractionation experiments demonstrated the proper localization of the pMal-c and pMal-p- expressed proteins to the cytosol and periplasm of the bacteria respectively. Control bacteria as well as the recombinant bacteria producing the metallothionein protein were incubated with solutions of 109Cd at concentrations of 0.2 microM, 1 microM, and 10 microM. The recombinant bacteria were able to accumulate significantly more 109Cd than control bacteria at all concentrations tested. Cadmium accumulation was rapid and highly selective. Maximum uptake was achieved at a pH of 7.0, with lower accumulation at lower or higher pH values. The pH-dependent uptake of cadmium by the recombinant bacteria was exploited to strip off the bound cadmium from the recombinant bacteria and to regenerate most of the cadmium-binding sites. These observations suggest the potential for using a metallothionein-based biosorbent for certain heavy-metal removal applications.

Effect of lead on metallothionein concentration in lead-resistant bacteria Bacillus cereus isolated from industrial effluent

“…Naturally occurring metal-binding peptides, such as MTs and polychelatins (Winklemann and Winge, 1994), are the main metalsequestering molecules used by cells to immobilise metal ions, offering selective, high affinity binding sites. Over expression of metal-binding proteins such as MTs in bacterial and yeast cells resulted in enhanced metal accumulation and thus offer promising strategy for the development of microbe-based biosorbents (Strouhal et al, 2003;Pazirandeh et al, 1995;Romeyer et al, 1990) for the removal and recovery of metals from contaminated water or soil. MTs have been found throughout the animal kingdom, in higher plants, in eukaryotic microorganisms, and in many prokaryotes.…”

Staphylococcal Surface Display of Metal-Binding Polyhistidyl Peptides

“…Such engineered bacteria have in fact been evaluated for removal of Cd 2ϩ from actual factory wastewater (2). Periplasmic expression of a Neurospora crassa metallothionein in Escherichia coli generated cells that were superior to bacteria with cytoplasmic metallothionein localization in terms of metal ion adsorption (29). Surface expression on E. coli cells of yeast or mammalian metallothioneins resulted in recombinant bacteria with increased ability to bind Cd 2ϩ ions (37).…”

On the Extraction of Rare Earth Elements from Geothermal Brines

“…An early study made an assessment for metal binding capacities of bacteria based on a dry weight basis, and estimated that binding capacity of bacteria is quite comparable to the binding capacities of commercial ion exchangers (10 −5 to 10 −3 mol metal/g) [81]. In various studies, several mesophilic bacteria have been engineered to show the metal-binding motifs on their cell surface with increased binding capacity and selectivity [82][83][84][85][86]. For example, binding of Gd 3+ with the bacteria has been demonstrated [87].…”