The Role of a Plant Metallothionein (MT) in Separating Cadmium (Cd) and Zinc (Zn) in E.Coli Using Isotope Process Tracing

Zinc (Zn) is an essential micronutrient, while cadmium (Cd) is a highly toxic pollutant. Due to their chemical similarity, both metals are absorbed by plants through the same pathways, including membrane proteins and vascular tissues. Plants mitigate Cd toxicity by chelating Cd with thiol-rich organic ligands and proteins, such as metallothioneins (MTs). Recent studies have shown that Zn and Cd isotopes are inversely fractionated in cereals like wheat and rice, with grains accumulating light Zn and heavy Cd isotopes. As Cd-thiol complexes are more stable than Zn-thiol complexes, we hypothesize that thiols separate Cd from Zn in living organisms, reflected in the ‘isotope fingerprint’ of these metals.

To test this hypothesis, we analyzed in a first step the Cd and Zn isotope composition in chickpea metallothionein (cicMT2). This is a model metallothionein and thiol for in vitro studies. To this end, cicMT2 was recombinantly expressed in E. coli cells with a GST tag for purification purposes, which was subsequently cleaved to obtain the native protein sequence. Metal-free cicMT2 was incubated with Cd(II) and Zn(II) ions for different equilibration times. After separating the unbound metal ions using size exclusion chromatography, the protein samples were measured for Cd and Zn isotope composition using a multi-collector ICP-MS. The isotope fractionation between the free and complexed metals at isotope equilibrium was Δ^114/110Cd_free-MT = 0.56 ± 0.21‰, reached after 8 h of incubation, and Δ^66/64Zn_free-MT = 0.87 ± 0.41‰ within the first 10 min of incubation. Our findings indicate that cicMT2 significantly fractionates Cd and Zn isotopes, showing a preference for lighter Cd and Zn isotopes in the cicMT2 complex.

In a second step, we recombinantly expressed cicMT2 in the living model organism E. coli and compared the growth and metal uptake with a wild-type (non-producing MT) strain under different Cd and Zn concentrations. E. coli expressing cicMT2 accumulated over four times more Cd and Zn than the wild-type strain without reducing the growth rate. At high Cd concentrations, the separation of Zn and Cd in E. coli with cicMT2 (Zn:Cd = 1.04) is 19% more pronounced when compared to the wild-type (Zn:Cd = 0.88). This first data suggests that the model thiol cicMT2 plays a role in separating Zn from Cd in a living organism. Until the conference, the isotope composition of Zn and Cd in E. coli will be determined to further elucidate the role of cicMT2 to separate Zn from Cd.