Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil

Abstract

Here we describe targeting of the mouse metallothionein I (MT) protein to the cell surface of the heavy metal-tolerant Ralstonia eutropha (formerly Alcaligenes eutrophus) CH34 strain, which is adapted to thrive in soils highly polluted with metal ions. DNA sequences encoding MT were fused to the autotransporter β-domain of the IgA protease of Neisseria gonorrhoeae, which targeted the hybrid protein toward the bacterial outer membrane. The translocation, surface display, and functionality of the chimeric MTβ protein was initially demonstrated in Escherichia coli before the transfer of its encoding gene (mtb) to R. eutropha. The resulting bacterial strain, named R. eutropha MTB, was found to have an enhanced ability for immobilizing Cd2+ ions from the external media. Furthermore, the inoculation of Cd2+-polluted soil with R. eutropha MTB decreased significantly the toxic effects of the heavy metal on the growth of tobacco plants (Nicotiana bentamiana). 

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Figure 1: Genetic structure of pMTβ-1 and production of MTβ in E. coli.
Figure 2: Accumulation of Cd2+ by E. coli and R. eutropha.
Figure 3: Structure of the transposon TnMTβ-1 and production of MTβ in R. eutropha.
Figure 4: Remediation of Cd2+ toxicity on plant growth by R. eutropha MTB.

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Acknowledgements

We thank Jon Beckwith and Max Mergeay for materials and strains used in this work, Sofía Fraile for her excellent technical work, and N. van der Lelie for inspiring discussions. We are grateful to Roser Gonzàlez-Duarte for her participation and continuous support to this work. M.V. held a predoctoral fellowship from the Generalitat de Catalunya (Spain). This work was supported in part by EU contracts BIO4-CT97-2123 and BIO4-CT97-2183 and by the Spanish CICYT grants BIO98-0808 and PB96-0225.

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Correspondence to Víctor de Lorenzo.

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Valls, M., Atrian, S., de Lorenzo, V. et al. Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil. Nat Biotechnol 18, 661–665 (2000). https://doi.org/10.1038/76516

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