Resumo

This work describes the construction of recombinant Escherichia coli UT5600 and Cupriavidus metallidurans CH34 strains, carrying genetic information for the cell surface display of either MerR or ArsR, the proteins showing the highest affinity and specificity for Hg²⁺ or As⁵⁺ ions, respectively. The merR and arsR genes were isolated from C. metallidurans CH34 total DNA, inserted into a plasmid vector which contains a system for expressing and anchoring heterologous proteins on Gram-negative bacteria cell surface, giving rise to the pCM-Hg and pCM-As recombinant plasmids. MerR and ArsR were produced under transcriptional control of the pan promoter which allowed constitutive expression of these proteins. The recombinant strains presented MerR or ArsR on the cell surface and enhanced accumulation of Hg²⁺ or As⁵⁺ ions, respectively, confirmed by SDS-PAGE and TEM. The recombinant strains proved to be more resistant to mercury or arsenic than the parental strains: the recombinant E. coli strains UT5600/pCM-Hg (resistance up to Hg²⁺ 25 μM) and UT5600/pCM-As (resistance up to As⁵⁺ 25 mM) showed 100% increased resistance relative to the control strains UT5600 (12,5 μM and 12,5 mM, respectively); the recombinant strain CH34/pCM-Hg showed resistance up to Hg²⁺ 12,5 μM, 100 % higher than the wild type CH34 strain (Hg²⁺ 6,2 μM), and the recombinant CH34/pCM-As strain presented resistance up to As⁵⁺ > 1000 mM, higher than 100 % when compared to the wild type CH34 strain (resistance up to As⁵⁺ 250 mM), thus being the most As⁵⁺ resistant Gram-negative bacterium ever described. The pCM-Hg and pCM-As plasmids promoted MerR and ArsR expression on the cell surface of both Gram-negative bacteria, the resulting recombinant strains presented enhanced resistance to Hg²⁺ or As⁵⁺ ions and enhanced capability and specific adsorption of these ions. The new plasmids can be used to transform other Gram-negative bacteria, with a view to increase their ability to survive in the presence of these toxic ions. The new recombinant bacteria here described are excellent candidates to be used in bioremediation processes of mercury and arsenic contaminated effluents.