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Recombinant tilapia (Oreochromis mossambicus) fish metallothionein (MT) was used as a surface biosorbent for mercury removal in Escherichia coli. Fish MT conferred better resistance than did mouse or human MT. When tilapia MT (tMT) was fused with an outer-membrane protein, outer membrane protein C (OmpC), the membrane-targeted fusion protein, OmpC-tMT, gave enhanced resistance compared with cytoplasmic tMT expressed in the same host cell. The cytoplasmically expressed tMT showed high mercury adsorption (4.3 +/- 0.4 mg/g cell dry weight). The cell surface that expressed E. coli showed about 25% higher adsorption ability (5.6 +/- 0.4 mg/g) than the cells expressing cytoplasmic MT, attaining almost twice the level of adsorption of the control plasmid (3.0 +/- 0.4 mg/g). As MTs are also known for their ability to scavenge hydroxyl-free radicals, it was also shown that tMT exhibited better radical-scavenging activities than glutathione. These results suggest that fish MT has potential for the development of a bioremediation system for mercury removal that protects the harboring E. coli host by free-radical scavenging.

Overexpression of a mercuric ion binding protein, MerP, from the mercury resistance operon genes of Gram-positive bacterial strain Bacillus megaterium MB1 and from Gram-negative bacterial strain Pseudomonas aeruginosa K-62 was found to enhance the mercury resistance level of Escherichia coli host cells, even though they share only 27.3% identity. Immunoblot analysis showed that MerP (BMerP) from Bacillus could be expressed on the membrane fraction of E. coli cells. Treated with 10 microM Hg2+, a recombinant strain harboring the BMerP gene significantly improved, showing a 27% increase in mercuric ion adsorption capacity, 16% better than that of a Pseudomonas merP gene (PMerP)-harboring strain. While multiple heavy metals co-existed, the mercuric ion adsorption capacity of the BMerP-harboring E. coli was not affected while that of the PMerP-harboring strain decreased. These results suggest that BMerP can act as a bio-adsorbent compartmentalizing the toxic mercuric ion on the cell membrane and enhancing resistance.

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