| Bacillus thuringiensis, isolated from a heavy metal contaminated soil in Southeastern Kansas, can grow in the media containing 200 mug/ml toxic Cr(VI). Previous work in our laboratory had shown that B. thuringiensis contains a cytoplasmic NADH-linked chromium reductase activity. After cell lysis by sonication the soluble chromium reductase activity was partially purified and shown to produce insoluble, green, precipitated chromium [Cr(III)] compound in vitro. During bacterial growth in Cr(VI) containing media, precipitated chromium-containing aggregates can be identified within the cytoplasm of both B. thuringiensis and certain recombinant Escherichia coli.; A 1.3 kb Sac I DNA restriction fragment from a 9.2 kb plasmid within B. thuringiensis (pBt9.2), confers resistance to Cr(VI) when inserted into a variety of expression vectors and cloned in a number of E. coli strains. Both original B. thuringiensis and E. coli recombinants containing the 1.3 kb sequence contain an intracellular Cr(VI) reductase activity.; A BLAST search of GenBank reveals that the 1.3 kb sequence has a 95% nucleotide identity with that of transposase A of the mobile DNA element Tn10. Use of PROPSEARCH reveals that the translated protein has a structure at the carboxy terminus similar to the heme binding motif of cytochrome P450. It is proposed that the Cr(VI) reductase contains a metal binding site of transposase A and conserved sequence of cytochrome P450 that involve in electron transfers that generate reducing power to reduce Cr(VI) to Cr(III). Finally, one additional investigation using PCR has identified the jemC, another metal resistance determinant from tn10, on the pBt9.2.; We have thus identified a novel chromium reductase and its gene in B. thuringiensis. Evidence suggest that the activity arose as a result of gene transfer (Tn10 was originally identified in Shigella dysentriae ) and recombination with a Bacillus element. Whether additional Tn10 DNA is a part of the pBt9.2 is under investigation. Various strategies for use of this information in bioremediation are also under investigation. Among these are insertion of the 1.3 kb Sac I element into another bacteria and plants, and use of oligonucleotides to assay for the presence of chromium reductase gene in Cr(VI) contaminated soil. |
