Engineering Deinococcus geothermalis for bioremediation of high-temperature radioactive waste environments

Abstract

Deinococcus geothermalis is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile Deinococcus radiodurans, which is being engineered for in situ bioremediation of radioactive wastes. We report that D. geothermalis is transformable with plasmids designed for D. radiodurans and have generated a Hg(II)-resistant D. geothermalis strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally, D. geothermalis is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55 degrees C.

FIG.1.

Transformation of D. geothermalis with pMD66 and resistance of pMD66-transformed D. geothermalis to acute gamma radiation. (A) D. geothermalis/pMD66. Total DNA from the indicated strains was uncut or digested with PstI before electrophoresis, blotting, and probing of the blot with a whole-plasmid radiolabeled pMD66 probe. Abbreviations: DEIRA, D. radiodurans; DEIGEO, D. geothermalis. (B) The copy number of pMD66 in D. geothermalis/pMD66 is about threefold higher than its chromosomal copy number. (C) Survival of D. geothermalis/pMD66 following acute gamma radiation. Symbols: open squares, D. geothermalis plated on TGY at 37°C; solid triangles, D. geothermalis/pMD66 plated on TGY-KAN at 37°C; solid diamonds, D. radiodurans plated on TGY at 32°C.

FIG.2.

Construction and characterization of Hg(II)-resistant-reducing D. geothermalis. (A) pMD727 (5) was transformed into D. geothermalis, giving strain MD865. (B) Southern blot hybridization of EcoRI-digested total DNA from D. geothermalis (wild type, mer negative) and MD865 (D. geothermalis/mer⁺) with a radiolabeled mer probe. pMD727 contains a unique EcoRI (E) site. Molecular size standards: λ/HindIII, as in Fig. 1A and B. Wild-type strain abbreviations are as in Fig. 1. (C) Hg(0) volatilization assays at 32 and 40°C for D. geothermalis, D. radiodurans, MD865 and MD735 (D. radiodurans/mer⁺), and TGY (growth medium, no cells). (D) Growth curves for MD865 and MD735 in TGY plus 50 μM merbromin [Hg(II)] at 50°C.

FIG. 3.

Metalloreduction by wild-type D. geothermalis. (A) Fe(III)-NTA reduction coupled to oxidation of organic substrates at 45°C in the absence of oxygen. (B) Cr(VI) reduction in aerobic or anaerobic conditions as measured by loss of Cr(VI) from solution at 40°C. (C) Reduction of U(VI) in the presence or absence of AQDS as measured by loss of U(VI) at 40°C.