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Engineering Deinococcus Radiodurans R1 Phosphate Metabolism for Metal Precipitation in Radioactive Waste

Alexandra D. Holland and Mary E. Lidstrom. Chemical Engineering, University of Washington, R&T building, 4th floor, 616 NE Northlake Place, Seattle, WA 98105

The capacity of the non-pathogenic Gram negative bacterium Deinococcus radiodurans to withstand extreme levels of chronic radiation makes it an attractive candidate for bioremediation of low-level radioactive waste. For this purpose, the native strain should be engineered to perform metal bioprecipitation. The resulting concentrated sludge would provide an inexpensive metal sequestration method from the low level waste, which would then be suitable for vitrification.

The ability to precipitate actinide phosphate complexes has been observed in three different Gram negative bacterial systems, which are however not yet fully understood. Metal phosphate complexes were observed in: polyphosphate accumulating Pseudomonas aeruginosa, through overexpression of the Polyphosphate Kinase ( ppk) gene; in polyphosphate accumulating sludge bacteria at the onset of the inorganic phosphate release; and in a Citrobacter sp., upon acid phosphatase-mediated phosphate release in the periplasm, where actinide phosphate complexes nucleated. These led to the hypothesis that actinide precipitation could be achieved in Deinococcus through a two-step process involving polyphosphate accumulation and phosphate release in the periplasm.

The phosphate metabolism genes needed for the hypothesized precipitation mechanism have been annotated in Deinococcus. As a first step, a chromosomal insertion vector was used to overexpress the native ppk gene, in order to achieve polyphosphate (PolyP) accumulation in a phosphate rich environment. Upon transfer into a phosphate-free metal-laden environment, the cytosolic PPX protein should degrade the PolyP into free phosphate. Subsequently, phosphate should be gradient driven into the periplasm through the bi-directional PIT phosphate transporter, leading to a local phosphate concentration high enough for nucleation of actinide phosphate complexes at the membrane.

Overexpression of native ppk or deletion of various PolyP-utilizing enzymes did not result in PolyP accumulation in D. radiodurans R1, even though biochemical studies of the purified Deinococcus rPPK revealed an active protein in both directions. The role of PolyP in stress response was investigated. As in E. coli and a Synechocystis sp., phoU gene deletion led to constitutive PolyP accumulation and impaired growth, but soon generated second-site mutations that alleviated the desired phenotype and restored growth. phoU deletion in the ppk mutant did not accumulate PolyP and had no growth phenotype. A phosphate uptake-release strategy was achieved in a quadruple mutant, using a variation of the exogenous IPTG inducible Pspac/lacI system (LeCointe et. al. 2004) and the tightly regulated phosphate-starvation inducible alkaline phosphatase promoter: the high level PgroES promoter was used to drive lacI, the Pspac promoter was inserted in the native ppx promoter site to achieve inducible expression (higher background than PphoA); a conditional PPK mutant was generated by driving ppk with PphoA; finally, the phoU gene was deleted. The resulting strain does not accumulate PolyP in phosphate-replete conditions. Upon phosphate starvation, the ppk gene is induced; subsequent phosphate addition leads to rapid PolyP accumulation and phosphate depletion from the medium. Metal and IPTG addition in the phosphate-free medium leads to PolyP degradation, and phosphate release in the periplasm, and metal-phosphate precipitation on the outer membrane, thus effectively sequestering the metals from solution.