| The legacy of the cold war includes sites across the U.S. storing mixed waste for a lack of a feasible treatment option. Simplification of the waste is a necessary first step in treatment. Bioremediation offers a potential solution for many of these wastes. Although bacteria that degrade toxic organics are ubiquitous in nature, these bacteria would mutate or die in mixed waste. Deinococcus radiodurans R1, however, is extremely resistant to ionizing radiation-induced mutation and mortality. The goal of this work is to equip D. radiodurans to degrade toxic organics in mixed waste.; A stable integrative insertion system in the pullulanase gene has been designed for heterologous gene expression in the D. radiodurans genome. Two broad range oxygenases, toluene-2,3-dioxygenase (Tod) and a toluene dioxygenase, biphenyl hydroxylase hybrid system (Tod: Bph) have been successfully expressed with this system. Both have been expressed to high levels, and the Tod insertion was stable for over 100 generations without antibiotic selection. The level of expression of these oxygenases has never before been achieved in D. radiodurans, and represents a major step towards an optimized mixed waste treatment strain.; The next step involved studies of solvent resistance. First, the structure of the D. radiodurans S-layer was studied by mutating the two S-layer genes, hpi (DR2508) and slpA (DR2577). A combination of phenotypic and microscopic experiments indicated for the first time that slpA is expressed and is necessary for cell envelope integrity as well as normal growth and resistance to external stresses, while hpi, which makes up the hexagonal surface array, seems much less critical during normal growth and for resistance to solvent or shear force stress.; Other factors affecting resistance of D. radiodurans to solvents were also studied. D. radiodurans has been found to be highly solvent-resistant. In addition, targets for manipulation of the solvent response were identified to customize future strains to specific wastes. Combined with successful stable expression of a broad spectrum oxygenase at high levels in D. radiodurans HMR158.105, we have developed the basis of a long-term treatment strategy for the class of mixed wastes amenable to bioremediation. |
