Different enzymes are involved in anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation in Thiobacillus denitrificans

Thiobacillus denitrificans is a widespread, obligate chemolithoautotrophic bacterium that is capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation. Both of these processes can mediate the mobility of uranium in contaminated aquifers and thereby influence the long-term efficacy of in situ reductive immobilization of uranium at DOE sites. T. denitrificans has been found at uranium-contaminated sites, including a contaminated aquifer at Oak Ridge National Laboratory. We previously reported that two membrane-associated, diheme, c-type cytochromes (a c₄ cytochrome, Tbd_0187, and a c₅ cytochrome, Tbd_0146) were involved in nitrate-dependent U(IV) oxidation in T. denitrificans. To date, these are the only genes identified to be involved in this process. In this poster, we report on work with T. denitrificans focused on determining whether the enzymes that were demonstrated to be involved in anaerobic, nitrate-dependent U(IV) oxidation are also involved in nitrate-dependent Fe(II) oxidation. Using a genetic system in T. denitrificans that enables us to create insertion mutants and complement them in trans, we constructed a series of insertion mutants. These included strains with mutations in the genes known to be associated with U(IV) oxidation (Tbd_0146 and Tbd_0187) as well as other genes encoding membrane-associated c-type cytochromes (a group of proteins that we hypothesize to be catalyzing Fe(II) oxidation). Anaerobic cell suspension assays were carried out to determine whether any of these mutants were defective in nitrate-dependent Fe(II) oxidation. We observed that the Tbd_0146 and Tbd_0187 mutants were not defective in nitrate-dependent Fe(II) oxidation, nor were any of the other c-type cytochrome mutants tested (including a Tbd_0146-Tbd_0187 double mutant). The finding that different enzymes are associated with nitrate-dependent Fe(II) and U(IV) oxidation has led us to pursue genome-wide studies in T. denitrificans to determine the genes associated with nitrate-dependent Fe(II) oxidation.