HR: 12:05h
AN: B12A-08    [Abstracts]
TI: Anaerobic, Nitrate-Dependent Oxidation of Uraninite by the Chemolithoautotroph Thiobacillus denitrificans: Cell Suspension and Whole-Genome Transcriptional Studies
AU: * Beller, H R
EM: beller2@llnl.gov
AF: Lawrence Livermore National Laboratory, 7000 East Ave., PO Box 808, L-542, Livermore, CA 94551 United States
AU: Chakicherla, A
EM: chakicherla1@llnl.gov
AF: Lawrence Livermore National Laboratory, 7000 East Ave., PO Box 808, L-542, Livermore, CA 94551 United States
AU: Legler, T C
EM: legler2@llnl.gov
AF: Lawrence Livermore National Laboratory, 7000 East Ave., PO Box 808, L-542, Livermore, CA 94551 United States
AU: Letain, T E
EM: letain2@llnl.gov
AF: Lawrence Livermore National Laboratory, 7000 East Ave., PO Box 808, L-542, Livermore, CA 94551 United States
AU: Coleman, M
EM: coleman16@llnl.gov
AF: Lawrence Livermore National Laboratory, 7000 East Ave., PO Box 808, L-542, Livermore, CA 94551 United States
AU: Kane, S R
EM: kane11@llnl.gov
AF: Lawrence Livermore National Laboratory, 7000 East Ave., PO Box 808, L-542, Livermore, CA 94551 United States
AB: Background: In-situ, reductive immobilization of uranium in aquifers, whereby relatively soluble U(VI) species are reduced to poorly soluble uraninite (UO₂) by aquifer bacteria, has been the subject of intensive research effort recently. This study explored the possibility that a widespread soil bacterium, Thiobacillus denitrificans, could catalyze anaerobic U re-oxidation in the presence of nitrate, a common co-contaminant with uranium at U.S. DOE sites. Whole-genome, cDNA microarray studies (representing all 2832 ORFs of the 2.9 Mb genome) were conducted to identify genes upregulated during nitrate-dependent U(IV) oxidation (relative to control conditions of nitrate-dependent thiosulfate oxidation). Methods: Washed cell suspension experiments were carried out under strictly anaerobic conditions and at circumneutral pH with UO₂ and T. denitrificans cells grown under denitrifying conditions and harvested in late exponential phase. Experiments included both sterile controls and live, no-nitrate controls. For microarray analysis, RNA was isolated from cells exposed to either UO₂ or thiosulfate under strictly anaerobic, denitrifying conditions. For all samples analyzed with microarrays, chemical analyses were used to confirm that the applicable metabolic activity [i.e., denitrification and either U(IV) or thiosulfate oxidation] was occurring. Reverse transcription, quantitative PCR was used to confirm selected microarray results. Results: In the cell suspension experiments, T. denitrificans cells oxidatively dissolved UO₂ in nitrate-dependent fashion: U(IV) oxidation required the presence of nitrate ( P<0.01) and was strongly correlated to nitrate consumption (r² = 0.98). However, U(IV) oxidation and denitrification appeared to be dependent on H₂. The microarrays identified 333 genes as upregulated under U(IV)-oxidizing conditions using RMA statistical analysis and a 2-fold ( P<0.0001) cutoff. Notably, 16 of these genes, which were upregulated 5- to 22-fold, were among the top 25 upregulated genes not only for UO₂ oxidation but also for nitrate-dependent oxidation of FeCO₃ and dissolved Fe²⁺. Conclusions: This is the first report of anaerobic U(IV) oxidation by an autotrophic bacterium. Preliminary microarray data analysis indicates that a small subset of genes is highly upregulated under conditions of nitrate-dependent U(IV) and Fe(II) oxidation.
DE: 0418 Bioremediation
DE: 0448 Geomicrobiology
DE: 0461 Metals
DE: 0471 Oxidation/reduction reactions (4851)
SC: Biogeosciences [B]
MN: Fall Meeting 2005