State Water Resources Research Institute Program

Project Id: 2010SD175B
Title: Protein-Based Mechanisms of Uranium Detoxification in Subsurface Bacteria
Project Type: Research
Start Date: 3/01/2010
End Date: 2/28/2011
Congressional District: SD First
Focus Categories: Radioactive Substances, Toxic Substances, Groundwater
Keywords: DMRB, In Situ Bioremediation, Proteomics, Subsurface Water
Principal Investigators: Sani, Rajesh Kumar (South Dakota School of Mines and Technology); Sani, Rajesh Kumar (South Dakota School of Mines and Technology)
Federal Funds: $ 16,771
Non-Federal Matching Funds: $ 33,542
Abstract: Uranium (U) contamination in the subsurface has become a global problem in aquifers, water supplies, and related ecosystems. It has been established that in situ microbial enzymatic U(VI) reduction to U(IV) provides an attractive alternative remediation strategy since U(IV) precipitates as uraninite (UO2), a relatively insoluble U mineral. For example, sulfate-reducing bacteria (SRB) can immobilize U in the subsurface through enzymatic reduction of highly soluble U(VI) to much less soluble U(IV). Published data suggest that U(VI) is likely be reduced by a variety of non-specific periplasmic proteins e.g., cytochromes; however, my group's published and unpublished results suggest that SRB can also reduce U(VI) using cytoplasmic proteins. Results also show that U(VI) is much more toxic to SRB than previously thought, and in response to U(VI) some proteins are induced in SRB, which likely detoxify U(VI). It has been established that membrane proteins play essential roles in many mechanisms conferring metal resistance in bacteria. These microbial protein-based mechanisms for U(VI) detoxification are very poorly understood in dissimilatory metal-reducing bacteria (DMRB) including SRB, even though these organisms can be very important in U(VI) sequestration in subsurface soils and groundwater.

It is vital, therefore, to characterize these mechanisms to understand natural attenuation of subsurface U or to rationally design in situ bioremediation strategies for U contaminated subsurface environments. I propose a fundamental molecular research approach to elucidate protein-based U detoxification mechanisms in a well-characterized SRB - Desulfovibrio desulfuricans G20. To determine these mechanisms of anaerobic U(VI) detoxification in SRB, the specific objectives are:

  1. Isolation and identification of induced proteins from D. desulfuricans G20 under U(VI)-stressed conditions and
  2. Elucidation of U detoxification mechanisms.

SRB constitute major groups of organisms responsible for heavy metal immobilization and groundwater detoxification. The chosen organism (D. desulfuricans G20) for this proposed research have complete genome sequence, which is critical to the success of the project, since using genomic information, U-induced proteins can be identified. Knowledge of U response mechanisms will provide an improved understanding of the potential impacts of U on environmental microorganisms and their impacts on the ultimate fate and transport of U in groundwater.

Using the general procedures, the induced membrane and soluble proteins of D. desulfuricans G20 will be characterized using 2-D gel electrophoresis and Matrix-Assisted Laser Desorption Ionization -Mass Spectrometry (MALDI-MS). While there may be involvement of constitutive proteins, we believe that induced proteins are more likely to be involved in U(VI) detoxification (and reduction). This has also been shown for Pseudomonas fluorescens, where induced proteins were critical in the survival of this organism in the presence of heavy metals. The proteins that are repressed or unexpressed in response to U, are not likely to play a significant role in U detoxification. Therefore only proteins that are induced in response to U(VI) will be identified. The proposed research will improve the understanding of protein-based U detoxification (and reduction) mechanisms in subsurface microorganisms (especially in SRB) and will provide a platform to elucidate biochemical pathways of detoxification and metal reduction in other DMRB such as fermentative and iron-reducing bacteria and in complex subsurface microorganisms present at the U-contaminated sites including the Field Research Center (TN), Hanford site (WA). Finally these results will be applicable in strategies to reduce environmental and human health risks from radionuclide contaminated sites.

Progress/Completion Report, 2010, PDF

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