Thrust area 1: The surface structure of Shewanella and the localization of outer membrane-associated proteins hypothesized to be involved in electron transfer from metal-reducing bacteria to metal oxide surfaces.
EMSL Project ID
13892
Abstract
A multidisciplinary team of scientists is investigating the mechanisms of electron transfer from metal-reducing bacterial to Fe oxide surface as part of a PNNL/DOE-BER Grand Challenge in Biogeochemistry (BGC) being led by Drs. Zachara and Fredrickson of PNNL. Dr. Terrence Beveridge is a leading expert on the analysis and characterization of prokaryote cell ultrastructure by electron microscopy, the analysis of outer membrane lipopolysaccharide, and microbial-mineral/metal interactions in general. Dr. Beveridge has contributed to the planning phase of the grand challenge and was a member of the Advisory Committee. His continued participation and research efforts in the BGC are anticipated to provide key information regarding the surface structure of Shewanella and the localization of outer membrane-associated proteins hypothesized to be involved in electron transfer from metal-reducing bacteria to metal oxide surfaces. To complete the objectives of this project, the following tasks need to be conducted at the University of Guelph under the direction of Dr. Beveridge. These tasks include:1. Determination of the location of mtrA, mtrB, and mtrC in S. oneidensis MR-1 cells relative to their predicted location (periplasm for mtrA, OM for mtrB, C), based upon genome sequence and fractionation studies, using state-of-science immunogold microscopy techniques. Antibodies for use in immunolocalization analyses will be provided by PNNL (contacts: A. Beliaev and L. Shi). In addition to polyclonal antibodies, it may be possible to utilize genetic constructs where a V5 tag has been inserted into the gene of the mtr proteins. V5 antibodies are commercially available. A major goal of this task is to assess whether these proteins are exposed to the outside of the OM where they can potentially contact metal oxides for purposes of electron transfer.
2. In addition to analyses of cells in the absence of metal oxides (task 1), a second task will focus on investigating the relative location of putative OM electron transfer proteins in cells that are in direct contact with and reducing metal oxides. The cultivation of Shewanella for these experiments will be done in a manner consistent with approaches agreed upon in advance by the BGC team to allow for maximum comparability of results between different laboratories. Y. Gorby (PNNL) is serving as coordinator for the BGC cultivation efforts and should be the point of contact for cultivation.
3. If polyclonal antibodies generated against mtr proteins are cross-reactive with homologous proteins in other select strains of metal-reducing Shewanella, including S. putrefaciens CN32 and S. baltica, a third optional task will focus on conducting immunolocalization analyses similar to those in task 1 to determine whether results obtained with MR-1 can be extended to other strains.
Anticipated contract period: November 1, 2004 through September 30, 2007.
Project Details
Project type
Grand Challenge
Start Date
2005-03-02
End Date
2007-06-01
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members