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Biological Sciences

Characterization of Metal-Reducing Microbial Systems by High-Resolution Proteomic Measurements

Sponsor: DOE Office of Biological and Environmental Research
Contacts: Mary Lipton

Exploiting microbial function for purposes of bioremediation, energy production, carbon sequestration and other missions important to DOE requires an in-depth and systems level understanding of the molecular components of the cell that confer its function. Inherent to developing this systems-level understanding is the ability to acquire global quantitative measurements of the proteome (i.e., the proteins expressed in the cell). We have obtained these types of measurements in a high throughput manner for the metal reducing bacteria Shewanella oneidensis and Geobacter sulfurreducens by applying our state of the art proteomics technologies based on high-resolution separations combined with Fourier transform ion cyclotron resonance mass spectrometry. S. ondeidensis MR-1, a Gram-negative, facultative anaerobe and respiratory generalist is of interest to the DOE because it can oxidize organic matter by using metals such as Fe(III) or Mn(III, IV) as electron acceptors. T his bacterium can also reduce soluble U(VI) to the insoluble U(IV) form, which prevents further U mobility in groundwater and subsequent contamination of down-gradient water resources. Geobacter sulfurreducens also is a dissimilatory metal-reducing bacterium that can reduce soluble U(VI) to insoluble U(IV). Such microbial reduction shows significant promise for in situ bioremediation of subsurface environments contaminated with U, Tc, and other toxic metals such as chromium.

Shewanella

In collaboration with the Shewanella Federation, we have characterized global cellular responses to changes in electron acceptors in S. oneidensis MR-1 cultures from a broad range of growth conditions, specifically the presence and absence of oxygen. This in-depth characterization requires not only a qualitative survey of the protein expression patterns, but also an understanding of how the levels of expression change with culture condition. To this end, we have applied quantitative proteomics approaches to characterize protein expression profiles in Shewanella. Relative changes in protein abundance have been determined by both 14N/15N and absolute peak intensity. Each method of protein quantitation has been applied to cells grown aerobically and anaerobically to determine the proteins important for electron transport within the cell. Proteins involved in the electron transport chain, as well as Fe(III) reduction have been observed to increase in expression under anaerobic conditions, while proteins involved in pyruvate and malate synthesis have been observed to decrease in expression under anaerobic conditions. All hypothetical and conserved hypothetical proteins are being evaluated for expression under these conditions, as well.

Geobacter

Proteomics efforts with G. sulfurrenducens complement other projects under the DOE Microbial Genome Program and have focused on creating a database of characteristic peptide mass and elution time tags, which serve as unique 2D markers for subsequent peptide identifications. This collaborative effort with Derek Lovely and the Geobacter project (www.geobactor.org) has produced initial global protein determinations with protein expression in most functional categories assigned by The Institute for Genome Research (TIGR). Thus far, quantitative analyses of protein expression patterns in Geobacter have been derived only by using an absolute peak intensity method. Characterization of the multiple cytochrome proteins in the organism has shown interesting changes in these proteins when they are exposed to different electron acceptors. Additionally, extension of these studies to protein expression pattern clustering is revealing interesting trends in protein expression as a result of the variation in solubility among the electron acceptor.

Schematic of the cellular envelope of Geobacter sulfurreducens.
Schematic of the cellular envelope of Geobacter sulfurreducens. In the process of making energy in the form of ATP, this metal reducing bacteria obtains elections from organic sources (producing CO2 as waste). Electron carrying proteins then transfer the electrons from proteins in the cytoplasm through electron carrier proteins in the inner membrane, periplasm, and outer membrane until the electrons are finally transfered to any available electron acceptor (primarily Fe(III) in natural envrironments).

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