Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Proteomics and Metabolomics
115
Characterization of Metal Reducing Microbial Systems by High Resolution Proteomic Measurements
Mary S. Lipton1* (Mary.Lipton@pnl.gov), Ruihua Fang1, Dwayne A. Elias1, Margie F. Romine1, Alex Beliaev1, Matthew E. Monroe1, Kim K. Hixson1, Yuri A. Gorby1, Ljiljana Pasa-Tolic1, Heather M. Mottaz1, Gordon A. Anderson1, Richard D. Smith1, Jim K. Fredrickson1, Derek Lovley2, and Yanhuai R. Ding2
1Pacific Northwest National Laboratory, Richland, WA and 2University of Massachusetts, Amherst, MA
Exploiting microbial function for purposes of bioremediation, energy production, carbon sequestration and other missions important to the U.S. Department of Energy (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 application of our state of the art proteomics technologies based upon high-resolution separations combined with Fourier transform ion cyclotron resonance mass spectrometry. S. oneidensis 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. This 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.
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 were determined by both 14N/15N and absolute peak intensity. Each method of protein quantitation was 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 were observed to increase in expression under anaerobic conditions, while proteins involved in pyruvate and malate synthesis were observed to decrease in expression under anaerobic conditions. All hypothetical and conserved hypothetical proteins are under evaluation for expression under these conditions, as well.
Other projects under the DOE Microbial Genome Program have already sequenced the G. sulfurreducens genome and initiated a functional genomics study to elucidate genes of unknown function in this organism. Proteomic efforts with this microorganism that complement this work have focused on creating a database of characteristic peptide mass and elution time tags, which serve as a unique 2D markers for subsequent peptide identifications. Initial global protein expression determinations have shown protein expression in most functional categories as assigned by TIGR. Thus far, quantitative analyses of protein expression patterns in Geobacter have been derived only by the 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 the clustering of the protein expression patterns is revealing interesting trends in proteins expression as a result of the variation in solubility among the electron acceptor.
The accuracy and precision of making these proteomic measurements is intricately linked to the analytical instrumentation, as well as to the efficiency of the sample processing methods. Advances in automation of sample processing will reduce variation among digested samples. Additionally, improved methods for quantitation and the application of increasingly sophisticated bioinformatics tools for data analysis will greatly improve the types and quality of the proteomic data available in the future.
* Presenting author
