Proteomic Analysis of Model Metal-reducing Microbial Communities
EMSL Project ID
39705
Abstract
The sulfate- and Fe(III)- reducing bacteria (SRB and IRB) are primarily responsible for metals and radionuclide immobilization in porous subsurface sediments. Desulfovibrio vulgaris and Geobacter sulfurreducens in particular are model organisms for DOE research towards environmental cleanup. While electron transfer mechanisms to metals by individual microorganisms is reasonably well understood, more detailed systems biology studies are needed. These organisms do not live alone in metal-reducing subsurface environments, but rather are only two components of a large and diverse microbial community. While they can reduce most metals at contaminated sites, the carbon usually ends up as methane. Hence we have added Methanococcus maripaludis S2 to the community. It survives from the H2 and acetate given off by D. vulgaris during lactate oxidation. By obtaining a more thorough understanding of the protein expression levels and patterns involved in a community setting, and hence mechanisms of extracellular electron transfer and compensatory pathways that emerge compared to pure laboratory culturing, new avenues for exploration are likely to be revealed. When combined with our other analyses, we anticipate elucidating previously unknown mechanisms for living syntrophically during metal-reduction. We will also use this information to determine if similar mechanisms apply in other organisms while furthering the goal of improving the metabolic model of D. vulgaris. The inclusion of comprehensive proteomic data to our community metabolic and mRNA microarray data will enhance our understanding of the differences between individual, co- and community cultures, but will also further building a metabolic model (i.e. virtual cell) of D. vulgaris encompassing all stress responses and cell to cell interactions from pure culture to model community. The proteomics facility at EMSL is uniquely poised to complete the proposed research and will contribute to broader scientific objectives currently funded by the US DOE office of science. The work to be conducted at EMSL will include processing of frozen cell pellets shipped from ORNL. This involves bead beating cell lysis, tryptic digestion, sample cleanup with C18 columns and MS analysis using the bottom-up approach using the nanoLC-LTQ platform. PMT database construction will be required for Mc. maripaludis but only under one growth condition. There are mature AMT tag databases for D. vulgaris and G. sulfurreducens stemming from Dr. Elias' post-doctoral work. Once the MS runs are completed, we wish to compare the presence/absence of peptides with quantitative comparisons of peptide and protein abundances. For this analysis, we wish to primarily use the raw abundance comparison known as 'spectral counting' with the peptide counting as a secondary measure. The resources requested are cell pellet preparation and triplicate MS analysis and bioinformatic processing of each biological sample all within the proteomics core of EMSL over the 1 year period. We anticipate duplicate biological replicates of the pure, co- and tri-cultures as described above. Thus we anticipate the construction of the Mc. maripaludis AMT tag database to require ~75 MS/MS runs (25 for all proteins; i.e. global, as well as 25 for the soluble protein fraction and 25 for the insoluble protein fraction). Once this database is established, we anticipate 3 MS runs per pure culture (D. vulgaris/Mc. maripaludis/G. sulfurreducens), 3 MS runs per co-culture (D. vulgaris/Mc. maripaludis; D. vulgaris/G. sulfurreducens), and 3 MS runs of the tri-culture. Each will be performed with biological duplicates for a total of 12 biological samples, 75 MS/MS runs and 36 MS runs.
Project Details
Project type
Exploratory Research
Start Date
2010-11-16
End Date
2011-11-20
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members