Proteomic Analysis of Differential Growth in Desulfovibrio spp. for a Systems Biology Level Investigation.
EMSL Project ID
25418
Abstract
Sulfate-reducing bacteria makeup a substantial portion of subsurface microbial communities in general and at DOE contaminated sites in particular. They are capable of reducing, and thus immobilizing, soluble U(VI) by enzymatic reduction. The most well-studied of this group are Desulfovibrio vulgaris and D. desulfuricans G20 due in part to having sequenced genomes. Each species has repeatedly been shown to enzymatically reduce U(VI), with the primary candidate in both cases being the type I periplasmic tetraheme cytochrome c3. However, to take advantage of their bioremediative potential, a more thorough understanding of the underlying regulation and alternate pathways for metal-reduction is essential. To accomplish this, we deleted the cytochrome c3 in each species to determine if removal of the gene abolished U(VI)-reduction activity or if activity was present in the mutant. We observed reduced activity and inferred that alternate pathways exist. Analysis of the G20 mutant showed that different electron donors varied in their ability to transfer electrons to U(VI) for reduction, with a 90-25% decrease compared to wild type. The ortholog in D. vulgaris was constructed for comparison and displayed a different phenotype, underscoring the need to study both bacteria.To fully understand such mutational effects and identify the proteins involved in these compensatory pathways, a systems biology approach that includes physiological and metabolic data, mRNA microarrays, and proteomic analysis is highly desirable. Numerous studies have utilized physiological data alone to assess alterations under diverse nutritional and metabolic (respiratory vs. fermentative) regimes as well as changes post-uranium exposure, with several utilizing microarrays. However, comprehensive examinations that include differential expression at the protein level are few. To this end, we wish to undertake a systems level approach to investigate electron transfer and energy generation in these model bacteria. We have coupled our expertise in the genetic and physiological investigation of Desulfovibrio with transcriptome analysis of D. desulfuricans G20 at Oak Ridge National Laboratory (ORNL). These analyses were performed with cells cultured using the same electron donors previously used to assess U(VI)-reduction. The results indicate differential expression in proteins for both central carbon metabolism and electron flow, and are consistent with our metabolic data.
The goal of the proposed work is to extend these efforts to include comprehensive proteome analysis by utilizing the AMT tag approach. Since AMT tag databases already exist for D. desulfuricans G20 and D. vulgaris, the time and cost to accomplish this work are reduced. The comparative determination of metabolism, gene expression and protein abundance in wild type and mutant strains contributes directly to the aims of the Biological Interactions and Dynamic science theme. The use of these data to make sense of our earlier results showing electron donor specificity on the rate and extent of U(VI)-reduction in the G20 mutant falls under the aims of the Geochemistry /Biogeochemistry and Subsurface Science theme. The identification and quantification of the proteins involved in alternate pathways of U(VI)-reduction will allow for analysis of their regulation and provide for more reliable predictions of immobilization in DOE contaminated sites areas pre-dominated by sulfate-reduction.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-05-25
End Date
2010-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Expression profiling of hypothetical genes in Desulfovibrio vulgaris leads to improved functional annotation
Dwayne A. Elias, Aindrila Mukhopadhyay, Marcin P. Joachimiak, Elliott C. Drury, Alyssa M. Redding, Huei-Che B. Yen, Matthew W. Fields, Terry C. Hazen, Adam P. Arkin, Jay D. Keasling and Judy D. Wall. 2926–2939 Nucleic Acids Research, 2009, Vol. 37, No. 9 Published online 17 March 2009
doi:10.1093/nar/gkp164
Keller KL, BJ Rapp-Giles, ES Semkiw, I Porat, SD Brown, and JD Wall. 2013. "New Model for Electron Flow for Sulfate Reduction in Desulfovibrio alaskensis G20." Applied and Environmental Microbiology 80(3):855-868. doi:10.1128/AEM.02963-13