Proteomic Analysis of Extracellular Appendages Displayed in Desulfovibrio vulgaris
EMSL Project ID
30461
Abstract
Sulfate-reducing bacteria makeup a substantial portion of subsurface microbial communities at DOE contaminated sites in particular. They are capable of reducing and immobilizing, soluble U(VI) by enzymatic reduction. The most well-studied and model organism is Desulfovibrio vulgaris due in part to having a sequenced genome. This organism has repeatedly been shown to enzymatically reduce U(VI), with the primary candidate being the type I periplasmic tetraheme cytochrome c3. However, to take advantage of its bioremediative potential, a more thorough understanding of the underlying regulation and alternate pathways for metal-reduction is essential. Encompassed in this aspect is the observation of extracellular appendages that are present only under limited electron-acceptor; in this case sulfate. These "nanowires" have been shown to be important in metal-reduction in Shewanella oneidensis MR-1 and previous proteomic analysis at PNNL revealed several proteins that support this assessment. In D. vulgaris, the extracellular space was recently shown to be comprised of >90% protein and <10% carbohydrates. This suggests that the cell/cell and cell/surface appendages are primarily proteinaceous and it is our ascertion that they play a role in extracellular electron transfer. Because of the demonstrated importance of the type I cytochrome c3, we have a deletion of this gene in hand, and it was shown to have reduced activity, suggesting that alternate pathways exist. This was conducted under electron-acceptor excess and limited conditions and so the question arises as to the localization of the cytochrome c3 and other cytochromes under each condition, and secondly, whether these compensatory pathways are only intracellular.To fully understand the importance of the proteins present extracellularly, a systems biology approach is highly desirable. 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 this model bacteria. We have coupled our expertise in the genetic and physiological investigation of Desulfovibrio with transcriptome analysis of D. vulgaris at Lawrence Berkeley National Laboratory (LBNL). 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 the AMT tag database already exists for D. vulgaris, the time and cost are reduced. The comparative determination of metabolism, gene expression and protein presence and abundance in the extracellular space in wild type and mutant strains contributes directly to the aims of the Biological Interactions and Dynamic science theme. The use of these data will also help to make sense of our earlier results showing condition specificity for appendage formation. The identification and quantification of the proteins involved will allow for analysis of their regulation and provide for more reliable predictions of U immobilization in DOE contaminated sites areas predominated by sulfate-reduction.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2008-08-20
End Date
2009-09-30
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