Meta-Proteomic Analysis of Hg and U Contaminated Microbial Communities
EMSL Project ID
40081
Abstract
The sulfate- and Fe(III)- reducing bacteria (SRB and IRB) are primarily responsible for the biotransformation of metals and radionuclides in porous subsurface sediments. Desulfovibrio spp. and Geobacter spp. in particular are model organisms for a significant portion of DOE related research towards environmental cleanup. However, while the components and mechanisms involved in electron transfer to some metals such as U(VI) within the individual microorganisms is reasonably well understood, the biochemical mechanism(s) for the methylation of Hg is almost completely unknown. Mercury methylation is an unwanted process because methylmercury (MeHg) is far more neurotoxic than Hg and can bioaccumulate in the food chain. Hence, more detailed systems biology studies are needed to maximize our understanding of the potential of these types of organisms and their interactions with Hg and U. As part of the Hg Science Focus Area (SFA) at Oak Ridge National Laboratory (ORNL), a comprehensive and multidisciplinary effort is underway to understand the processes of Hg methylation and MeHg demethylation in-situ, at the organismal level by discovering the enzymes responsible in these model organisms, and at the molecular level by determining the mechanism of action within the active site of the known proteins for Hg(II)-reduction and other related activities. To this end, metagenomic analyses will be performed on five sites where Hg contamination is present and there is existing evidence for the bacterial methylation of Hg. Samples will also be taken for metatranscriptomic analyses to tease out the plausibly dominant metabolic processes in Hg methylating vs non-methylating sites. All of this information will be combined with our existing comprehensive datasets for geochemistry, microbial community structure via FLX454 pyrosequencing, and functional gene arrays (FGA) that encompass over 23,000 genes. The inclusion of comprehensive metaproteomic data to our community metabolic and mRNA datasets will not only enhance our understanding of the in-situ microbial community at all systems biology levels, but will also serve to determine the complement of proteins found within the 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 group of known Hg methylating bacteria and the processes of methylation and demethylation are of considerable importance to the DOE OBER in particular. By obtaining a more thorough understanding of the protein expression levels and patterns involved in a community setting that is Hg contaminated but not producing MeHg vs one that is both contaminated and has evidence of MeHg generation, new avenues for exploration and elucidation of the genes and proteins responsible are likely to be revealed. When combined with our other analyses, we anticipate elucidating previously unknown mechanisms for these activities.
The work to be conducted at EMSL will include the processing of frozen sediment samples 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 for the metaproteome will be required and the metagenome will be supplied at the earliest time, but certainly within 6 months of this date. 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 for sediment sample preparation and triplicate MS analysis and bioinformatic processing of each of the five samples all within the proteomics core of EMSL over the two year period. We anticipate duplicate sample replicates. Thus we anticipate the construction of the meta-AMT tag database to require ~150 MS/MS runs (50 for all proteins; i.e. global, as well as 50 for the soluble protein fraction and 50 for the insoluble protein fraction).
Project Details
Project type
Large-Scale EMSL Research
Start Date
2010-10-01
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members