Protemic Analysis of Naturally Ocurring Methane Oxidizing Archaeal Communities
EMSL Project ID
5104
Abstract
Microbial mediated anaerobic methane oxidation (AOM) in marine sediments represents a significant but poorly understood carbon sink throughout the world's oceans. Mounting geochemical and isotopic evidence indicates an essential coupling between methane oxidation and sulfate reduction in these sediments. Methane oxidizing archaea (MOA) have yet to be cultured. However, environmental shotgun and fosmid libraries constructed from samples enriched for MOA and sulphate-oxidizing bacterial groups have recently been constructed. In collaboration with the US Department of Energy Joint Genome Institute (JGI) more than 22 Mb shotgun and 6.4 Mb fosmid sequence has been determined and partially assembled. Analysis of DNA sequences obtained from shotgun and fosmid libraries has identified several important metabolic pathways with the potential to enable AOM, including constituents of both sulfate reduction and methanogenic apparatus.Although considerable progress has been made in predicting the genomic blueprint for MOA group members present in these libraries, the static nature of the data cannot capture the dynamics of gene expression and function in relation to the process of AOM. For uncultured groups such as MOAs, proteomic analysis would provide the best means of examining function, and coupled to the genomic database, would be a powerful truthing mechanism for in silico annotation efforts and genome assignment. Accurate annotation in turn would provide a platform for robust biochemical pathway modeling and reconstruction. In this way, proteome analysis from partially reconstructed environmental genomes could provide a bridge between genetics and geochemistry in naturally occurring MOA populations.
In addition, recent advances in microcosm enrichment of specific MOA groups opens another major road of inquiry into the metabolic and physiologic processes underlying AOM. Our group has been successful in enriching for MOA under controlled laboratory conditions. Moreover, we have been able to incubate those sediments using a variety of conditions, thereby providing the opportunity for experimental manipulation. For example, our MOA enrichment system offers us the opportunity to conduct perturbation studies (e.g. by altering the concentration of methane or limiting sulfate) and use proteomics to examine changes in protein expression as a function of these perturbations. We believe that by coupling genomic data and the enrichment system to proteome analyses, a novel and unique intellectual window into the process of AOM would be opened. Interestingly, preliminary LC-MS/MS analysis of MOA enriched environmental protein extracts has identified mass tags for the three subunits of methyl coenzyme M reductase (mcr) holoenzyme mcrA, mcrB and mcrG, thereby supporting the feasibility of this approach. Accordingly, we propose to produce a MOA-enriched library of protein extracts from both environment and laboratory-incubated samples. We initially intend to use the existing genomic dataset to identify mass tags in a comparative look at both environmental and laboratory-based experimental systems.
Project Details
Project type
Exploratory Research
Start Date
2005-01-01
End Date
2006-11-08
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
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