S25: “Omics” analyses of resident microbes in extreme environments such as petroleum reservoirs and deep sea oil plumes

A systems biology approach using metagenomic, transcriptomic and proteomic analyses to characterize resident microorganisms in extreme environments, such as those in oil reservoirs, or in deep sea oil plumes, is hampered by low cellular abundances. To address this issue, we are employing pressure, in combination with and in comparison to, conventional extraction techniques, to enhance cell lysis thereby increasing the yield of nucleic acids and proteins from concentrated biomass. Thus far, we have developed a protocol that utilizes pressure to successfully extract proteins from biomass concentrated on filters. Specifically, a proteomics pipeline was developed that employs direct extraction from microbial biomass concentrated on filters using Urea and pressure lysis.  Proteins are then subjected to pressure assisted tryptic digestion.  Finally, peptides are analyzed using 2D-LC-MS/MS.  Translated metagenomic sequences that are generated using the Illumina sequencing platform are then used to identify proteins (proteogenomics).  The goal of this work is to develop protocols that allow for unprecedented insights into the in situ microbial processes in environments with intrinsically low biomass, such as oil reservoirs, or in the deep sea oil plumes that have resulted from the oil spill in the Gulf of Mexico.  Within these deep oil plumes characterizing the microbial community and examining their role in bioremediation is essential and requires sufficient macromolecule concentrations to allow for a systems biology analysis.