| The major goal of this Microbial Observatory project is uncovering the diversity of C1-metabolizing populations in the environment, using Lake Washigton sediment as a model, via detection of an ancient H4MPT-linked C1 transfer metabolic pathway and obtaining insights into the evolution of this pathway in prokaryotes, particularly in the bacterial domain of live, via phylogenetic analyses. We have employed comparative genomics to build and expand the databases of genes involved in C1-transfer reaction in the bacteria. Based on gene alignment, we have designed novel tools for environmental detection of four of the genes: fae, mtdB, mch and fhcD. In addition, we have constructed a large insert metagenomic library carrying a total 1.3Gb of sediment DNA and analyzed this library for the presence and distribution of these genes. A variety of C1-utilizing microbes have been isolated from Lake Washington in pure cultures, and a number of novel taxa have been described. In addition, we have tested for community members actively metabolizing C1 compounds via detection of specific RNA isolated directly from the lake sediment. Another approach we have utilized to characterize active members of the methylotrophic community in the site was the stable isotope probing (SIP) approach, in which lake sediment samples were exposed to a variety of 13C-labeled C1 compounds, DNA was extracted, and 13C-labeled (heavy) DNA was separated and analyzed via PCR-amplification of 16SrRNA and functional genes. In collaboration with JGI our group has recently initiated a functional metagenomics project that involves group-targeted environmental sequencing of the enriched C1-utilizing communities from Lake Washington. 13C-DNA from five separate microcosms exposed, respectively, to methane, methanol, methylamine, formaldehyde and formate were used to construct five separate shotgun libraries, and these are in the process of being sequenced. |