Highly Divergent Genes for Methanopterin-Linked C ₁ Transfer Reactions in Lake Washington, Assessed via Metagenomic Analysis and mRNA Detection

Abstract

The origins and the evolutionary history of tetrahydromethanopterin-linked C 1 transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea ( Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.

Real-time detection of actively metabolizing microbes by redox sensing as applied to methylotroph populations in Lake Washington

“…Lake Washington sediment core samples were collected as described previously (Kalyuzhnaya et al, 2005b). Sediment cores were transported to the laboratory on ice and stored at 4 1C.…”

“…The upper layer of the sediment of this lake is known for high rates of methane metabolism and is an attractive model for studying environmental cycling of C 1 compounds. The C 1 -utilizing bacterial community has been characterized so far using a number of culture-reliant (Auman et al, 2000;Miller et al, 2005;Kalyuzhnaya et al, 2005aKalyuzhnaya et al, , 2006a) and culture-independent methods, including surveys of functional genes, detection of specific RNA transcripts and stable isotope probing (Costello and Lidstrom, 1999;Kalyuzhnaya et al, 2005b;Nercessian et al, 2005). The picture emerging from these studies shows a complex multimember community that includes species known for methylotrophic capability (Methylomonas, Methylobacter, Methylocystis, Methyloversatilis, Methylotenera etc.)…”

“…), with specific groups revealing specific nutritional preferences (Kalyuzhnaya et al, 2005c;Nercessian et al, 2005). In addition, species are present unidentified phylogenetically, being only detected at the level of functional genes (Kalyuzhnaya et al, 2005b). In an attempt to connect predicted function to phylogenetic identity, we have previously developed a method including fluorescent in situ hybridization (FISH) staining combined with flow cytometry (FC) and cell sorting (Kalyuzhnaya et al, 2006b).…”

Respiration Response Imaging for Real-Time Detection of Microbial Function at the Single-Cell Level

“…Sediment samples were collected on 3 March 2009 and on 27 July 2009 as described previously (9). Sediment cores were transported to the laboratory on ice and stored at 9°C.…”

“…Thus, this helps in identification of cells involved in a targeted metabolic process. We used methanol as an example of a single-carbon compound for detection of methylotrophic abilities among bacterial populations from the well-studied sediment of Lake Washington in Washington (2,3,(8)(9)(10)(11) and demonstrated that it is possible to use RRI for direct functional profiling of environmental samples.…”