NEW YORK (GenomeWeb News) – Functional targeting can refine metagenomic studies, revealing new organisms and providing genetic clues about their role in an ecosystem, according to new research.
In a paper appearing in Nature Biotechnology’s advanced, online publication yesterday, a team of researchers used DNA labeling to target methylotrophs — microorganisms that break down one-carbon compounds such as methane — in sediment samples pulled from the floor of Seattle’s Lake Washington that contained thousands of microbes.
“Our approach was simply to define our community before sequencing,” senior author Ludmila Chistoserdova, a microbiologist at the University of Washington, told GenomeWeb Daily News.
From the sequence they garnered, the team was able to stitch together a nearly complete genome for a microbe called Methylotenera mobilis and begin analyzing the function of this previously unknown bug. And, the researchers noted, such functional metagenomics techniques will likely prove useful in other environments as well.
Although metagenomic sampling offers the potential to capture all of the DNA in a particular environment, Chistoserdova explained, it also has drawbacks. Because environmental samples usually contain complex communities, metagenomic sequencing frequently turns up bits and pieces of DNA that don’t quite fit together, she said. That makes it difficult to determine what sorts of organisms are in a sample, what they’re doing in the environment, how they fit together, and how they’re related to one another.
By linking sequence to function, Chistoserdova and her colleagues were able to start honing in on relationships between certain community members and understanding the functions and interactions of these players.
The researchers — including collaborators from the University of Washington, US Department of Energy’s Joint Genome Institute, the IBM Thomas J. Watson Research Center, Lawrence Berkeley National Laboratory, Combimatrix, and the Los Alamos National Laboratory — focused on methylotrophs, microorganisms with a role in carbon cycling. “They consume C1 compounds and some of these are very potent greenhouses gases, such as methane,” Chistoserdova said.
After collecting mud samples from more than 200 feet below the surface of Lake Washington, Chistoserdova and her team mixed them with five 13C-labeled C1 compounds: methane, methanol, methylamine, formaldehyde, and formate. They subsequently separated the DNA by weight using density gradient ultracentrifugation, constructed a shotgun library for each substrate, and sequenced each using ABI PRISM 3730 sequencers.
“It took a lot of mud,” Chistoserdova said. Still, the effort paid off. In the process, the researchers managed to score an unexpected find: sequence covering almost the entire genome of M. mobilis, a previously unknown and uncultured organism.
In the past, Chistoserdova noted, it would have been virtually impossible to put together the genome of one species out of the roughly 5,000 in the mud sample, especially since M. mobilis accounted for less than half a percent of the species in the sample.
Based on information gleaned from its genome, the researchers speculated that M. mobilis probably prefers a microanaerobic environment and acts as a denitrifier. Chistoserdova and her team are currently doing follow up experiments to test these and other hypotheses about M. mobilis. They are also studying another bug from the lake mud sample — an organism called Methylobacter tundripaludum, for which they have put together a partial genome.
The team also compared the M. mobilis genome with that of another methylotroph called Methylobacillus flagellatus. That analysis suggested that the two microbes share genes involved in some central functions and in methylotrophy but diverge in those other biochemical functions. “We learned that they are quite different even though there are a core set of genes that are similar,” Chistoserdova said.
Although the researchers focused on lake sediments for this study, Chistoserdova noted that functional metagenomics should work in other environments, too, as long as there is sufficient DNA available and appropriate enrichment conditions are selected. “If you are interested in a certain function,” she explained, “you just need to pick a substrate that will work.”
For her part, Chistoserdova said that she is interested in pursuing additional functional metagenomic studies, including some aimed at assessing methylotroph transcriptomes. That should be possible using techniques similar to those described in this study, she said, by sequencing RNA rather than DNA.