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At ABRF, Metagenomic Approaches Offer Glimpse of Microbial Makeup, Role in Environment

ST. LOUIS (GenomeWeb) – Though they present challenges, metagenomic tools are enabling researchers to characterize microorganisms living in even the most extreme environments and begin to understand what role those microbes play there, according to speakers at this year's Association of Biomolecular Resource Facilities meeting.

Susannah Tringe of the US Department of Energy's Joint Genome Institute and her colleagues have sequenced microorganisms living in restored wetland to determine how they fit into the carbon cycle of that setting. At the same time, other researchers are using similar tools to study whether microorganisms enable deep-sea mussels to cope with an oily habitat as well as to better understand which microorganisms can live in extreme environments like hypersaline lakes or gas craters.

Still, metagenomics presents technical challenges. An ABRF committee established at last year's annual meeting, dubbed the Metagenomics Research Group, is taking on some of these issues.

The University of Vermont's Scott Tighe reported that the group has begun to develop bacterial reference standards for metagenomic studies based in part on a reference developed by the ABRF's Nucleic Acids Research Group in 2013. The standard currently includes the whole genomes of six biosafety-level one bacteria — including Pseudomonas fluorescens, Escherichia coli, Micrococcus luteus, Halobacillus halphilus, and Staphylococcus epidermidis — that have variable GC content and relatively simple genomes.

In the future, Tighe said the Metagenomics Research Group plans to develop reference standards that include more complex microbial genomes as well as ones that represent other kingdoms.

The working group, he noted, wants to drive the field towards standardization, and it is teaming up with both academic and industry partners to do so. By next year, he added, the group hopes to have a whole-cell standard available.

"These microbial standards, we need [them]," Tighe said during a session devoted to the working group's efforts. "If there's a way we can build them, we want to do that."

Speakers also gave a peek into what such metagenomic studies can uncover.

In her plenary talk, JGI's Tringe described how she and her colleagues, including researchers from the US Geological Survey, have been sequencing microbial communities to understand their contribution to the carbon cycle of wetlands, especially restored wetlands.

Though wetlands make up a small portion of the world's landmass, they store about a third of terrestrial carbon and act as a carbon sink, removing it from the environment. However, wetlands, in some instances, produce methane, which is also a greenhouse gas.

Tringe and her team have been studying former wetlands in the San Francisco Bay area. At one reclaimed site in particular called Twitchell, the researchers noticed that peat accretion occurred fairly rapidly across the region, though the amount of methane produced in the area varied with distance from the water. This, they hypothesized, could be due to increased production by methanogens or due to the presence of different methanogens than at other sites.

By sequencing the community microbes, she and her team found differences in gene abundance — there was a higher abundance of methanogen genes at sites with higher levels of methane. However, there wasn't a difference in RNA abundance.

At the same time, the high methane regions were enriched for hydrogenotrophic methanogenesis versus acetoclastic methanogenesis. There does seem, Tringe said, to be a difference in how methane is produced.

The University of Georgia's Matthew Saxton, meanwhile, has been studying the microbes associated with deep-sea mussels living in an oily, deep-sea environment. These Bathymodiolus typically reside in beds near hydrothermal vents and cold seep sites, but the researchers also found them near a natural oil and gas seepage site in the Gulf of Mexico.

Mussels pull water across their gills, and in this habitat, the water comes with the oily addition of polycyclic aromatic hydrocarbons that then become concentrated in the mussels, Saxton noted in a separate session.

He and his colleagues collected two Bathymodiolus brooksi and one B. childressi from the Gulf. By exposing tissues from these mussels to a 14C tagged oil substrate substitute comprised of hexadexane and naphthalene, the researchers found B. brooksi could oxidize both hexadexane and naphthalene of the oil-like mixture, while B. childressi could oxidize hexadexane.

Saxton and his colleagues sequencedtheir mussels and associated microbes and then reconstructed 16S rRNA genes from the raw reads to separate the host and symbiont reads.

From this, they found that B. brooksi had both methanotropic and thioptropic symbionts, and B. childressi had methanotropic symbionts. Saxton noted that while the sulfur oxidizers looked like others that have been found before, they did appear to harbor gene variants associated with PAH degradation, suggesting that they may play a role in that process.

They also found that commensal gill-associated bacteria also appeared to include potential oil-degrading bacteria like Pseudoalteromonas.

In addition to these efforts, the ABRF group has launched its own research project to study microbes found in extreme environments using whole-genome shotgun sequencing. It has, Tighe reported, identified a number of forbidding environments to sample, including the super salty Lake Hillier in Australia and the Doors to Hell gas crater in Turkmenistan, as well as feces collected from penguins living in Antarctica.

This project, Tighe said, would enable the research group to mimic the worse-case scenarios faced by metagenomic researchers.

They've collected and begun to analyze some samples, and were able to generate sequences from the Australia and Turkmenistan environmental samples and the penguin feces, finding Streptomyces with high GC content in the Turkmenistan samples and Gillisia, Geobacillus kaustophilus, and Clostridium perfringes in the penguin feces samples.

By relying on whole-genome sequencing for these projects, Tighe noted that they hope to get, where possible, species-level information that can sometimes be elusive in metagenomic studies.