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Metagenomic Study Reveals Diverse Relationships Amongst Deep Sea, Methane-Oxidizing Microorganisms

NEW YORK (GenomeWeb News) – A metagenomic approach is revealing new physical associations between species of deep sea microorganisms in methane vents — information that may ultimately help scientists unravel the functional interactions between these bugs.
Researchers from the California Institute of Technology used a so-called “magneto-FISH” strategy, which combines fluorescence in situ hybridization with immunomagnetic cell capture, to pull out groups of bacteria associated with anaerobic methane-oxidizing archaea — single-celled prokaryotes that oxidize methane in the absence of oxygen.
The results, appearing this week in the Proceedings of the National Academy of Sciences, suggest that the relationships between these sediment archaea and bacteria are much more diverse than previously recognized. They also provide insights into nutrient cycling in these deep sea sediment communities.
“There were groups we didn’t expect,” Caltech geobiologist Victoria Orphan, senior author on the paper, told GenomeWeb Daily News. “It suggests that there’s a bit more versatility in this syntrophy than previously realized.”
Although people don’t tend to think of the ocean as a methane source, Orphan said, anaerobic metabolism, decaying organic matter, and geothermal sources can all produce ocean methane, which could potentially end up as greenhouse gas in the atmosphere. Roughly 80 percent of this methane is sequestered deep in the ocean by methane-oxidizing microorganisms.
Orphan and her colleagues are studying a group of these microorganisms, called anaerobic methane oxidizing archaea, or ANME, which do “the lion’s share of methane oxidation in marine environments.” So far, all of the members of this group remain uncultured and are recognized only genetically — in particular, by 16S rRNA sequences. They archaea have been classified into three main groups: ANME-1, 2, or 3.
ANME apparently live in syntrophic associations with sulfate-reducing bacteria, in which the microorganisms depend on one another for survival. But characterizing these associations has been difficult. In an effort to identify and understand these interactions, Orphan and her team used magneto-FISH to nab bacteria associated with an ANME sub-group called ANME-2c. These archaea seem to form shelled, structured architectures, Orphan explained, with ANME archaea on the inside and associated bacteria on the outside.
First, the researchers collected deep sea sediments from regions near methane seeps in California’s Eel River Basin using the Woods Hole Oceanographic Institute deep-sea submersible, Alvin. Then, they added fluorescently labeled 16S rRNA probes to the sediment. These targeted specific ANME subgroup in which they were interested. Finally, the team amplified the fluorescence signal in the cells and isolated ANME-2c and interacting cells using paramagnetic beads coated with antibodies to the fluorescent probe.
These microorganism associations could then be analyzed by metagenomic sequencing, using 454 Life Sciences’ pyrosequencing technology, and by PCR and microscopy approaches. Through these analyses, Orphan and her colleagues found a surprisingly diverse group of bacteria interacting with the ANME-2c methane-oxidizing archaea.
For instance, pyrosequencing revealed many reads associated with the sulfate-reducing bacteria Deltaproteobacteria, including one new sulfate reducing Deltoproteobacterium species, and Methanomicrobiales. The researchers also detected a Betaproteobacterium species that wasn’t previously known to reduce sulfate.
Their results also provide new clues about methane cycling and the nutrient composition in these deep sea environments. Unexpectedly, the researchers detected enzymes in the ANME communities that are involved in nitrogen fixation, a process that brings in and fixes new nitrogen. And using labeled dinitrogen, Orphan and her team showed that the methane-oxidizing archaea themselves take up new nitrogen.
In the future, Orphan said, the group plans to target other ANME subgroups with magneto-FISH, so they can better understand co-metabolism between different syntrophic species, along with the evolution of deep sea syntrophic relationships, nutrient cycling, and much more.
“It was like opening Pandora’s box,” Orphan said. “There are so many new objectives to pursue.”

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