IDENTIFYING THE ACTIVE MICROBIAL COMMUNITY IN YELLOWSTONE RIVER WATER AMENDED WITH METHYLATED SUBSTRATES

Rivers connect ecosystems and play a key role in nutrient cycling. Despite their importance, most microbiology research done on rivers has failed to account for microbial activity, instead focusing solely on marker gene or metagenomic surveys. Metagenomic studies are useful for profiling microbial communities and defining their metabolic potential, but they fail to provide information about the activity of the extant microbes or determine under which conditions that metabolic potential is realized. This results in a systemic disconnect between our understanding of the diversity of microbial communities and their biogeochemical functions. To bridge this gap, our work introduces the use of BioOrthogonal Non-Canonical Amino acid Tagging coupled to Fluorescence Activated Cell Sorting (BONCAT-FACS) to examine Yellowstone River samples. BONCAT-FACS depends on the incorporation of an amino acid analog into the proteins of translationally active cells. This amino acid analog can then be tagged with a fluorescent dye. Using a cell sorter, the fluorescently labeled cells can be separated, allowing for the identification of the anabolically active community. In this work, BONCAT-FACS was preceded by incubating Yellowstone River water with methylated substrates (methylamine, glycine betaine) implicated in aerobic methane production, under light and dark conditions. We demonstrate that BONCAT-FACS is a powerful tool to determine the members of the riverine microbial community actively involved in carbon cycling and, in this instance, potentially involved in methane production.

Primary Presenter: William Christian, Montana State University (wcchrist@mtu.edu)

Authors:

Jennifer Crandall, Montana State University  (jennifer.crandall1@student.montana.edu)

Andrew Montgomery, Montana State University  (andymontgomery2009@gmail.com)

Sylvia Nupp, Montana State University  (sylvia.nupp@student.montana.edu)

Qian Wang, Montana State University  (qian.wang2@montana.edu)

Roland Hatzenpichler, Montana State University  (rolandhatzenpichler@gmail.com)

Tim McDermott, Montana State University  (timmcder@montana.edu)