UW-Madison lab decodes lake bacterial cycles with RNA sequencing

by Shelby Lyon of the Journal Sentinel –

When a sheriff’s deputy found a van parked at the edge of Sparkling Lake in Vilas County at 2 a.m. on July 7, chances are he expected to find something nefarious afoot. Instead, he got a crash course in fresh water ecology from graduate student Alex Linz.

“The police car pulled up behind us, and I thought ‘oh no!'” Linz said. “But I explained how we were studying bacterial cycles in the lake and that we had been collecting an overnight sample. He actually got really excited and wanted to know more about the bacteria. ”

Linz is a member of Trina McMahon’s lab at the University of Wisconsin-Madison that studies how bacterial activity changes throughout the day. The lab just wrapped up its biggest project of the year in the wee hours of a recent Saturday morning.

Gene Expression in Oligotrophic, Dystrophic and Eutrophic Systems, dubbed GEODES for short, is a massive three-lake, two-week water-sampling experiment. Members of the McMahon lab already have logged their samples from two northern lakes near Minocqua: Trout Bog Lake and Sparkling Lake. Then they set about retrieving the first of 13 samples from a sheltered spot on Lake Mendota.

“These bacteria are wild and can’t be grown in a lab, so, if we want to study them, we have to do it in the lake,” Linz said.

McMahon, a professor of bacteriology and civil and environmental engineering at UW-Madison, hopes the results from the project will provide insight into how bacteria metabolize different compounds to keep lakes healthy and contribute to the global carbon cycle.

“You can kind of think of it like breathing, with the CO2 going in and out of the water balanced by the bacteria,” McMahon said. “Oceans get the most attention, but lakes play a critical role in the carbon cycle and climate change.”

Each experiment spanned the course of 48 hours with samples taken every four hours. That meant a lucky pair of lab members had to take the boat out into the middle of the lake for the overnight time points at 1 a.m. and 5 a.m.

“We go out about a half-hour before a time point to check water temperature, dissolved oxygen and acidity because we want to see if those factors, in addition to sunlight, drive changes in the bacteria,” Linz explained. “Then we drop a 39-foot tube, which acts kind of like a soda straw, to collect water. We plug the end and haul up the water into buckets, then pass the water through a filter with teeny, tiny pores to get bacteria and RNA.”

And because RNA can degrade in as little as five minutes, the team carried liquid nitrogen to flash freeze the samples and lock in the RNA levels from the time of collection.

In addition to the periodic RNA samples, a single DNA sample was taken from each lake as a reference. Since each bacterial cell has a DNA genome, samples can be sequenced and matched to a known genome database to catalog all the different species that were present.

Unlike DNA, RNA is transcribed, or made, based on how much of a particular protein is needed. Sequencing the RNA from different time points reveals which of the bacteria present in the lake were active. It can even uncover which genes were being expressed and, by extension, which functions were necessary to the bacteria in a particular environment.

Sparkling Lake contains low levels of nutrients, illustrating the oligotrophic “O” in GEODES. Few organisms live there, which makes the water sparklingly clear.

Trout Bog Lake is dark with carbon compounds and tannic acid, but it’s low on pretty much everything else. This type of lake is dystrophic.

Finally, Lake Mendota, which is replete with nutrients like phosphorus and nitrogen from agricultural runoff, is a classic example of a eutrophic lake.

While sequencing has become cheaper over the years, the sheer number of samples from all three lakes and the complexity of microorganisms in the water would make sequencing the DNA and RNA too expensive and time consuming for the lab members to perform themselves.

“I don’t think people realize that there are thousands of different bacterial species in a lake,” McMahon said. “That’s (exponentially) more organisms than the fish and species people are more familiar with.”

So Linz submitted a proposal to the Joint Genome Institute, a Department of Energy program in California that provides free sequencing services to scientists conducting huge DNA and RNA projects called metagenomics and metatranscriptomics.

GEODES was selected from hundreds of proposed projects, meaning the lab will receive around $150,000 in sequencing and support services free of charge.

Linz expects to get up to 2 terabytes of data representing the genetic sequences from all the DNA and RNA collected in the experiment.

“I’ll probably spend the rest of my Ph.D. analyzing the results,” Linz said.

The enormous amount of data will help the researchers figure out how bacteria behave in their natural habitats and interact with other species to form the foundation of the lake ecosystem.

“Microbes rule the planet,” McMahon said. “They are essential for keeping the water clean and providing energy. The next time you go swimming, you should realize all the beneficial things bacteria do. It’s only a few species that are bad.”

Source –  Milwaukee Wisconsin Journal Sentinal

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