Life From the Pit
How Montana’s toxic lake may be the key to a new antibiotic
By Courtney Brockman
The circumstances that led Don and Andrea Stierle to begin studying the Berkeley Pit were simple: They were out of money. The professors and natural products organic chemists — a husband and wife team — had worked everywhere, from the waters off the coast of Bermuda to the forests of the Pacific Northwest.
Their quest: find new microorganisms containing drug potential.
But now, with limited funding, they looked no farther than a mile from their lab at Montana Tech of the University of Montana in Butte for their next project.
“We were luckier than most people in our field, because we had the Berkeley Pit,” Andrea says.
The Berkeley Pit, part of the largest Superfund site in North America, is filled with a soupy, brownish liquid comprised of high concentrations of iron, copper and other metal salts. The pit’s walls are rich in iron pyrite — or fool’s gold — which produces an unceasing acidic broth. Because of the lake’s low pH of 2.5, no one had thought before to look for life within such a toxic environment.
But a colleague collecting samples found something growing on a stick submerged in the pit’s “uninhabitable” waters. He brought it to the Stierles’ lab for identification, and the blue-green blob turned out to be an alga. It was the summer of 1995.
This was a time when other scientists sought life in deep sea vents and other less-than-hospitable environments. Thirty years before, Thomas Brock had found life in the hot, acidic pools of Yellowstone National Park.
“We knew these organisms growing in extreme environments had secrets to share,” Andrea says.
But the Stierles never guessed that they would still be making important discoveries from the pit’s microbes more than 20 years later.
The Berkeley Pit sits in the Boulder Batholith near Butte, a city built by copper. During Butte’s heyday in the late 1800s and early 1900s, the world demanded copper wires to transmit electricity, mines pulled metals day and night, and miners dropped deeper into the Earth as the ore in the rock diminished.
Butte switched to open-pit mining in 1955, which allowed large quantities of rock to be removed, concentrated and processed. The result was the Berkeley Pit — a mile-wide, half-mile-deep crater that has become Montana’s deepest lake.
Pumps kept the Pit dry until they shut down in 1982, and water flowed back in within a year. Today, the water has risen to just 71 feet below the critical 5,410-foot mark, where it can seep into surrounding aquifers.
At one point, boats dropped long cables with collecting vials down to the sediment of the pit to take water samples, but monitoring ceased in 2013 after sloughing caused a 30-foot tidal wave to take out the pier and dump an estimated 820,000 tons of material into the pit.
The Stierles’ pit water samples were gathered by the Bureau of Mines and Geology in July 1995 and spring 1996.
“And something happened between those two collections,” Andrea says.
Exhausted from their long migration from the Arctic and attracted to what looked like a body of fresh water, 342 snow geese landed in the pit during a snowstorm on Nov. 15, 1995. Exposure to the toxic water killed them.
The 1996 pit water samples yielded a new murky, charcoal-colored yeast that absorbed metals from the water — something reported only once before by a Japanese scientist who had taken rectal swabs from a goose. The Stierles theorized that the geese expelled the yeast to make themselves more aerodynamic for take-off.
“To put it indelicately, they all pooped,” Andrea says.
A year later, the yeast was gone from the water samples. When thousands of other snow geese died in the pit in November 2016, the Stierles were unable to obtain a sample. But what they have found in the previous samples will keep them busy for a lifetime.
The Stierles have used several techniques for isolating fungi and bacteria from the pit water.
“We didn’t know whether these guys were just incidental — if they just happened to land in the pit, and we just happened to grab them — or if they could thrive under these hostile conditions,” Don says.
Fungi in the pit could have come from the air as spores, from bird feathers or from rotting, carbon-containing mining timbers from old drifts and shafts. When the Stierles created broths with pit water in them, they found that 90 to 95 percent of the fungi grew just as well in the extreme broth as in a conventional broth. Some grew even faster.
The Stierles cultivated each of the fungi under a variety of conditions to test their secondary metabolism.
Most bacteria and fungi produce secondary metabolites, or natural products, they can survive without — such as the caffeine in a coffee plant or the nicotine of a tobacco plant. Compounds such as these may help fight diseases in humans. But little is known about the secondary metabolism of organisms living in extreme environments.
In the early pit fungi samples, Don discovered some of the compounds produced could potentially target metastatic cancer, inflammation and post-stroke brain damage. But there was no antibiotic activity.
Last fall, the Stierles looked at two new fungi. They had little bioactivity.
“They were the most boring organisms we had ever looked at,” Andrea says.
They then decided to grow them together to see if they would produce a new compound in competition with each other.
Each petri dish where two or more fungi are co-cultured becomes a fighting ring for resources. All fungi and bacteria, which contain more genes than usually expressed, have the potential to produce many more compounds when challenged by other organisms.
“It’s cool turning on these genes that are obviously there and produce antibiotics that weren’t turned on until they got associated with a competitor,” Don says.
In the pit, where life is sparse, fungi do not have to compete with other organisms for resources. But when two are put into a broth in close proximity, they become aware of each other through chemical quorum sensing — the fungi’s form of communication.
“It’s going to be worth the energy investment to create an antibiotic to kill that other guy, so that you get all the ‘cookies’ — all the nutrients,” Andrea says.
In a lab at UM in Missoula, where the Stierles moved in 2009, the two fungi sit in petri dishes — one a nondescript dull brown and the other containing a slight hint of green. When the two were grown together, something interesting happened.
The fungi produced several new classes of compounds, and one of which resembled a known antibiotic. The Stierles sent it to colleague Nigel Priestley, a UM organic chemistry professor, who tested it against a suite of pathogenic bacteria and yeasts.
Priestley found the compound was active against Bacillus anthracis, a couple of Streptococcus strains and a few yeasts. But it was more active against drug-resistant strains of Staphylococcus aureus MRSAs — community and hospital-acquired antibiotic-resistant bacteria — than anything else.
Although structurally similar to other antibiotics, the Stierles and Priestley noticed the compound did not contain the sugar groups of most. The discovery excited them.
“We really aren’t generating new antibiotics faster than old ones are becoming obsolete due to resistance,” Priestley says. “Any time we can get a new structural class of antibiotics, there is hope that a new antibiotic will eventually make it to the clinic.”
Antibiotic resistance has been known since the day Alexander Fleming accidentally discovered penicillin in the early 1940s.
According to Fleming’s notes, a spore of the fungus Penicillium fell as a contaminant onto a petri dish streaked with Staphylococcus aureus, creating a zone of inhibition that killed the bacterium. But within the cleared zone, Fleming noted there were a few colonies of Staph growing that already had become resistant to the penicillin produced by the Penicillium spore.
Today, more than 23,000 people in the U.S. die annually from drug-resistant bacteria. Groups all over the world are looking at different ways to fight bacteria and come up with new modes of targeting bacteria, such as using bacteriophages — viruses that kill bacteria.
“Any time you’re dealing with disease, it’s a chemical, biological chess match,” Andrea says. “And each side is kind of bringing a new arsenal, a new strategy into the game. As chemists, as scientists, this is our game. This is what we bring to it.”
The Stierles have isolated 12 antibiotic compounds from the two co-cultured Berkeley Pit fungi. Most antibiotics find something unique to target in a pathogenic bacterium, such as an enzyme, to disrupt protein synthesis or another process.
“To our surprise, it didn’t touch protein synthesis. It did not bind to the ribosome. It is nothing like any known macrolide antibiotic,” Andrea says.
For now, the Stierles are trying to learn how exactly the compound works as an antibiotic. First they must test if the compound is toxic to mammalian cells and whether it can be metabolized and made into an orally available drug. Then, they must find a pharmaceutical company interested in investing in the drug.
One pharmaceutical company has contacted them, and they are collaborating with UM’s Division of Biological Sciences to eventually do a genomic study of the two fungi. They also have submitted a grant with a new biotech company to look at the gene clusters within the fungi.
The Stierles, who believe collaboration is essential, have received their funding through individual grants and support from UM’s Center for Biomolecular Structure and Dynamics. And their lab on campus is just big enough to do the work needed.
“The University of Montana has been a good move for us,” Andrea says.
The Stierles visit Butte, where they still have a house, on most weekends, and sometimes find themselves at the edge of the Berkeley Pit, just as they did 37 years ago during their honeymoon.
“It is a bleak, bizarre landscape,” Andrea says. “And it’s hauntingly beautiful in some ways.”
Above the mine sits the Granite Mountain Memorial, dedicated to the 168 lives lost in one of the worst hard-rock mining disasters in the United States in 1917. The last names match the names of many present-day Butte families.
But, despite its toxicity, the Stierles believe the Berkeley Pit is a lesson on how to approach problems.
“We like the idea of reminding people that even contaminated sites have value,” Andrea says. “A toxic waste dump can harbor fungi and bacteria that produce compounds that could cure cancer, fight inflammation and even be the next antibiotic. So it’s the unexpected; it’s looking at something in a way that nobody else has looked at it before.” •
