Fungal Pests Reassessed

Fungal Pests Reassessed

BTI researcher maps the gene expression of a fungus to better understand how the microorganism eats.

By Allison Kronberg

Some fungi have developed a bad reputation as pests eating wood from the buildings where people live and work. But BTI researcher Jonathan Schilling is challenging old assumptions and finding new reasons to study the ubiquitous microorganisms.

Research in Schilling’s fungal biology lab suggests that plant- or wood-degrading fungi may find their way into a variety of new technologies, from insulation and indoor air filtration, to biofuel production.

“Since I’ve been at the University of Minnesota, I’ve branched out from looking at these organisms as pests to looking at their potential in biotechnology, as well as their importance in nature,” Schilling says.

The United States Department of Energy has funded Schilling to study specific mechanisms that control how fungi eat. “The research could help biofuel companies understand the steps a fungus takes when it degrades plants,” Schilling says.

According to Schilling, the companies have an interest in finding alternative ways to make fuel from biomass, because current processes are costly and require multiple biochemical steps. Some fungi are able to consolidate those steps, and if companies are able to tap into that ability, it could make biofuels cheaper for consumers.

A recent DOE grant, is called Connectomics — a term borrowed from neurology. The term is derived from a method of study usually used to map the real-time expression of the nerves in the human brain. Schilling has applied this method to the gene expression of fungal systems.

To produce the maps, Schilling and his lab grow a fungus named Postia placenta on wood wafers. The team then cuts the wafers into slices, like a loaf of bread, and analyzes each slice. When they put the data back together, it produces a connectome map, which shows how the fungus expresses genes and operates differently at various growth regions.

But the fungus works very slowly, so commercial use may not be likely just yet.

Instead, Schilling hopes future researchers will take advantage of the maps, using the information on fungal genome and gene expression to create new technologies or improve existing ones.

“On the science side, we’re providing a service,” Schilling says. “I offer tools. Other researchers need to go out and solve practical problems.” Schilling is hopeful that a PNAS paper, which he anticipates will be published in early fall, will be a jumping off point for others to engage with and find applications for his research.

Schilling and his students work with biomaterial companies throughout the country on research ranging from the study and application of fungi as an adhesive for window and door companies, to illuminating the characteristics of Scoby — the fungus used to produce the unconventional tea remedy, kombucha. This fall he will be teaching a new course in applied mycology. foster a new generation of scientists interested in fungi. The variety of application shows that fungal “pests” have the potential to create biotech solutions, too.

Mushrooms in the Marketplace

When they aren’t researching biotech applications of fungi, students in the Mycology Club engage with local communities by selling edible mushrooms at the University of Minnesota Farmers Market.

Standing in the pouring rain might not make a lot of people happy, but as graduate student Gerald Presley turned a small umbrella-like wine cap mushroom upside down to explain how its gill margin would expand as it matured, he had a smile on his face. More rain means more fungi — and that’s good for a fungal garden.

Presley is a graduate student in lab of BTI member Jonathan Schilling (BTI/Plant Pathology). He is also president of the UMN Mycology Club. The student group hosts workshops, lectures, and activities related to fungi. They also tend the fungal garden.

The garden has hundreds of logs blooming with fungi, so the group has also been able to raise money and interact with the local community by selling mushrooms not used    for research at the Farmers Market.

“It’s always good to find a practical application outside of the laboratory,” Presley says. “It’s been great for us.”

The club first starting selling mushrooms at the farmers market in 2013, two years after it was founded. It sells a variety of edible fungi, including shitake, wine cap, and oyster mushrooms. The Mycology Club has since teamed up with the certified organic student farm Cornercopia, which sells the mushrooms at its stand, as well.

“People like the mushrooms,” Presley said. “So they’ll come to the stand… and then they might buy some of [Cornercopia’s] stuff, too.”

Cornercopia locations — outside the Andrew Boss Meat Lab in St. Paul and in  Gateway Plaza on the East Bank — are ideal because they attract customers beyond the student body. Local chefs have even begun purchasing student-grown mushrooms for their restaurants.

The stand has shoppers talking about mushrooms. “We hear people say things like, ‘Oh, what I do with mushrooms is…’ or ‘I found a bunch of morels’ or ‘I’ve got this spot with a bunch of chanterelles this year,” Presley says. “People who come up and actually talk to us, they’ve always got their story, and it’s a chance for us to teach.”

The Mycology Club sells the mushrooms for about $10 per pound, earning about $200 over the past two seasons. The small profit, along with grant money and proceeds from selling mushroom spawn, are more than enough to support the low-cost group. “It’s kept the club alive,” Presley explains.

The club uses the money to fund biannual barbeques, teaching workshops, and guest speakers. When Presley and other student officers graduate, they will tap the 300 students on the Mycology Club’s email list so the club can continue to sell fungi grown by a new crop of mycologists.

Science, It’s What’s for Dinner

Science, It’s What’s for Dinner

New BTI member David Baumler aims to develop novel biotech for protecting local and global food supplies.

By Estelle Smith

If it’s on the shelf at the grocery store, it must be safe to eat… right? Hopefully, that answer is yes. Yet a dazzling array of microorganisms — not all of them friendly — enjoy human grub in our gastrointestinal tracts as much as we do. How can science help to guarantee the safety of our foods and bodies against an army of opportunist bugs?

David Baumler, a faculty member in the Department of Food Science and Nutrition and new member of the BioTechnology Institute, develops technologies that could improve the safety and nutritional value of the food supply chain, both locally in the Midwest and across the world. “I go where the problems facing people are,” says Baumler, who hopes to engineer solutions for major health and economic problems.

Baumler’s background includes biochemical investigation of everything from pathogenic foodborne bugs and biofuel-producing bacteria to ancient microbes that thrive in the most extreme conditions on the planet. “I cross paths with BTI faculty who work on topics like bioremediation and fermentation,” says Baumler. His wide-ranging experience allows him to ask questions that cut across academic disciplines, often with promising results.

For instance, Baumler was the first to build a computational model of the metabolism of a pathogenic E. coli strain based on approximately 20 bacterial genomes — a small number compared to those available today, but an important technological leap at the time. “We tied together all the genes that produce reactions within the organism,” says Baumler. “We could then simulate almost an entire cell on a computer.” He continues to build computational models in an ever-expanding variety of contexts and organisms.

Such models provide insights that lead directly to important applications. “We look at all the information in the genome to figure out what an organism really needs to grow better,” says Baumler. If you know what an organism needs to survive, you can provide it with an optimized environment when you want it to flourish. Alternatively, you can remove what it needs if you want to kill it.

Some of Baumler’s current projects tackle the challenge of warding off prevalent food-borne pathogens like Listeria and Salmonella. One recent project even revisited a malicious
microbe from the middle ages. European collaborators dug  up corpses of bubonic plague victims and sequenced bacterial genomes trapped in their teeth. Based on these
genomes, Baumler built a model that helped reveal fatal secrets buried in the past.

Another of Baumler’s projects aims to cultivate new probiotics that could keep livestock healthier. Minnesota’s beloved turkey flocks, for example, haven’t been plumping up properly — to the tune of 30 million dollars per year in lost revenue. Baumler hopes to fatten the birds by replenishing an important bug that appears to have flown the coop of their microbiomes.

Dairy, another economic boon for the Midwest, has its own set of microbial mishaps. Milk quality and flavor can be compromised by germs, but when and where do they enter the product chain? In parallel with a massive initiative launched by IBM and the Mars candy company, Baumler will spearhead an effort to record comprehensive genetic data on dairy products at every stage of their journey from the cow to your fridge. This information could enable food makers to detect problems as they arise rather than after the fact.

To address epidemic malnutrition, Baumler also mines his personal collection of over 400 varieties of chili peppers in search of vitamin-rich (but not too spicy) varieties. “Chilies are a culturally accepted food in every country in the world,” he says, noting that 30% of all children suffer from insufficient vitamin A. “We could breed new strains and share seeds with everybody around the world to alleviate vitamin deficiencies.”

Baumler —  who has even been known to dress in a chili pepper outfit and sing the praises of his favorite plant — is a big picture thinker with an animated approach to teaching and research. “For me and my students, BTI is opening doors for networking, new information, new funding opportunities, and new collaborations,” he says. “As a BTI member with a background in food science and nutrition, I hope to bring new types of research questions to other BTI members, as well.”

Bio-machines and Nanospheres

Bio-machines and Nanospheres

Mapping Selenium Respiration Pathways in Gram-negative Bacteria

Imagine for a moment, the conditions necessary to sustain life. What comes to mind? Water? Oxygen? Sunlight? Think again. Many of the world’s smallest organisms have evolved and adapted to live under extreme conditions where these basic building blocks are scarce or absent altogether.

Visiting MnDRIVE scholar Clive Butler is determined to crack the code in one such extremophile, the bacterium Thauera selenatis. Like many microorganisms, T. selenatis exhibits remarkable respiratory flexibility. In the absence of oxygen, it’s able to substitute compounds based upon nitrogen or sulfur in a process called anaerobic respiration, which is familiar to most of us as the “rotten egg” smell from the anaerobic respiration of sulfur.

More importantly, T. selenatis is part of a small class of organisms that can breathe selenium oxides; a nonmetal trace element required for some protein synthesis. This trait alone makes the organism worthy of attention, but the bug is also a remarkably efficient bio-machine able to secrete tiny selenium nanoparticles with unexpected physical and optical characteristics. Currently very expensive and highly toxic to produce, these nanoparticles are sought after in scientific research and advanced manufacturing.

To better understand how T. selenatis is able to accomplish this remarkable feat, Butler is spending his research sabbatical working in the lab of UMN microbiologist, Jeff Gralnick (Microbiology/BTI). Gralnick and Butler have known each other for several years and follow each other’s research. Butler, an associate professor at the University of Exeter, is a biochemist looking at protein structure and function who is also interested in exploring molecular genetics and the ability to clone and manipulate an organism’s genetic code. When the opportunity for a study leave arose, Butler approached Gralnick about a possible collaboration. Leveraging Gralnick’s expertise in molecular genetics, the pair set out to create knockout mutants in a related organism called Aeromonas hydorphila, also reported to respire selenate and iron, which could help shed light on the inner working of T. selenatis and some of the iron-reducers studied in the Gralnick lab.

Unlike the mutants in science-fiction movies, these genetic mutants are bacterial strains engineered to express or suppress specific genes and traits. They help scientists isolate biochemical processes to better understand their sequence and function.

“Getting an idea of those mechanisms will give us a leg up on how this organism lives and how it survives,” says Butler. Later this month when he returns to his lab in Exeter, Butler will use the techniques learned in the Gralnick lab to investigate the missing links in the selenium respiration cycle for T. selenatis.

Butler traces his interest in science to a childhood accident that damaged his right arm. The original injury, which occurred when Butler was seven, led to tissue damage from internal swelling and required extensive reconstructive surgery. Over the next ten years as Butler’s arm continued to grow, complications arose, and the process repeated itself.

“From seven to sixteen,” Butler explains, “I spent my life in and out of hospitals around doctors and medical professionals. I became quite interested in science, medicine, and biological processes in general. Why don’t these muscles work anymore? Why doesn’t the arm do what it should do? In school, science was one of the few things I was pretty good at. Sports or the trades were out of the question. In today’s society, we’re more accepting of limitations, but this happened in the seventies, so it tended shut some doors and open others.”

By the time he entered university, his interest had shifted to biochemistry.

“One of things the arm brought home is just how dependent we are on oxygen. At a human level, if we don’t have oxygen we suffocate and die, but oxygen is also necessary at a cellular level. One of the great things about bacteria is they are really flexible with how they use energy. They often find themselves in a situation where they are deprived of oxygen and have to find a way to conserve energy and live and grow. They have to adapt. That adaptability is intriguing from a personal perspective because the injury forced me to adapt and move on with my life.”

Right handed before the accident, Butler learned to use his left hand for most daily tasks. Today he considers himself a lefty. If someone throws a ball his way, he instinctively uses his now dominant left hand. With the encouragement of his father, an amateur painter, Butler began to draw as a way of building eye-hand coordination. He became a rather accomplished draftsman and continues to draw and paint in his spare time, though science remains his primary focus.

His interest in selenate respiration began about 15 years ago after the first strain of T. selenatis was isolated from selenium-contaminated drainage waters in California’s San Joaquin Valley, by the late Prof. Joan Macy. Like many organisms that thrive in extreme environments, T. selenatis evolved to take advantage of available resources. In this case, a soluble and potentially toxic selenium oxide known as selenate.

Because selenium is an essential micronutrient necessary for some enzyme and amino acid production, many micro-organisms have evolved selenium uptake mechanisms. However, few have evolved as T. selenatis, which uses selenium in the respiratory chain that converts organic nutrients into cellular energy.

An important micronutrient, selenium is a basic building block for the 21st amino acid, selenocysteine, which is found in nearly every life form on earth. It’s widely used in feed supplements and has important industrial applications in glass production and the manufacture of circuit boards, batteries, and solar cells. Some studies suggest it may even have anti-microbial effects, but despite its beneficial role, selenium is not entirely trouble-free. Selenium oxides are frequently a byproduct of mining and fossil fuel production. They are highly soluble and toxic in large quantities and represent a potential environmental risk. Soon after its discovery, scientist began looking at T. selenatis as a potential tool for bioremediation of selenate contamination in the water district surrounding the San Joaquin Valley.

Understanding the selenate reduction pathways could allow scientists to develop highly efficient microbial filters for use in bioremediation and the recovery of selenium from mining or industrial processes. Equally intriguing, however, is the potential to develop microbial factories or bio-machines capable of producing selenium nanoparticles perhaps combined with other materials like cadmium.

“In theory, using synthetic biology you could insert both transport and production proteins into an organism like E. coli to create a biological machine that would produce a range of nanomaterials,” explains Butler. “The advantage would be getting the organism to select the materials, process them, and spit them out, which means you don’t have to go through the downstream processes of breaking up the cell to extract material.”

Early on, Butler hypothesized that selenium was processed at the very heart of the cell in the cytoplasm, where it would be available both for cell respiration and protein synthesis. Over several years, Butler and his group at the University of Exeter observed and documented the two-step process whereby T. selenatis absorbs selenate (Se042-) from the surrounding environment and converts it to elemental selenium. The first phase, selenate reduction, takes place in the cell’s outer compartment, the periplasm, and was fairly common among selenate reducing bacteria. The second phase, in which the byproduct selenite (Se032-) was further reduced to elemental selenium, took place in the cytoplasm at the very heart of the cell.

“Most respiratory processes involving oxygen or nitrogen produce aqueous or gaseous end products, making elimination from the cell possible through diffusion,” Butler explains. “Organisms that produce non-soluble respiratory products must evolve a mechanism to avoid entombment of the waste product within the cell.”

Once inside the cell, Butler points out, it is typically very difficult to excrete elemental solids like selenium. The alternative is the slow accumulation of selenium within the cell wall, which would eventually overwhelm the cell and destroy the bacteria. This clearly wasn’t the case in T. selenatis.

When Butler’s group began tracking the accumulation of selenium within T. selenatis, they found that selenium deposits formed inside the cell in the early-growth phase, but shifted to the outside during later stages of cell growth. Under an electron microscope, they also found that the selenium had formed into distinct nanospheres that were not quite round and strikingly uniform in size of 130 nanometers (± 28nm). The particles were quite different from structures observed when mineral accumulation takes place outside of the cell where there is little or no regulation of size or shape.

The evidence suggested that selenium particles were formed inside the cell membrane. This behavior left Butler with an even more puzzling question. How was the cell able to consistently regulate particle size within a very narrow band observed in the lab? The answer, Butler discovered, lay in a novel protein called SefA.

SefA is part of a family of proteins, which Butler refers to as minerallins, that play a role in mineral formation. SefA has been found in other selenium respiring organisms. It has also been isolated from dental plaque and likely plays a role in both the formation and transport of mineral structures, though as Butler admits, this idea is still theoretical.

In T. selenatis, SefA appeared to play an important role in regulating both nanosphere assembly and particle size. A series of physiological experiments and biochemical assays linked SefA to the selenite reduction stage. SefA production rose when selenite levels where high and SefA even accompanied the export of selenium particles from the cell.

Butler’s confirmed his hunch about SefA through in vitro experiments. His team found that the were able to control selenium particle size by regulating the amount of SefA protein in solution. Evidence for SefA’s role in particle formation was further strengthened by synthetic biology experiments in which the research team inserted SefA producing genes into a strain of E. coli. The new strain behaved as expected, and produced nanoparticles similar to those found in T. selenatis. 

Exactly how SefA regulates particle size is still a mystery. Butler suspects that SefA may be coating the particles, and perhaps forming a protein cage with an optimal size of 130 nm. 

“We have done some experiments placing tags on the SefA protein to interfere with its function. If you put a tag on one end, you get much smaller particles. If you put a tag on both ends, there is no regulation at all. The protein itself is definitely controlling size, which is probably due to the way it assembles the particles. Unless we get down to the atomic structures, we won’t know for sure.”

Using atomic force microscopy, Butler’s team hopes to probe the surface and test its rigidity. If the particles are solid, it presents a very different set of challenges than if the particles are made of a sponge-like materials that could be compressed as they cross the cell wall.

Butler also hopes to use high-resolution optical microscopy to study single cells, and observe as they respire selenate to determine how particles leave the cell. They may be secreted randomly across the membrane, but he has also observed pearl like particles streaming off the cell wall, which suggests a single point of secretion. If this proves true, it raises new questions. “What controls the flow?” Butler asks. “How does it that happen in a microbe with no digestive track or organ-level development?” 

Back in Exeter, Butler will continue to create his knockout mutants, producing bacterial strains that selectively eliminate proteins he suspects are involved in selenium transport. With luck, he will identify the elusive proteins responsible for moving selenium nanoparticles across the cytoplasm and outer membrane without damaging the cell. Someday, researchers may use this knowledge to create the perfect bio-machine. Until then, Butler’s work continues to evolve as he studies the biochemical processes of one very small and highly adaptable creature.

Managing Microbes in Brazil’s Agricultural South

BTI Director travels to Brazil to mentor students researching bioremediation of agriculture chemicals.


BTI Director Michael Sadowsky understands both French and Spanish, but hardly a word of Portuguese. Yet this summer he launched a two-year collaborative research initiative in Ponta Grossa, Brazil, as part of the Brazilian Government’s Science Without Borders Project.

Sponsored by Brazilian National Council for Scientific and Technological Development (CNPQ), Brazil’s equivalent of the National Science Foundation, the program allows Sadowsky to share his expertise in microbiology and bioremediation with colleagues and students in this Southern Brazilian industrial and agricultural center.

In Brazil, Sadowsky works alongside his long-time colleague Marcos Pileggi, a professor of biology and evolution at the Universidade Estadual de Ponta Grossa. Along with Pileggi’s students, the pair will conduct research on bacterial pesticide degradation that could help protect Brazil’s environment and save money for local farmers and pesticide manufacturers.

According to Sadowsky, Brazilian farmers receive concentrated chemicals from pesticide and herbicide companies, often in five-gallon buckets. Farmers then transfer the liquid to large tanks and add water to dilute the chemicals before spraying.

But not all of the pesticide is used and the toxic residue can’t be thrown away or dumped in the environment. Some manufactures provide a pick-up service for the remaining liquid, and store it in large tanks where it degrades over time. But if the biodegradation is incomplete, they burn it — and burning liquid is very expensive.

With Sadowsky’s help, Pileggi and his lab of 10 undergraduate and graduate students hope to identify bacteria that will naturally and efficiently degrade the concentrated liquid waste. During Sadowsky’s first 10-day trip, the group created a research plan and took initial steps toward isolating bacteria capable of degrading leftover pesticides and herbicides.

“Bacteria are the most versatile tool we have for degrading compounds in the environment,” Sadowsky said. “We are trying to find more environmentally friendly ways of doing things, and very often that involves old-fashioned microbiology.”

The team put the chemicals in a tank and added different types of bacteria to see which microbes would survive and grow using the pesticides and herbicides as a food source. The technique (called enrichment) was developed in the late 1800s and is still considered the most effective way to find bacteria that can breakdown chemical compounds.

Sadowsky, who also serves as co-Director of MnDRIVE’s microbial bioremediation initiative, began international scientific work in the 80s, and he has visited Brazil a few times before, but this is his first fully funded research trip abroad.

“Brazil is a good place to perform the research,” Sadowsky said, “because some of the herbicides and pesticides the lab is now studying have been banned in the United States.”

The seed for the collaboration was planted almost five years ago when Pileggi came to Minnesota to research pesticide degradation with Sadowsky’s lab. Now, upon Pillegi’s request, the two professors have swapped roles.

“This is a great experience for me,” Sadowsky said. “Hopefully we’ll develop some new technologies out of this research. But that’s going to take some time, and we’ll have to see if it really comes to fruition.”

Sadowsky’s trip has a strong educational focus as well, and supports Science Without Border’s goal of increasing the number of Brazilian Ph.D.’s  through international scientific research and collaboration.

“Mike is helping me organize my Environmental Microbiology Laboratory with more focus and efficiency,” Pileggi said. “He pushed us a lot, and we enjoyed it.”

Students from the Universidade Estadual de Ponta Grossa, can continue their training at institutions like the Federal University system, which grants Ph.D.’s.

Sadowsky will travel to Brazil six times over the next two years. Once the project is complete, he hopes to invite some of the Brazilian students to the United States where they can receive advanced training in new and emerging technologies.

Primordial Peptides

UMN researcher Burckhard Seelig wins the prestigious Simons Investigator Award and joins the Collaboration on the Origins of Life

University of Minnesota researcher Burckhard Seelig (BMBB, BTI) has a longstanding interest in how the earliest forms of life may have come into existence. This year, his efforts were rewarded with a 5-year, one million dollar grant from the New York-based Simons Collaboration on the Origins of Life. One of two scientists invited to join the Collaboration in 2015, Seelig is part of a 21-member multi-disciplinary team looking at potential scenarios for how life could have started from non-biological matter, and the planetary conditions that could have supported the origin of life.

The goal of this Simons Collaboration is to fund an interactive community of investigators using systems reaching across disciplines, technologies, and institutions to advance our understanding of the processes which led to the emergence of life.

“This collaboration is a unique opportunity,” said Seelig. “There are a number of physicists, chemists, and biochemists, like me, but there are also planetary scientists and geobiologists. So, based on our knowledge of early planetary conditions, you can ask what kind of chemistry could have existed and talk to the chemist to find out what kind of reactions could have occurred. Then you can talk to the biochemists to see what you could make from those chemicals.”

In a field where much of the work is dominated by hypotheses, Seelig works experimentally to investigate the missing link between early non-biological amino acids, building blocks for complex proteins, and the modern alphabet of 20 amino acids that make up life’s universal genetic code.

“Today’s genetic code uses 20 amino acids. It did not start with all 20 right away, that’s for sure, but which ones exactly and in what order? This subject has been mostly theoretical. In our lab, we can actually make proteins using likely earlier versions of the genetic code and we can test them,” explained Seelig.

Dialing back the clock, his lab will test ever smaller alphabets of amino acids for their ability to produce functional proteins necessary for the survival of protocells at the origin of life. “If you have an alphabet of only early amino acids, can you make proteins as functional as those we have today? Probably not. But you can see what functions they have and ask what minimum alphabet is necessary to make a functional folded protein. So far, we don’t know. That’s what we’re trying to investigate with this project,” said Seelig. “The further you go back in time,” says Seelig, “the noisier the picture gets because we have less and less reliable information. We will never be able to really tell how life began, but what we hope to do is come up with realistic scenarios about how parts of this process could have happened. In our case, it’s about proteins.”

The award will fund two postdoctoral researchers and help support basic research, providing a welcome balance for the lab’s ongoing applied research on the synthesis of designer enzymes for medical applications and use in the pharmaceutical industry.

Dr. Seelig is a faculty member in the College of Biological Sciences Department of Biochemistry, Molecular Biology and Biophysics and a member of the University of Minnesota BioTechnology Institue.