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Scientists strike on Pay Dirt with new microbial research technology



  Scientists Meet Pay Dirt with New Microbial Research Technique
BONCAT is a highly effective microbial sorting tool that allows scientists to differentiate between active and microbial organisms. The tool has now been adapted for use on soil samples and will provide information on how soil microbiomes affect large environmental cycles. Picture credits: Susan Brand and Marilyn Chung / Berkeley Lab

Leonardo da Vinci wrote a long time ago in the European Renaissance that we humans "know more about the movement of celestial bodies than about the ground under their feet". Five hundred years and countless technological and scientific advances later, his feeling still holds true.

However, this could change soon.

In a report from Nature Communications a team of scientists from the Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) described the first successful use of a technique called BONCAT to isolate active microbes in a soil sample Achievement that could enable a tidal wave of new research.

"Soils are probably the most diverse microbial communities on the planet," said Estelle Couradeau, lead author of the study. "In every ounce of soil there are billions of cells from tens of thousands of species that together carry out important nutrient cycles on Earth, they are the backbone of terrestrial ecosystems, and healthy soil microbiota are the key to sustainable agriculture." We now have the tools to to see who these species are, but we do not yet know how they do what they do.This proof-of-concept study shows that BONCAT is an effective tool for linking active microbes to environmental processes. "

Over the past two years, Couradeau, her co-authors, and many other US researchers have collaborated in a Berkeley Lab-led scientific research called ENIGMA (for ecosystems and networks integrated into genes and molecular arrays). to penetrate deeper into the inner workings of soil microbiome. ENIGMA's projects are not only a high priority for biologists and energy and earth scientists, because they help to close gaps in our knowledge about the functioning of the environment, but also, because these basic findings could help applied scientists to make microbiome better for To use the improvement of dry resistance in crops remove pollutants from the environment and produce sustainable fuels and other organic products.




BONCAT is a highly efficient microbial sorting tool that allows scientists to distinguish between active and microbes. The tool has now been adapted for use on soil samples and will provide information on how soil microbiomes affect large environmental cycles. Credit: Susan Brand and Marilyn Chung / Berkeley Lab

However, as most soil microbes do not grow in laboratory cultures, and because of their overwhelming abundance of natural habitats, they investigate which microbial species cause what is incredibly difficult. "There are many barriers to measuring microbial activity and interactions," said Trent Northen, principal author and director of biotechnology at ENIGMA. "For example, soil microbiota, which remove waste from underground reservoirs, are found hundreds of feet below the surface, and in some ecosystems, up to 95% of the microbes are inactive at any one time."

Because direct observation is disabled In the table, microbiologists usually collect environmental samples and rely on indirect approaches such as DNA sequencing to characterize the communities. However, most of the common techniques can not distinguish active microbes from those that are slumbering in the soil and sediment, or from the abundance of free-floating DNA pieces.

BONCAT, short for Bioorthogonal Non-Canonical Amino Acid Tagging, was invented in 2006 by Caltech geneticists to isolate newly formed proteins into cells. In 2014, Rex Malmstrom, Danielle Goudeau, and other members of the Joint Genome Institute (JGI) of the US Department of Energy (DOE), a Berkeley Lab-managed office of the Office of Science, partnered with Victoria Orphan's Caltech lab to merge BONCAT into one new form tool to identify active, symbiotic clusters of tens to hundreds of marine microbes in the ocean sediment. After further refinement of their approach, BONCAT Fluorescent Activated Cell Sorting (BONCAT + FACS), they were able to detect single active microbes.

As the name suggests, BONCAT + FACS allows scientists to sort unicellular organisms by presence or absence of fluorescent label molecules that bind to a modified version of the amino acid methionine. When liquid containing the modified methionine is introduced into a sample of microbes, the modified methionine is incorporated into cells only by those who form new proteins – the hallmark of the activity.

It's also far more efficient and reliable than previous methods In microbial identification, the entire process takes only a few hours – meaning that active cells can be labeled, even if they do not replicate.

  Scientists meet Pay Dirt with new microbial research technology.
Charles Paradis, now a post-doctoral student, is holding a soil core sample at the Los Alamos National Laboratory taken from the Oak Ridge Field Research Site in Tennessee. The BONCAT + FACS Optimization Tests reported in the current study used samples such as these. Credit: Lance E. King / Y-12 National Security Complex

Given that some soil microbes grow notoriously slowly, many scientists were immediately interested in using BONCAT + FACS on terrestrial soils. After three months of experimenting and optimizing, the team of ENIGMA and JGI researchers developed a protocol that works smoothly and, above all, provides highly reproducible results.

"BONCAT + FACS is a powerful tool that provides a sophisticated method for determining which microbes are active in a community at any given time," said Malmstrom, who is also the author of the current study. "It also opens the door to experiment to assess which cells are active under condition A and which cells become active or inactive when switching to condition B."

BONCAT + FACS will provide researchers with an opportunity to collaborate on JGI's user programs. Northen and Malmstrom have already received several suggestions from research groups willing to work with the tool, including groups from the Berkeley Lab who want to use BONCAT to study how environmental change stimulates groups of microbes. "With BONCAT, we can immediately see how microbiota respond to both normal habitat fluctuations and extreme climatic events such as drought and floods, which are becoming more common," Northen said.

According to Couradeau, the team expects the approach to catalyze a number of other important and interesting research lines, including improving agricultural land practices, assessing antibiotic susceptibility to non-cultivable microbes and studying the completely unknown role of candidatus Dormibacteraeota, a strain of soil bacteria. All over the world, apparently slumbering most of the time.

Malmstrom referred to the diversity of scientists within ENIGMA and JGI to reflect how he and his colleagues achieved a goal that many have pursued. "This is a true example of team science because no single person has or will ever have the expertise to do it all."


See and sort: Development of new techniques for visualizing the activity of uncultivated microbial cells


Further information:
Nature Communications (2019). DOI: 10.1038 / s41467-019-10542-0

Provided by
Lawrence Berkeley National Laboratory




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Scientists beat Pay Dirt with new microbial research technology (2019, 24 June)
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