June 24, 2019 -- Researchers use bioorthogonal non-canonical amino-acid tagging (BONCAT) to successfully isolate active microbes present in soil samples. These findings were published in Nature Communications by a team of scientists from the Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab).
Soils are considered the last scientific frontiers and contain one of the most diverse microbial communities on Earth. Researchers at the University of Vienna note that microbial diversity requires redundancy in key processes which ensures ecosystem stability. Functional redundancy is embodied in non-active, dormant microorganisms can be recruited to participate in a given function upon resuscitation with environmental cues.
"In every gram of soil, there are billions of cells from tens of thousands of species that, all together, perform important Earth nutrient cycles,” said Estelle Couradeau, first author of the study. “They are the backbone of terrestrial ecosystems, and healthy soil microbiomes are key to sustainable agriculture. We now have the tools to see who these species are, but we don't yet know how they do what they do. This proof-of-concept study shows that BONCAT is an effective tool that we could use to link active microbes to environmental processes."
Direct observation of microbial populations is off the table, therefore microbiologists typically collect environmental samples and rely on indirect approaches such as DNA sequencing to characterize the communities. However, most of the commonly used techniques fail to differentiate active microbes from dormant ones or free-floating microbial DNA found in soil and sediment.
BONCAT as a novel approach derived from “click chemistry” by Nobel laureate and synthetic chemist K. Barry Sharpless in 2001. This method describes reactions that create only byproducts that can be removed without chromatography, are stereospecific, simple to perform and can be conducted in easily removable or benign solvents. BONCAT is used to detect the process of translation or protein modification in cells and can broadly be broken into two steps: labeling of newly synthesized proteins and tagging of azidohomoalanine- and homopropargylglycine-containing proteins.
In 2014, the U.S. Department of Energy (DOE) Joint Genome Institute (JGI), an Office of Science user facility managed by Berkeley Lab, collaborated with a Caltech lab to adapt BONCAT into a tool that could identify active, symbiotic clusters of dozens to hundreds of marine microbes within ocean sediment. With this new methodology, called BONCAT Fluorescent Activated Cell Sorting (BONCAT+FACS), detection of individual active microbes was possible.
BONCAT+FACS allows scientists to sort single-cell organisms based on the presence or absence of fluorescent tagging molecules, which bind to a modified version of the amino acid methionine. When fluid containing the modified methionine is introduced to a sample of microbes, only those that are creating new proteins will incorporate the modified methionine into cells, which can then be detected.
"With BONCAT, we will be able to get immediate snapshots of how microbiomes react to both normal habitat fluctuations and extreme climate events - such as drought and flood - that are becoming more and more frequent," said Trent Northen, lead author and director of biotechnology for ENIGMA.
ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) digs deeper into the inner-workings of soil microbiomes. ENIGMA's projects are a high priority for biologists and energy and Earth scientists not only because they help fill gaps in our knowledge of how the environment functions, but also because these fundamental insights could help applied scientists more effectively harness microbiomes to improve drought-resistance in crops, remove contaminants from the environment, and sustainably produce fuels and other bioproducts.
This new BONCAT technology could accelerate the development of a variety of other tools and techniques in agriculture and microbiology to help us understand the living world around us and work towards a sustainable future.
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