New RNA barcoding method tracks gene transfer in microbial communities

New RNA barcoding method tracks gene transfer in microbial communities

In the microscopic world of bacteria, gene transfer is a powerful mechanism that can alter cellular function, promote antibiotic resistance, and even shape entire ecosystems. Now an interdisciplinary group of researchers at Rice University has developed an innovative RNA “barcoding” method to track these genetic exchanges in microbial communities, providing new insights into the way genes move across species. The results were recently published inNatural biotechnology.

We have long known that bacteria swap genes in ways that impact human health, biotechnology, and environmental stability. However, mapping which microbes are involved in gene transfer has been challenging. This new technique gives us a direct way to record this information in the cells themselves. “

James Chappell, Associate Professor of Biosciences and Bioengineering

Traditional methods for studying gene transfer involve tagging mobile genetic elements with fluorescent proteins or antibiotic resistance genes. While effective, these approaches require isolation and growth of microbes in a laboratory, limiting their use in complex environments.

To address this challenge, an interdisciplinary team from Rice's Chappell, Joff Silberg and Lauren Stadler research laboratories have created a new synthetic biology tool. This team consisted of Matthew Dysart, Kiara Reyes Gamas, Lauren Gambill, Prashant Kalvapalle, Li Chieh Lu and August Staubus.

The rice team's new method, called RNA-addressing modification (RAM), gets around these hurdles by using a synthetic catalytic RNA (Cat-RNA) to "barcode" ribosomal RNA (rRNA) in living cells.

By writing genetic information directly into 16S rRNA—a molecule commonly found in bacteria—researchers were able to track which microbes acquired foreign DNA without disturbing their natural environment. As a targeted sequencing of 16S rRNA, this method is also the gold standard for identifying various bacterial species that can utilize established and easy-to-use protocols and analysis software.

“This is a game-changer for creating a mobile DNA atlas,” said Silberg, the Stewart Memorial Professor of Biosciences and professor of bioengineering. “Instead of writing information randomly in bacterial DNA, which is persistent and laborious to read, we write information in a region of RNA that is highly conserved throughout the tree of life, making the information cheap and easy to read aloud.”

To achieve this, the researchers designed a small ribozyme-based RNA molecule (also called catalytic RNA) that retained a unique barcode of 16S rRNA during gene transfer. This Cat RNA was introduced into a model microbial community using conjugative plasmids, which are naturally occurring gene carriers in bacteria.

The experiment involved introducing these barcoding plasmids into E. coli donor bacteria, which then transferred their genetic material to various microbes in a wastewater community. After 24 hours, the researchers extracted total RNA and sequenced the barcoded 16S rRNA.

“What we saw was remarkable,” said Stadler, an associate professor of civil and environmental engineering. “Approximately half of the bacterial taxa in the wastewater community could harbor the plasmids, allowing us to create a detailed map of horizontal gene transfer events.”

The study also showed that RAM can be used to measure the differences in host ranges between DNA plasmid types. With tens of thousands of different DNA plasmids in natural environmental microbes, RAM provides a simple and inexpensive method to understand the relationship between plasmids and their hosts.

“RAM can be used to track the movement of multiple genetic elements throughout an entire microbial community,” Chappell said. “This allowed us to track the movement of multiple plasmids in a single experiment and could be extended to study the dynamics of plasmid transfer in microbial communities and interactions between mobile genetic elements.”

The RAM method has potentially wide-ranging applications in medicine, biotechnology and environmental sciences. One of the most pressing concerns is antibiotic resistance, as tracking the spread of resistance genes and wastewater could help predict and prevent outbreaks of drug-resistant infections. In the field of bioremediation and waste management, this technology can develop microbiomes that efficiently break down pollutants while ensuring that beneficial genetic modifications are retained. In synthetic biology and biotechnology, the ability to produce microbiomes for specific tasks such as the production of biofuels or pharmaceuticals also relies on safe and controlled gene transfer.

“The potential here is enormous,” said Stadler. "We now have a way to study how bacteria share genes in their natural habitat without having to grow them in a laboratory. This opens the door to a new wave of microbial research and synthetic biology applications."

In the future, this barcoding technique could also be expanded and applied to other forms of gene switching such as transduction (via bacteriophages) and transformation (direct DNA uptake). Furthermore, optimizing Cat RNA stability and increasing the number of unique barcodes may enable even finer resolution when tracking microbial interactions.

“With further development, RNA barcoding could become a universal tool for storing information in environmental communities beyond additional microbial behaviors,” Silberg said.

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