Rice research team creates universal RNA barcoding system for tracking gene transfer in bacteria

(Press-News.org) In the microscopic world of bacteria, gene transfer is a powerful mechanism that can alter cellular function, drive 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 how genes move across species. The findings were recently published in Nature Biotechnology.

“We’ve long known that bacteria swap genes in ways that impact human health, biotechnology and environmental stability,” said James Chappell, associate professor of biosciences and bioengineering. “But mapping which microbes participate in gene transfer has been challenging. This new technique gives us a direct way to record this information inside the cells themselves.”

Traditional methods for studying gene transfer involve labeling mobile genetic elements with fluorescent proteins or antibiotic resistance genes. While effective, these approaches require isolating and growing microbes in a lab, limiting their use in complex environments.

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

The Rice team’s new method, called RNA-addressable modification (RAM), bypasses these hurdles by using a synthetic catalytic RNA (cat-RNA) to “barcode” ribosomal RNA (rRNA) inside living cells.

By writing genetic information directly into the 16S rRNA — a molecule universally found in bacteria — the researchers could track which microbes acquired foreign DNA without disrupting their natural environment. Additionally, as targeted sequencing of 16S rRNA is the gold standard for identifying different species of bacteria, this method can leverage 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 permanent and arduous to read out, we write information in a region of RNA that is highly conserved across the tree of life, making the information cheap and easy to read out.”

To achieve this, the researchers designed a small ribozyme-based RNA molecule (also called catalytic RNA) that attached a unique barcode to 16S rRNA upon 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, associate professor of civil and environmental engineering. “Around half of the bacterial taxa in the wastewater community could pick up the plasmids, giving us a detailed map of horizontal gene transfer events.”

The study also showed that RAM can be used to measure differences in host ranges between DNA plasmid types. With tens of thousands of different DNA plasmids found in natural environmental microbes, RAM provides an easy and cost-effective method to begin to understand the relationship between plasmids and their hosts.

“RAM can be used to track the movement of multiple genetic elements across 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 possible far-reaching applications in medicine, biotechnology and environmental science. One of the most pressing concerns is antibiotic resistance, as tracking how resistant genes spread in hospitals and wastewater could help predict and prevent outbreaks of drug-resistant infections. In the field of bioremediation and waste management, this technology has the potential to engineer microbiomes that efficiently break down pollutants while ensuring that beneficial genetic modifications remain contained. Additionally, in synthetic biology and biotechnology, the ability to program microbiomes for specific tasks, such as producing biofuels or pharmaceuticals, relies on safe and controlled gene transfer.

“The potential here is enormous,” Stadler said. “We now have a way to study how bacteria share genes in their natural habitat without needing to grow them in a lab. That opens the door for 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 exchange such as transduction (via bacteriophages) and transformation (direct DNA uptake). Additionally, optimizing cat-RNA stability and increasing the number of unique barcodes could allow even finer resolution in tracking microbial interactions.

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

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