RNA Barcoding Reveals Hidden Phage-Host Relationships in Microbial Communities

A Rice University team has used an RNA-based barcoding system to identify previously unknown relationships between bacteriophages and their bacterial hosts—findings that could advance efforts to engineer microbiomes for medicine, environmental remediation, and industrial biotechnology. The study is published in Nature Communications. 

Bacteriophages, or phages, are viruses that infect bacteria and are the most abundant biological entities on Earth. They shape microbial ecosystems by killing bacteria, altering their metabolism, and transferring genes between organisms. Scientists are interested in using phages as alternatives to antibiotics and as tools for microbiome engineering, but pinpointing which phages infect which bacteria in real-world environments has been difficult. Traditional methods require laboratory culturing, are labor-intensive, and often cannot distinguish between a virus merely attaching to a cell and successfully transferring DNA.

"Phages are everywhere, and they play an enormous role in shaping microbial communities and moving genes between bacteria," said corresponding author Lauren Stadler. "But identifying which phages interact with which hosts in real-world microbial communities has been a long-standing challenge. This work gives us a scalable way to directly observe those interactions."

The Rice team adapted a synthetic biology platform called RNA-addressable modification, originally developed to track gene transfer through bacterial conjugation. The system uses an engineered ribozyme that inserts a unique barcode into a bacterium's 16S ribosomal RNA after it receives DNA from a phage. Recipient organisms can then be identified through targeted RNA sequencing.

The researchers incorporated the barcoding system into bacteriophage P1, a virus known to transfer DNA among enteric bacteria and thought to contribute to the spread of antibiotic resistance genes. Testing the approach in wastewater collected from a Houston-area treatment plant, the team found that P1 transferred genetic material to members of the order Aeromonadales, including Aeromonas hydrophila—a common wastewater bacterium that had never previously been identified as a P1 host.

"Finding a completely new host group in a complex environmental sample demonstrates the power of this approach," Stadler said. "There are likely many important phage-host relationships that remain hidden simply because we haven't had the tools to observe them easily and without laborious methods."

The team also used the technology to examine how changes in viral tail fibers— the protein structures phages use to recognize and attach to bacteria—influence which microbes a phage can target. By engineering phage-derived particles with alternative tail fibers, they showed that each variant targeted a distinct set of microbes within wastewater communities.

"These experiments allowed us to see how relatively small genetic changes in a phage can dramatically alter which bacteria it interacts with," Stadler said. "That information is incredibly valuable for designing phages with specific functions, whether the goal is delivering beneficial genes or selectively eliminating harmful bacteria."

Because the approach relies on amplicon sequencing rather than labor-intensive culturing, it could enable large-scale studies of viral ecology across diverse microbiomes.