Tracking Horizontal Gene Transfer in Microbiomes Using RNA Memory

Horizontal gene transfer (HGT) is a primary means by which microbes acquire new DNA and offers a potentially powerful method for microbiome engineering. Despite the potential of HGT for engineering microbes within communities, its dynamics and drivers in natural environments are not well understood. To harness HGT as a gene delivery mechanism, we require tools for in situ quantification and characterization of gene transfer in complex communities. Here, we introduce RNA-Addressable Memory (RAM), an RNA-based genetic memory system that enables high-throughput and quantitative measurements of HGT in microbial communities. RAM uses an engineered ribozyme to attach an RNA barcode onto specific RNA sequences. We encoded RAM on a plasmid such that after entering a recipient cell, RAM generates a ribozyme that targets a conserved region of 16S rRNA and inserts a barcode. Barcoded rRNAs can be amplified to include species-specific variable regions, sequenced, and analyzed to identify transconjugants. We demonstrated the RAM plasmid’s ability to track HGT in a wastewater microbiome. We showed that 140 out of 279 amplicon sequence variants (ASVs) from the sample participated in HGT with an E. coli donor carrying a conjugative plasmid. Further, we detected barcoding in low abundance community members, including those present at < 0.1% abundance. RAM can enable cellular programming and biosurveillance of synthetic DNA across diverse environments, deepen our understanding of microbial processes in situ, and expand the synthetic biology toolbox for engineering unculturable organisms.