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<p>Gene transfer can be studied using genetically encoded reporters or metagenomic sequencing but these methods are limited by sensitivity when used to monitor the mobile DNA host range in microbial communities. To record information about gene transfer across a wastewater microbiome, a synthetic catalytic RNA was used to barcode a highly conserved segment of ribosomal RNA (rRNA). By writing information into rRNA using a ribozyme and reading out native and modified rRNA using amplicon sequencing, we find that microbial community members from 20 taxonomic orders participate in plasmid conjugation with an <i>Escherichia coli</i> donor strain and observe differences in 16S rRNA barcode signal across amplicon sequence variants. Multiplexed rRNA barcoding using plasmids with pBBR1 or ColE1 origins of replication reveals differences in host range. This autonomous RNA-addressable modification provides information about gene transfer without requiring translation and will enable microbiome engineering across diverse ecological settings and studies of environmental controls on gene transfer and cellular uptake of extracellular materials.</p>

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Information storage across a microbial community using universal RNA barcoding

  • 期刊论文

  • 发表日期：

    2025年3月18日

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  • Prashant B. Kalvapalle,
  • August Staubus,
  • Matthew J. Dysart,
  • Lauren Gambill,
  • Kiara Reyes Gamas,
  • Li Chieh Lu,
  • Jonathan J. Silberg,
  • Lauren B. Stadler,
  • James Chappell

摘要

Gene transfer can be studied using genetically encoded reporters or metagenomic sequencing but these methods are limited by sensitivity when used to monitor the mobile DNA host range in microbial communities. To record information about gene transfer across a wastewater microbiome, a synthetic catalytic RNA was used to barcode a highly conserved segment of ribosomal RNA (rRNA). By writing information into rRNA using a ribozyme and reading out native and modified rRNA using amplicon sequencing, we find that microbial community members from 20 taxonomic orders participate in plasmid conjugation with an Escherichia coli donor strain and observe differences in 16S rRNA barcode signal across amplicon sequence variants. Multiplexed rRNA barcoding using plasmids with pBBR1 or ColE1 origins of replication reveals differences in host range. This autonomous RNA-addressable modification provides information about gene transfer without requiring translation and will enable microbiome engineering across diverse ecological settings and studies of environmental controls on gene transfer and cellular uptake of extracellular materials.