Kalvapalle Prashant B, Staubus August, Dysart Matthew J, Gambill Lauren, Reyes Gamas Kiara, Lu Li Chieh, Silberg Jonathan J, Stadler Lauren B, Chappell James
Systems, Synthetic, and Physical Biology Graduate Program, Rice University, Houston, TX, USA.
Department of BioSciences, Rice University, Houston, TX, USA.
Nat Biotechnol. 2025 Mar 18. doi: 10.1038/s41587-025-02593-0.
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.
可以使用基因编码报告基因或宏基因组测序来研究基因转移，但这些方法在用于监测微生物群落中移动DNA的宿主范围时，受到灵敏度的限制。为了记录有关废水微生物群落中基因转移的信息，使用一种合成催化RNA对核糖体RNA（rRNA）的高度保守片段进行条形码标记。通过使用核酶将信息写入rRNA，并使用扩增子测序读出天然和修饰的rRNA，我们发现来自20个分类目的微生物群落成员参与了与大肠杆菌供体菌株的质粒结合，并观察到跨扩增子序列变体的16S rRNA条形码信号存在差异。使用具有pBBR1或ColE1复制起点的质粒进行多重rRNA条形码标记，揭示了宿主范围的差异。这种自主的RNA可寻址修饰提供了有关基因转移的信息，而无需翻译，并且将能够在不同的生态环境中进行微生物组工程，以及研究基因转移和细胞摄取细胞外物质的环境控制。
