High-throughput suppressor selection of a type I toxin-antitoxin system in Helicobacter pylori

As the number of bacterial genomes and transcriptomes increases, so does the number of newly identified toxin–antitoxin (TA) systems. However, their functional characterization remains challenging, often requiring the use of overexpression vectors that can lead to misinterpretations. To fill this gap, we developed a systematic approach called FASTBAC-Seq (Functional AnalysiS of Toxin–Antitoxin Systems in BACteria by Deep Sequencing). Combining life/death phenotypic selection with next-generation sequencing, FASTBAC-Seq allows the rapid identification of loss-of- function (toxicity) mutations in toxin-encoding genes belonging to TA loci with nucleotide resolution. Here, we apply this new tool to study aapA3/IsoA3, a member of a new family of type I TA systems hosted on the chromosome of the major human gastric pathogen Helicobacter pylori. Overall design: Amplicon DNA sequencing of strains transformed with PCR products of a wild-type or mutated toxin-antitoxin system; in both cases three biological replicate samples were sequenced (6 samples in total)

随着细菌基因组与转录组的数量持续增长，新发现的毒素-抗毒素（toxin–antitoxin, TA）系统的数量也随之增加。然而，对其进行功能表征仍存在较大挑战，通常需要借助过表达载体开展研究，而该手段往往会导致解读偏差。为填补这一研究空白，我们开发了一种名为FASTBAC-Seq（基于深度测序的细菌毒素-抗毒素系统功能分析，Functional AnalysiS of Toxin–Antitoxin Systems in BACteria by Deep Sequencing）的系统性研究方法。该方法将生死表型选择与下一代测序（next-generation sequencing）相结合，能够以核苷酸分辨率快速鉴定属于TA基因座的毒素编码基因中的功能丧失（毒性）突变。本研究将这一新型工具应用于 aapA3/IsoA3 的研究——该系统是寄居在主要人类胃病原体幽门螺杆菌（Helicobacter pylori）染色体上的新型I型TA系统家族成员之一。实验设计概述：对分别携带野生型或突变型毒素-抗毒素系统PCR产物的转化菌株进行扩增子DNA测序；两组均设置三个生物学重复样本，总计完成6个样本的测序。