FASTBAC-Seq: Functional Analysis of Toxin-Antitoxin Systems in Bacteria by Deep Sequencing.

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 of in vivo results. 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 present the setup used on the first-time application of FASBACT-Seq to characterize a member of the aapA/IsoA family of type I TA systems hosted on the chromosome of the major human gastric pathogen Helicobacter pylori. We propose FASBACT-Seq as a powerful tool for the functional characterization of TA systems that can in addition uncover key elements for the understanding of gene expression regulation in bacteria.