DNA–protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA–protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA–protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.

DNA-蛋白质相互作用是基本细胞过程的核心，但在细菌基因组规模上广泛实施的测量这些相互作用的技术非常费力，而且只能捕获结合事件的快照。我们设计了一种使用双链 DNA 特异性胞嘧啶脱氨酶毒素 DddA 绘制体内 DNA-蛋白质相互作用位点的简便方法。在 3D-seq（DddA 测序）中，含有与目标 DNA 结合蛋白融合的 DddA 的菌株在与 DNA 结合融合蛋白占据的位点相邻的 DNA 序列中积累特征突变。深度测序能够检测这些菌株中突变频率增加的位点，从而生成 DNA-蛋白质相互作用位点的全基因组图谱。我们验证了铜绿假单胞菌和大肠杆菌这两种细菌中四种转录调节因子的 3D-seq。我们证明 3D-seq 易于实施、随时间记录结合事件签名的能力以及单细胞分辨率的能力。