Genome-wide protein–DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase

Abstract

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.

AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity

“…We identified 32 FleQ target promoters, in which 21 of them were reported previously, and 11 promoters were newly identified in this study (Supplemental File 4). In addition, 15 previously FleQ-targeted promoters identified from ChIP-Seq were devoid in our AIDmut-Seq results ( 7 ). We tested those newly found targets using EMSA and real-time RT-PCR.…”

“…ChIP-seq is the most commonly used method for genome-wide mapping of TF targets in bacteria. We then compared previously published ChIP-seq data ( 7 , 37 , 38 ) of several TFs (GacA, ExsA, FleQ, AmrZ) with the corresponding AIDmut-Seq results. AIDmut-Seq and ChIP-seq detected similar numbers of target promoters in PAO1 genome for GacA, ExsA, and FleQ, while ChIP-seq detected far more targets for AmrZ than AIDmut-Seq ( Fig.…”

“…Recently, another in vivo method named 3D-seq was developed by fusing the cytidine deaminase DddA to the 3′ end of TFs ( 7 ). TF-binding sites were directly mapped by searching C-to-T or G-to-A transition peaks on the genome.…”

“…The 10 kb SNP window also precludes 3D-seq in distinguishing close TF-targets on the genome. For example, the bis-(3′–5′)-cyclic dimeric GMP (c-di-GMP) dependent transcriptional regulator FleQ in Pseudomonas aeruginosa has three binding sites that are less than 200 bp apart on the cdrA promoter, while they exhibited only one summit in the 3D-seq map ( 7 , 8 ). In this study, we aimed to develop a simple method that is achievable by junior researchers and research groups with little experimental experience in molecular biology.…”

A comparison between full-length 16S rRNA Oxford Nanopore sequencing and Illumina V3-V4 16S rRNA sequencing in head and neck cancer tissues

“…The microbiome has been reported to influence numerous facets of tumour pathobiology biology including treatment efficacy, tumour immunity and tumour progression (1). Gemcitabine, a chemotherapeutic treatment for pancreatic, bladder and metastatic triple-negative breast cancers, can be transported into the cytoplasm of Gammaproteobacteria (class) using nucleoside transporter (NupC), where it gets inactivated by bacterial cytidine deaminase (41–43). Gut-derived Bifidobacterium spp.…”