Hfq-licensed RNA-RNA interactome in Pseudomonas aeruginosa reveals a keystone sRNA.

The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.

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

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.