Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2′-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.

Highlights d The phage antirepressor Qtip sequesters its partner cI repressor to the cell poles d Qtip-bound repressor is inhibited for DNA binding and autoproteolysis d Qtip recognizes the N-terminal domain of the cI repressor d Repressor recognition and polar localization are separable properties of Qtip Authors

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain that is necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA-binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter.

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DNA-damaging agents are the pervasive inducers of temperate phages in model bacteria. However, most bacteria in the biosphere are predicted to carry multiple prophages, a state called polylysogeny, making it unclear how co-residing prophages compete for host cell resources if they all respond to the identical trigger. We discover regulatory modules encoded on phage genomes that control prophage induction independently of the DNA damage cue. Genes specifying these pathways exist in linear plasmid-like phages at sites essential for phage propagation. The modules lack sequence similarity but display a shared regulatory logic of a transcription factor that activates expression of a neighboring gene encoding a small protein. The small protein inactivates the master repressor of lysis, leading to prophage induction. In some phages, the regulatory unit detects sensory information including quorum-sensing autoinducers, making lysis host-cell-density dependent. Exposure of the polylysogens studied here to different induction scenarios reveals that mixed phage populations emerge following DNA damage, however, induction through the SOS-independent module drives near-exclusive production of the phage sensitive to that specific cue. Considering the lack of potent DNA-damaging agents in natural habitats, we propose that additional phage-encoded sensory pathways that drive lysis play fundamental roles in phage-host biology and inter-prophage competition.