The DNA binding domain of the Vibrio vulnificus SmcR transcription factor is flexible and recognizes diverse DNA sequences

The quorum-sensing regulon in vibrios is controlled by the LuxR/HapR family of transcriptional regulators. In Vibrio vulnificus, this regulator is called SmcR, and it controls expression of numerous virulence behaviors, including biofilm formation and elastase production. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic, as is common for regulators that bind as dimers with helix-turn-helix N-terminal DNA binding domains. However, the LuxR/HapR/SmcR consensus site is highly degenerate and asymmetric with variations in sequence at each promoter. To determine the mechanism of DNA site recognition, we generated separation-of-function mutants of SmcR that either repress or activate transcription but not both. The SmcR N55I protein is defective at transcription activation due to loss of binding to most DNA binding sites in activated promoters but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R, and N142D are defective for interaction with RNAP alpha but retain functional DNA binding activity. Using X-ray crystallography, we show that the wild-type SmcR dimer and the three RNAP-interaction mutants exhibit two conformations of the helix-turn-helix DNA binding domain. Conversely, the SmcR N55I X-ray crystal structure is limited to only one conformation and is restricted in recognition of single base-pair variations in DNA binding site sequences. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNA polymerase and a multi-conformational DNA binding domain that permits recognition of variable DNA sites. Thus, the LuxR/HapR/SmcR proteins balance specificity for quorum-sensing targets and diversity to accommodate binding at hundreds of sites genome-wide. SignificanceThe cell-cell communication system called quorum sensing controls expression of genes required for virulence in Vibrio bacteria species, including the potent human pathogen Vibrio vulnificus. The master transcriptional regulator of quorum-sensing genes in vibrios belongs to the LuxR/HapR/SmcR family. These regulators directly activate and repress transcription of >100 genes via binding to degenerate sites in promoter regions. We used X-ray crystallography to determine the structure of mutant SmcR proteins. Our experiments reveal that SmcR recognizes diverse sequences via a DNA binding domain that samples multiple conformations to accommodate variations in palindromic DNA sequences. Significantly, the DNA binding domain of SmcR is completely conserved in LuxR/HapR/SmcR family proteins, suggesting that this mechanism is representative of quorum-sensing regulation in other vibrios.