VirB, a key transcriptional regulator of Shigella virulence, requires a CTP ligand for its regulatory activities

The VirB protein, encoded by the large virulence plasmid of Shigella spp., is a key transcriptional regulator of virulence genes. Without a functional virB gene, Shigella cells are avirulent. On the virulence plasmid, VirB functions to offset transcriptional silencing mediated by the nucleoid structuring protein, H-NS, which binds and sequesters AT-rich DNA, making it inaccessible for gene expression. Thus, gaining a mechanistic understanding of how VirB counters H-NS-mediated silencing is of considerable interest. VirB is unusual in that it does not resemble classic transcription factors. Instead, its closest relatives are found in the ParB superfamily, where the best-characterized members function in faithful DNA segregation before cell division. Here, we show that VirB is a fast-evolving member of this superfamily and report for the first time that the VirB protein binds a highly unusual ligand, CTP. VirB binds this nucleoside triphosphate preferentially and with specificity. Based on alignments with the best-characterized members of the ParB family, we identify amino acids of VirB likely to bind CTP. Substitutions in these residues disrupt several well-documented activities of VirB, including its anti-silencing activity at a VirB-dependent promoter, its role in generating a Congo red positive phenotype in Shigella, and the ability of the VirB protein to form foci in the bacterial cytoplasm when fused to GFP. Thus, this work is the first to show that VirB is a bona fide CTP-binding protein and links Shigella virulence phenotypes to the nucleoside triphosphate, CTP.