The role of the VirB ligand CTP in the molecular mechanism of transcriptional anti-silencing in <i>Shigella flexneri</i>

In bacteria, nucleoid-structuring proteins bind and constrain DNA, often leading to transcriptional silencing. In <i>Shigella spp</i> ., the histone-like nucleoid-structuring protein H-NS silences many genes on the large virulence plasmid. Upon a shift to human body temperature, VirB, a DNA-binding protein and key transcriptional regulator of the <i>Shigella</i> virulence cascade, is produced. VirB counteracts H-NS-mediated transcriptional silencing and belongs to a fast-evolving clade of the ParB superfamily. Like other ParB proteins, VirB binds the ligand CTP. While CTP is essential for the anti-silencing activity of VirB, the role of CTP in the mechanism of VirB-dependent anti-silencing has yet to be determined. This work shows that VirB does not require CTP for specific engagement of its DNA recognition site, but CTP is necessary for the formation of large VirB-DNA complexes, which likely form when VirB dimers adopt a sliding clamp conformation. Furthermore, CTP-binding mutants do not trigger a loss of negative supercoils from plasmid DNA, a VirB-dependent activity proposed to destabilize H-NS-DNA complexes. Together, our findings provide novel insight into the relationship between VirB, CTP, and DNA and reveal the role of the VirB ligand, CTP, in regulating <i>Shigella</i> virulence.