VirB, a transcriptional activator of virulence in Shigella flexneri, uses CTP as a cofactor

Abstract

VirB is a transcriptional activator of virulence in the gram-negative bacterium Shigella flexneri. It is encoded by the large invasion plasmid, pINV, and is thought to counteract the transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Mutations in virB lead to loss of virulence. Studies suggest that VirB binds to specific DNA sequences, remodels the H-NS nucleoprotein complexes, and changes DNA supercoiling. VirB belongs to the superfamily of ParB proteins which are involved in plasmid and chromosome partitioning often as part of a ParABS system. Like ParB, VirB forms discrete foci in Shigella flexneri cells harbouring pINV. Our results reveal that purified preparations of VirB specifically bind the ribonucleotide CTP. We show that VirB slowly but detectably hydrolyses CTP, which is mildly stimulated by the virS targeting sequences found on pINV. CTP and DNA binding promote VirB clamp closure. Curiously, DNA stimulation of clamp closure appears efficient even without virS sequences. These findings suggest that VirB acts as a CTP-dependent DNA clamp and may indicate that so far elusive factors might prevent offsite DNA clamping in vivo.