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

Abstract

AbstractVirB is a transcriptional activator of virulence in the gram-negative bacteriumShigella 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 invirBlead 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 inShigella flexnericells 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 thevirStargeting sequences found on pINV. CTP and DNA binding promote VirB clamp closure. Curiously, DNA stimulation of clamp closure appears efficient even withoutvirSsequences. These findings suggest that VirB acts as a CTP-dependent DNA clamp and may indicate that so far elusive factors might prevent offsite DNA clampingin vivo.