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

VirB is a transcriptional activator of virulence in the gram-negative bacterium Shigella flexneri encoded by the large invasion plasmid, pINV. It counteracts the transcriptional silencing by the nucleoid structuring protein, H-NS. Mutations in virB lead to loss of virulence. Studies suggested 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 and slowly but detectably hydrolyse it with mild stimulation by the virS targeting sequences found on pINV. We show that formation of VirB foci in cells requires a virS site and CTP binding residues in VirB. Curiously, DNA stimulation of clamp closure appears efficient even without a virS sequence in vitro. Specificity for entrapment of virS DNA is however evident at elevated salt concentrations. These findings suggest that VirB acts as a CTP-dependent DNA clamp and indicate that the cellular microenvironment contributes to the accumulation of VirB specifically at virS sites.

VirB是编码于大型侵袭性质粒pINV中的革兰氏阴性菌志贺菌Shigella flexneri的毒力转录激活因子。该蛋白可抵消核质结构蛋白H-NS导致的转录沉默。virB基因的突变会导致毒力丧失。研究指出，VirB可与特定的DNA序列结合，重塑H-NS核蛋白复合物，并改变DNA的超螺旋结构。VirB属于ParB蛋白超家族，这些蛋白常作为ParABS系统的一部分参与质粒和染色体的分配。与ParB相似，VirB在携带pINV的志贺菌细胞中形成离散的焦点。我们的研究结果揭示，纯化的VirB制剂可特异性地结合核苷三磷酸CTP，并在pINV上的virS靶向序列的轻微刺激下缓慢但可检测地水解它。我们证明，细胞中VirB焦点的形成需要virS位点及VirB中的CTP结合位点。然而，在体外，即使没有virS序列，DNA对夹闭的刺激也显示出高效的效应。然而，在较高的盐浓度下，virS DNA捕获的特异性则明显。这些发现表明，VirB作为一种CTP依赖性的DNA夹，并指出细胞微环境对VirB在virS位点的特异性积累起着重要作用。