TY - JOUR T1 - VirB, a key transcriptional regulator of virulence plasmid genes in <em>Shigella flexneri</em>, forms DNA-binding site dependent foci in the bacterial cytoplasm JF - bioRxiv DO - 10.1101/2020.11.20.392365 SP - 2020.11.20.392365 AU - Jillian N. Socea AU - Grant R. Bowman AU - Helen J. Wing Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/11/22/2020.11.20.392365.abstract N2 - VirB is a key regulator of virulence genes located on the large virulence plasmid (pINV) of the bacterial pathogen Shigella flexneri. VirB is unusual in that it is not related to other transcriptional regulators, instead, it belongs to a protein family that primarily functions in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing of virulence genes mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (&lt;10%) in cells cured of pINV. Moreover, in the context of the fusion, amino acid substitutions in the DNA binding domain of VirB resulted in the fluorescent signal becoming entirely diffuse. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed.Importance This study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm. ER -