VirB, a Key Transcriptional Regulator of Virulence Plasmid Genes in Shigella Flexneri, Forms DNA-Binding Site-Dependent Foci in the Bacterial Cytoplasm

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Journal of Bacteriology






VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri. VirB is unusual, as it is not related to other transcriptional regulators; instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning, which is exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the histone-like 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 antisilencer. To do this, a green fluorescent protein (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 using fluorescence microscopy in live wild-type S. flexneri cells and in an isogenic virB mutant. In contrast, foci were rarely observed (,10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA-binding domain. Finally, the 25-bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB-DNA interactions required for the transcriptional antisilencing 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 antisilencing 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 (pINV) 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 (i) provide novel insight into VirB-pINV interactions, (ii) suggest that VirB may have utility as a DNA marker, and (iii) raise questions about how and why this antisilencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.


Antisilencing; GFP fusion; H-NS; ParB/Spo0J; Plasmid partitioning; Shigella; Subcellular localization; Transcriptional silencing; VirB; Virulence plasmid





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