Abstract
The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB. Our previous in vivo studies at a different VirB-dependent promoter, icsP, found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB, virF, spa15, and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed.
Highlights
In Shigella species, expression of a set of virulence genes carried on the large virulence plasmid, pINV (~220 kb), is controlled by transcriptional silencing and anti-silencing
To test DNA sequences required for VirB binding in vivo, putative cognate DNA sequences were inserted into a unique BglII restriction site positioned immediately upstream and adjacent to the −35 promoter element of Ptac in pBT-empty
We hypothesized that if VirB binds to the inserted sequences, RNA polymerase would be sterically hindered from engaging promoter elements (Figure 1Aii) required for transcription initiation, which would result in low Ptac activity and low lacZ expression [14]
Summary
In Shigella species, expression of a set of virulence genes carried on the large virulence plasmid, pINV (~220 kb), is controlled by transcriptional silencing and anti-silencing. Since VirB belongs to the ParB protein superfamily [11], the putative VirB-binding site at the icsB promoter was initially identified due to its similarity to the Box A component of the binding site for P1 ParB (50 -(A/G)(A/T)G(G)AAAT-30 ) [9]. Despite this sequence being organized as an inverted repeat, only the Box A-like sequence, subsequently renamed Box 2, was shown to be required for VirB-dependent regulation of the icsB promoter and important for VirB-DNA binding in vitro [6,9].
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