Remote Regulation by VirB, the Transcriptional Anti‐Silencer of Shigella Virulence Genes, Provides Mechanistic Information

ABSTRACT Classical models of bacterial transcription show regulators binding close to promoter elements to exert their effect. However, the scope for long‐range regulation exists, especially by nucleoid structuring proteins, like H‐NS. Here, long‐range regulation by VirB, a transcriptional regulator that alleviates H‐NS‐mediated silencing of key virulence genes in Shigella species, is explored in vivo to test the limits of long‐range regulation and provide further mechanistic insight. VirB‐dependent regulation of the well‐characterized icsP promoter persists if its cognate site is repositioned 1 kb, 3.3 kb, and even 4.7 kb further upstream than its native position in a plasmid reporter. VirB‐dependent regulation diminishes with binding site distance. While increasing cellular VirB pools elevated promoter activity in all constructs with wild‐type VirB binding sites, it did not generate a disproportionate increase in promoter activity from remote sites relative to the native site. Since VirB occludes a constitutively active promoter (PT5) when docked adjacent to its −35 element, we next moved the VirB binding site far outside the promoter region. We discovered that VirB still interfered with promoter activity. These findings and those generated from molecular roadblocks engineered around a distally located VirB‐binding site are reconciled with the various models of transcriptional regulation by VirB.