Rational Design of an Artificial Genetic Switch: Co-Option of the H-NS-Repressed proU Operon by the VirB Virulence Master Regulator

Abstract

The H-NS protein represses the transcription of hundreds of genes in Gram-negative bacteria. Derepression is achieved by a multitude of mechanisms, many of which involve the binding of a protein to DNA at the repressed promoter in a manner that compromises the maintenance of the H-NS-DNA nucleoprotein repression complex. The principal virulence gene promoters in Shigella flexneri, the cause of bacillary dysentery, are repressed by H-NS. VirB, a protein that closely resembles members of the ParB family of plasmid-partitioning proteins, derepresses the operons that encode the main structural components and the effector proteins of the S. flexneri type III secretion system. Bioinformatic analysis suggests that VirB has been co-opted into its current role as an H-NS antagonist in S. flexneri. To test this hypothesis, the potential for VirB to act as a positive regulator of proU, an operon that is repressed by H-NS, was assessed. Although VirB has no known relationship with the osmoregulated proU operon, it could relieve H-NS-mediated repression when the parS-like VirB binding site was placed appropriately upstream of the RpoD-dependent proU promoter. These results reveal the remarkable facility with which novel regulatory circuits can evolve, at least among those promoters that are repressed by H-NS.

VirB-Mediated Positive Feedback Control of the Virulence Gene Regulatory Cascade of Shigella flexneri

“…This role places it at a point that is intermediate between the primary regulator, VirF, and the promoters of the structural genes. The significance of having a two-stage VirFVirB-dependent regulatory system for the S. flexneri virulence cascade is unclear but may reflect the evolution of a regulatory checkpoint within the cascade at virB (35,73).…”

“…1). Since ParB-like proteins can autoregulate their own genes via adjacently located cis-acting parS sequences (8,11,21,25,28,32,37), it was hypothesized previously that virB may have been disconnected from the VirB binding site at icsB through the insertion of the icsB-ipg-ipa-acp operon during the evolution of the 230-kbp plasmid (35). Might this mean that VirB once had a role in controlling the expression of its own gene, and if so, has the modern plasmid evolved an alternative mechanism that restores the VirB-mediated control of virB transcription?…”

“…The VirF binding sites are located immediately upstream of the region bound by H-NS, which extends from positions Ϫ20 to ϩ20 and includes the virB promoter. The inactivation of hns gene expression by mutation causes an increased expression level of the virB gene at 30°C, demonstrating that H-NS plays a role in controlling the thermal regulation of virB expression (6,35). However, there is an absolute requirement for the presence of the VirF protein for the full activation of virB, indicating that the AraC-like VirF protein is not simply an antirepressor that displaces H-NS but has a positive role to play in virB promoter activation (53,(70)(71)(72).…”

“…Modifications to local DNA topology in response to environmental stress play an important part in modulating S. flexneri virulence gene transcription (18,42,47,72). The chromosomally encoded H-NS protein controls much of this expression through the repression of many of the virulence promoters under conditions that are inappropriate for invasion (6,17,34,35,39). Virulence gene activation occurs via a regulatory cascade mediated by the plasmid-encoded VirF and VirB proteins (1,19,20) (Fig.…”

Shigella

“…Bioinformatic analysis suggest that VirB has been co-opted into its current role as an H-NS antagonist in S. flexneri. 98 The virB gene transcription is activated by VirF and is temperature-dependent. When bacteria are grown at 37°C, the transcription of virB is highly activated, while at 30°C the level of the transcription of virB decreases significantly.…”