The response regulator SsrB activates expression of diverse Salmonella pathogenicity island 2 promoters and counters silencing by the nucleoid‐associated protein H‐NS

Abstract

SummaryThe two-component system SsrA-SsrB activates expression of a type III secretion system required for replication in macrophages and systemic infection in mice. Here we characterize the SsrB-dependent regulation of genes within Salmonella pathogenicity island 2 (SPI-2). Primer extension and DNase I footprinting identified multiple SsrB-regulated promoters within SPI-2 located upstream of ssaB, sseA, ssaG and ssaM. We previously demonstrated that ssrA and ssrB transcription is uncoupled. Overexpression of SsrB in the absence of its cognate kinase, SsrA, is sufficient to activate SPI-2 transcription. Because SsrB requires phosphorylation to relieve inhibitory contacts that occlude its DNA-binding domain, additional components must phosphorylate SsrB. SPI-2 promoters examined in single copy were highly SsrB-dependent, activated during growth in macrophages and induced by acidic pH. The nucleoid structuring protein H-NS represses horizontally acquired genes; we confirmed that H-NS is a negative regulator of SPI-2 gene expression. In the absence of H-NS, the requirement for SsrB in activating SPI-2 genes is substantially reduced, suggesting a role for SsrB in countering H-NS silencing. SsrB activates transcription of multiple operons within SPI-2 by binding to degenerate DNA targets at diversely organized promoters. SsrB appears to possess dual activities to promote SPI-2 gene expression: activation of transcription and relief of H-NS-mediated repression.

Salmonella pathogenicity island 2 expression negatively controlled by EIIA Ntr –SsrB interaction is required for Salmonella virulence

“…1 B and C). Consistent with previous reports (3,6,23), deletion of the ssrB gene markedly impaired transcriptional activation of the ssaG gene at acidic pH (i.e., 11-fold reduction; Fig. 2A).…”

“…Among them, the SsrA/SsrB two-component system, which is encoded by the ssrA and ssrB genes located within SPI-2, seems to have the most direct effect on SPI-2 expression. The purified C terminus of SsrB binds to multiple promoters of SPI-2 genes encoding the TTSS and secreted effectors (3). As the SsrB protein is a response regulator, the DNA-binding activity, which is controlled by the phosphorylation state (4), the cognate sensor kinase, SsrA, should be activated to phosphorylate the SsrB protein within macrophages.…”

New insights into transcriptional regulation by H-NS

“…The SPI1-encoded transcriptional activator HilA not only promotes expression of SPI1 genes but also appears to counter H-NS silencing of the SPI4 pathogenicity island [66], which acts in concert with SPI1 during Salmonella interactions with the host intestinal mucosa [67]. A requirement for the transcriptional activator SsrB for SPI2 expression is reduced in the absence of H-NS, suggesting that SsrB both activates transcription and relieves H-NS-mediated repression [68]. This rationalizes the presence of SsrB binding sites both within the promoter region and downstream of the translational start site.…”

HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2

“…Because the two-component system SsrA/B is critical for the expression of the SPI-2 regulon in N-minimal medium (12,20,21), we next asked if it is also essential for expression of ssaG and pipB during the stationary phase in LB. Expression of the ssaG and pipB transcriptional fusions in a ssrB::kan strain was nearly abolished in either growth medium ( Fig.…”

“…The SPI-1 regulon is controlled by a regulatory cascade composed mainly of the SPI-1-encoded regulators HilD, HilA, and InvF (13)(14)(15)(16). SPI-2 genes are expressed when Salmonella is inside host cells (17)(18)(19) and is under the control of the SPI-2-encoded two-component system SsrA/B (12,20,21) [see supporting information (SI) Fig. S1 for details].…”