Dataset Information

Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription.

ABSTRACT: The response regulator SsrB activates expression of genes encoded within and outside of a pathogenicity island (SPI-2), which is required for systemic infection of Salmonella. SsrB binds upstream of the sifA, sifB, and sseJ effector genes and directly regulates transcription. SsrB also relieves gene silencing by the nucleoid protein H-NS. Single molecule experiments with magnetic tweezers demonstrated that SsrB displaces H-NS from DNA only when it is bound in a polymerization (stiffening) mode and not when H-NS is bound to DNA in the bridging mode. Thus, in contrast to previous views, the polymerization binding mode of H-NS is the relevant form for counter-silencing by SsrB. Our results reveal that response regulators can directly activate transcription and also relieve H-NS silencing. This study adds to the repertoire of mechanisms by which NarL/FixJ subfamily members regulate transcription. Because SsrB-dependent promoters are diversely organized, additional mechanisms of transcriptional activation at other loci are likely.

SUBMITTER: Walthers D

PROVIDER: S-EPMC3023485 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription.

Walthers Don D Li You Y Liu Yingjie Y Anand Ganesh G Yan Jie J Kenney Linda J LJ

The Journal of biological chemistry 20101108 3

The response regulator SsrB activates expression of genes encoded within and outside of a pathogenicity island (SPI-2), which is required for systemic infection of Salmonella. SsrB binds upstream of the sifA, sifB, and sseJ effector genes and directly regulates transcription. SsrB also relieves gene silencing by the nucleoid protein H-NS. Single molecule experiments with magnetic tweezers demonstrated that SsrB displaces H-NS from DNA only when it is bound in a polymerization (stiffening) mode a ...[more]

PMID: 21059643

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Project description:We performed chromatin immunoprecipitation on microarray (ChIP-chip) transcriptional profiling of the SsrB regulator of Salmonella enterica Typhimurium to better characterize its regulon and to identify the DNA-recognition element coordinating its specific interaction at cis-regulatory sites. SsrB, the response regulator of the two component regulatory system SsrA-SsrB encoded within the Salmonella Pathogenicity Island (SPI-2), directs transcriptional activation of the closely associated type three secretion system (T3SS) also encoded at this locus. Immunoprecipitaiton of SsrB translationally fused to a C-terminal FLAG-tag from formaldehyde cross-linked genomic DNA was performed under SPI-2 activating and non-activating conditions. Downstream analysis and experimental work identified specific interactions within SPI-2 at the previously identified ssrA, ssaB, sseA, ssaG, ssaM promoters and identified an additional cryptic promoter driving expression upstream of ssaR. Additionally, a previously unknown SsrB interaction site within ssaE was shown to be the major contributor driving the expression of the downstream genes and not the previously identified interaction site within the intergenic region of ssaE-sseA. Interactions were also identified outside of SPI-2 upstream of other T3SS-associated genes encoded within other genomic islands, and for other uncharacterized genes. Integration of this data with transcriptional microarray work, previously published DNase I footprinting data, bacteria 1-hybrid investigations and comparative genomics analyses of cis-regulatory regions within the orthologous Sodalis symbiosis region 3 (SSR-3) of Sodalis glossinidius enabled identification of a conserved 18bp palindrome which was experimentally validated as being required for transcriptional activation of SsrB dependant genes. SsrB-FLAG immunoprecipitations were performed under SsrB activating and non-activating conditions from formaldehyde cross-linked bacterial lysates. Nine immunoprecipitation reactions were pooled into three replicate samples for the activating condition and three reactions were pooled into one sample for the non-activating control condition. The immunoprecipitated DNA in addition to the non-immunoprecipitated control DNA for each of the four samples were labeled with Cy3 or Cy5 fluorophores respectively and were concurrently hybridized to four arrays on a single slide.

Dataset Information

Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription.

Publications

Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription.

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