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| Status |
Public on Feb 08, 2010 |
| Title |
ChIP-chip profiling of SsrB in Salmonella enterica sv. Typhimurium |
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 |
| Experiment type |
Genome binding/occupancy profiling by genome tiling array
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| Summary |
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.
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| Overall design |
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.
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| Contributor(s) |
Mulder DT, Tomljenovic AM, Coombes BK |
| Citation(s) |
20300643 |
| |
| Submission date |
Feb 04, 2010 |
| Last update date |
Mar 22, 2012 |
| Contact name |
Brian Coombes |
| E-mail(s) |
coombes@mcmaster.ca
|
| Phone |
905-525-9140
|
| URL |
http://www.coombeslab.com
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| Organization name |
McMaster University
|
| Department |
Biochemistry & Biomedical Sciences
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| Lab |
HSC-4H17
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| Street address |
1200 Main St. West
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| City |
Hamilton |
| State/province |
Ontario |
| ZIP/Postal code |
L8N 3Z5 |
| Country |
Canada |
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| Platforms (1) |
| GPL10008 |
Oxford Gene Technology Chip2 Salmonella typhimurium microarray SL1344 strain product code: 010021 |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA125691 |
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