Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Mapping the DNA-binding response regulators of Desulfovibrio vulgaris Hildenborough

ABSTRACT: D. vulgaris is a model sulfate reducing bacteria whose genome encodes a high number of two component systems, including 29 DNA-binding response regulators (RRs) whose functions are unknown, but are very likley to be important to the environmental lifestyle of the organism. We determined the gene targets for 24 of these RRs using purified His-tagged RR and sheared genomic DNA in an in vitro DAP-chip (DNA-affinity-purified - chip) assay. For each RR, one target was identified first using gel shift assays, and qPCR was used to ensure that the target was enriched in the RR-bound DNA before the samples were hybridized to a tiling array. Based on the peaks generated by the array analysis, we determined that at least 200 genes are regulated by two component systems in this organism. We also predicted binding site motifs and validated them for 15 RRs using gel shift assays. In vitro DAP-chip for 28 response regulators where RR-bound enriched DNA is compared to input total genomic DNA

ORGANISM(S): Desulfovibrio vulgaris

SUBMITTER: Lara Rajeev

PROVIDER: E-GEOD-25163 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Mapping the DNA-binding response regulators of Desulfovibrio vulgaris Hildenborough

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