Project description:Mapping the occupancy of FNR, HNS, and IHF throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anerobic growth conditions. We also mapped the binding of the ß subunit of RNA Polymerase under both aerobic and anaerobic growth conditions. As a control, we also performed ChIP-chip on FNR in a ∆fnr mutant strain of Escherchia coli MG1655 K-12. We also examined FNR immunoprecipitation at various FNR concentrations using IPTG and Ptac::fnr (PK8263). The ∆hns/∆stpA strains were also used. Descirbed in the manuscript Genome-scale Analysis of E. coli FNR Reveals the Complexity of Bacterial Regulon Structure
Project description:Mapping the occupancy of ArcA throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anaerobic and aerobic growth conditions. As a control, we also performed ChIP-chip onArcA in a ∆arcA mutant strain of Escherchia coli MG1655 K-12. Described in the manuscript The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli
Project description:Mapping the occupancy of FNR, HNS, and IHF throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anerobic growth conditions. We also mapped the binding of the M-CM-^_ subunit of RNA Polymerase under both aerobic and anaerobic growth conditions. As a control, we also performed ChIP-chip on FNR in a M-bM-^HM-^Ffnr mutant strain of Escherchia coli MG1655 K-12. We also examined FNR immunoprecipitation at various FNR concentrations using IPTG and Ptac::fnr (PK8263). The M-bM-^HM-^Fhns/M-bM-^HM-^FstpA strains were also used. Descirbed in the manuscript Genome-scale Analysis of E. coli FNR Reveals the Complexity of Bacterial Regulon Structure Mapping of occupancy of FNR, NNS, IHF and M-CM-^_ of RNAP in the genome of Escherchia coli MG1655 K-12 under aerobic or anaerobic growth conditions. Immunoprecipitated DNA compared to INPUT for each sample.
Project description:Determining how facultative anaerobic organisms sense and direct cellular responses to electron acceptor availability has been a subject of intense study. However, even in the model organism Escherichia coli, established mechanisms only explain a small fraction of the hundreds of genes that are regulated during shifts in electron acceptor availability. Here we propose a qualitative model that accounts for the full breadth of regulated genes by detailing how two global transcription factors (TFs), ArcA and Fnr of E. coli, sense key metabolic redox ratios and act on a genome-wide basis to regulate anabolic, catabolic, and energy generation pathways. We first fill gaps in our knowledge of this transcriptional regulatory network by carrying out ChIP-chip and gene expression experiments to identify 463 regulatory events. We then interfaced this reconstructed regulatory network with a highly curated genome-scale metabolic model to show that ArcA and Fnr regulate > 80% of total metabolic flux and 96% of differential gene expression across fermentative and nitrate respiratory conditions. Finally, based on the data we propose a feedforward with feedback trim regulatory scheme by showing extensive repression of catabolic genes by ArcA and extensive activation of chemiosmotic genes by Fnr. We further corroborated this regulatory scheme by showing a 0.71 r2 (p < 1e-6) correlation between changes in metabolic flux and changes in regulatory activity across fermentative and nitrate respiratory conditions. We also are able to relate the proposed model to a wealth of previously generated data by contextualizing the existing transcriptional regulatory network. We integrated transcription factor binding regions and mRNA transcript abundance to elucidate the ArcA and Fnr regulons experimentally. To measure transcription factor binding at a genome scale, we employed a ChIP-chip method to derivative strains of E. coli K-12 MG1655 harboring ArcA-8myc or Fnr-8myc under various conditions. The E. coli strains harboring Fnr-8myc and ArcA-8myc were generated as described previously [PMID 16454042]. A 12 chip study with two different strains under two different culture conditions.
Project description:Mapping the occupancy of ArcA throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anaerobic and aerobic growth conditions. As a control, we also performed ChIP-chip onArcA in a M-bM-^HM-^FarcA mutant strain of Escherchia coli MG1655 K-12. Described in the manuscript The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli Mapping of occupancy of ArcA in the genome of Escherchia coli MG1655 K-12 during anaerobic fermentation and aerobic respiration. Immunoprecipitated DNA compared to INPUT for each sample.
Project description:Investigation of whole genome gene expression level changes in a Escherichia coli MG1655 K-12 ∆arcA mutant, compared to the wild-type strain. The mutations engineered into this strain produce a strain lacking the ArcA protein. The results are further described in the manuscript The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli
Project description:Investigation of whole genome gene expression level changes in a Escherichia coli MG1655 K-12 ?fnr mutant, compared to the wild-type strain. The mutations engineered into this strain produce a strain lacking the FNR protein. WT strains were grown under aerobic and anaerobic growth conditions.
Project description:Investigation of whole genome gene expression level changes in a Escherichia coli MG1655 K-12 ?fnr mutant, compared to the wild-type strain. The mutations engineered into this strain produce a strain lacking the FNR protein. WT strains were grown under aerobic and anaerobic growth conditions. A six chip study using total RNA recovered from two separate cultures of Escherichia coli MG1655 K-12 WT (aerobic and anaerobic) and two separate cultures of the ?fnr mutant strain (anaerobic). Each chip measures the expression level of 4,661 genes from Escherichia coli MG1655 K-12 with eight 60-mer probes per gene, with each probe represented twice on the array.