ChIP-seq analysis of the cAMP binding protein (CRP) in Enteroaggregative Escherichia coli 042
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina MiSeq
ORGANISM(S): Escherichia coli
SUBMITTER: James Haycocks
PROVIDER: E-MTAB-14748 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Background: The biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, biofuels etc. However, high growth rate aerobic E. coli cultivations are accompanied by acetate excretion i.e. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear. Results: In the current work, growth rate dependent acetate overflow metabolism of E. coli was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 1/h) is the repression of acetyl-CoA synthethase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 1/h with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for E. coli chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of E. coli decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 1/h leading to total Acs inhibition and fast accumulation of acetate. Accumulation of acetate was also coupled to excretion of products such as orotate and N-carbomoyl-L-aspartate making it a novel carbon spilling mechanism in E. coli. Conclusion: This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in E. coli shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in E. coli is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node. Reference samples at specific growth rate (μ) 0.11 1/h were compared to the ones acquired at μ 0.21, 0.26, 0.31, 0.36, 0.40 and 0.48 1/h
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Project description:The transcriptional profile of Escherichia coli O157 treated with small molecule inhibitors of type III secretion was determined. Four variations of the small molecule inhibitor were assessed for global changes in transcription by treating cells with 20uM of inhibitor or an equivalent volume of DMSO (inhibitor solvent). Keywords: treatment, dose, Cy3, Cy5, 2-colour For each inhibitor, biological tripicates of treated (20uM inhibitor in DMSO) and untreated (DMSO only) cultures were grown in 25ml of MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2. RNA was extracted from 15ml of culture at OD 0.7 and labeled with Cy5 (treated) and Cy3 (untreated) (excepting ME0055 that was performed as a dye swap). Labeled untreated RNA was pooled prior to hybridisation. Treated RNA (biological replicates) and pooled untreated RNA was hybridised to microarray slides. Slides were scanned using an Axon 4100A scanner and data processed using Genespring GX.
2009-08-10 | E-GEOD-10319 | biostudies-arrayexpress
Project description:Comparative genomic hybridization between Escherichia coli strains to determine core and pan genome content of clinical and environmental isolates Two color experiment, Escherichia coli Sakai (reference), clinical and environmental Escherichia coli strains (testers): At least two replicates including a single dye swap for each reference-tester comparison
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Project description:In this study, we generate genomic maps of Mediator, Rad2, Pol II, TBP and TFIIH, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type strains from Saccharomyces cerevisiae. A related study involving ChIP-chip analysis of Rad2 occupany is also deposited at ArrayExpress under accession number E-MEXP-3875 ( http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-3875 ).
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Project description:In this study, we generate genomic maps of Mediator, Pol II, TBP and TFIIH, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type (WT) strains and med17-ts mutants from Saccharomyces cerevisiae. Some of the data, concerning WT strains are also deposited at ArrayExpress under accession number E-MTAB-1595 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1595). There are 2 series of experiment: 1- WT (see E-MTAB-1595) and mutants med17-98, med17-444, and med17-670 (this submission) 2- WT and mutant med17-444 (this submission).
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Project description:Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibits platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen related peptide (CRP-XL). We identified 1634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. 925 of these peptides show an increase of more than 2-fold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on a number of proteins including Syk, filamin and integrins. Adhesion and spreading on fibrinogen mediated by the major platelet integrin IIb3 is blocked by inhibition of deubiquitylases. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.
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Project description:To determine sites where RpoS binds (and hence likely plays a direct role in transcription), we used ChIP-seq to map the association of RpoS across the Escherichia coli chromosome during stationary phase growth in minimal medium. To facilitate ChIP, RpoS was C-terminally SPA-tagged at its native locus.
2016-12-23 | E-MTAB-5339 | biostudies-arrayexpress