ArcA phosphorylation at different aerobiosis levels (steady states)

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ArcA phosphorylation in chemostat cultures grown at different aerobiosis levels was quantitated by Phos-tag SDS-PAGE gel analysis and subsequent immunodetection of ArcA.

SEEK ID: https://fairdomhub.org/assays/216

Class: Experimental assay

Projects: SUMO

Investigation: Steady state studies for different oxygen availability in Escherichia coli

Study: The Escherichia coli steady state response to oxygen: from molecular interactions to regulatory modules

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Assay type: Experimental Assay Type

Technology type: SDS-PAGE

Organisms: Escherichia coli : MG1655 (del LAM;del rph-1 / pyrimidine starvation )

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Matthew Rolfe

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Views: 3221

Created: 30th Jan 2013 at 15:22

Last updated: 8th Nov 2017 at 14:21

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Projects (1)
Investigations (1)
Studies (1)
Strains (1)
SOPs (3)
Publications (3)

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Matthew Rolfe

Projects: SUMO

Institutions: University of Sheffield

Expertise: Transcriptomics, Molecular microbiology, E. coli, Salmonella

Tools: Transcriptomics, Chemostat culture, Molecular biology techniques (RNA/DNA/Protein), qRT-PCR

I am a post-doctoral research associate working in Sheffield in the SUMO consortium. My research focuses on transcriptional regulation in E. coli, with particular emaphasis on the transcriptomic analysis of steady-state chemostat cultures using both microarray and qRT-PCR approaches.

Previous experience, especially that gained during my PhD, involved work on Salmonella physiology and lag phase growth, focusing particularly on gene-expression and transcriptional regulation. Other techniques used ...

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SysMO

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

Projects: BaCell-SysMO, COSMIC, SUMO, KOSMOBAC, SysMO-LAB, PSYSMO, SCaRAB, MOSES, TRANSLUCENT, STREAM, SulfoSys, SysMO DB, SysMO Funders, SilicoTryp, Noisy-Strep

Web page: http://sysmo.net/

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SUMO

"Systems Understanding of Microbial Oxygen responses" (SUMO) investigates how Escherichia coli senses oxygen, or the associated changes in oxidation/reduction balance, via the Fnr and ArcA proteins, how these systems interact with other regulatory systems, and how the redox response of an E. coli population is generated from the responses of single cells. There are five sub-projects to determine system properties and behaviour and three sub-projects to employ different and complementary modelling ...

Programme: SysMO

Public web page: http://www.sysmo.net/index.php?index=55

Organisms: Escherichia coli, Escherichia coli K-12

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Steady state studies for different oxygen availability in Escherichia coli

SUMO

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Michael Ederer

Changing the oxygen availability leads to an adaptation of Escherichia coli at different biological levels. After pertubation of oxygen in chemostat experiments the microorganism(s) will come back to another steady state. This investigation deals with these stationary responses of Escherichia coli within the aerobiosis scale. The change for different biological variables, in different areas of the organism like the electron transport chain, the TCA cycle or globally is investigated by wildtype ...

Submitter: Michael Ederer

Studies: Basic regulatory principles of Escherichia coli’s electron transport cha..., Determination of the impact of specific enzyme reactions and regulatory ..., Quantitative analysis of catabolic carbon and electron fluxes in E. coli..., The Escherichia coli steady state response to oxygen: from molecular int...

Assays: Analysis of by-product formation rates in MG1655, Analysis of gene expression rates at different aerobiosis levels via RT-PCR, ArcA phosphorylation at different aerobiosis levels (steady states), Characterization of E. coli MG1655 and ∆sdhC and ∆frdA isogenic mutant s..., Determination of intracellular metabolite concentrations, Determination of intracellular redox state by means of NAD/NADH ratio, Determination of intracellular redox state by means of ubiquinones (oxd/..., FNR activity at different aerobiosis levels (steady state), Kinetic modelling of Escherichia coli's electron transport chain, Kinetic modelling of Escherichia coli's electron transport chain coupled..., Literature Data from Alexeeva et al., J. Bacteriol., 2000, 2002, 2003, Measurement of cytochrome numbers, Physiological measurements from Sheffield chemostat, Steady State Oxygen Response of E. coli WT and two Electron Transport Ch..., Transcriptional profiling of steady states at different aerobiosis levels

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Created: 17th Sep 2009 at 09:35, Last updated: 22nd Nov 2011 at 19:12

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The Escherichia coli steady state response to oxygen: from molecular interactions to regulatory modules

SUMO

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Michael Ederer

No description specified

Submitter: Michael Ederer

Investigation: Steady state studies for different oxygen avail...

Assays: ArcA phosphorylation at different aerobiosis levels (steady states), Determination of intracellular redox state by means of NAD/NADH ratio, Determination of intracellular redox state by means of ubiquinones (oxd/..., FNR activity at different aerobiosis levels (steady state), Literature Data from Alexeeva et al., J. Bacteriol., 2000, 2002, 2003, Measurement of cytochrome numbers, Physiological measurements from Sheffield chemostat, Steady State Oxygen Response of E. coli WT and two Electron Transport Ch..., Transcriptional profiling of steady states at different aerobiosis levels

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Created: 17th Sep 2009 at 11:03, Last updated: 14th Jan 2013 at 16:43

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MG1655

Submitter: Not available

Provider Name: Yale CGSC

Provider's strain ID: CGSC#: 6300

Organism: Escherichia coli

Genotypes: del LAM;del rph-1

Phenotypes: pyrimidine starvation

Comment: This culture was grown from lyophil Oct. 2003 and it tested Fnr+. The previous working culture was mixed fnr-/fnr+.

Created: 10th May 2012 at 11:16, Last updated: 18th May 2012 at 15:55

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Determination of protein phosphorylation levels using Phos-tag™ gels

SUMO

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Matthew Rolfe

The phosphorylation level of a particular protein can be determined using a procedure based upon western immunoblotting, with Phos-tag™ reagent present in the SDS-PAGE gel. The Phos-tag™ reagent, supplied in the form of Phos-tag™ acrylamide (Wako Pure Chemical Industries, AAL-107), causes proteins to be resolved both on the basis of size and phosphorylation state. This means that phosphorylated and de-phosphorylated forms of the same protein can be distinguished.

Creator: Matthew Rolfe

Submitter: Matthew Rolfe

Investigations: Steady state studies for different oxygen avail...

Studies: The Escherichia coli steady state response to o...

Assays: ArcA phosphorylation at different aerobiosis le...

Created: 18th Jan 2013 at 12:51

Evans Medium

SUMO

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Katja Bettenbrock

No description specified

Creator: Katja Bettenbrock

Submitter: The JERM Harvester

Investigations: Analysis of Escherichia coli with linear electr..., Dynamical studies for different oxygen availabi..., Steady state studies for different oxygen avail...

Studies: Analysis of Escherichia coli strains with linea..., Determination of the impact of specific enzyme ..., The Escherichia coli dynamical response to oxyg..., The Escherichia coli steady state response to o...

Assays: Analysis of by-product formation rates in MG1655, Analysis of gene expression rates at different ..., ArcA phosphorylation at different aerobiosis le..., Characterization of E. coli MG1655 and ∆sdhC an..., FNR activity at different aerobiosis levels (st..., Gene expression analysis of mutants with linear..., Physiological measurements from Sheffield chemo..., Transcriptional profiling of E. coli during ana...

Created: 16th Mar 2010 at 17:13

SUMO chemostat conditions

SUMO

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Sonja Steinsiek

This document describes the chemostat conditions to run comparable experiments in different bioreactors in different labs.

Creator: Sonja Steinsiek

Submitter: Sonja Steinsiek

Investigations: Analysis of Escherichia coli with linear electr..., Analysis of the glucose transport in Escherichi..., Dynamical studies for different oxygen availabi..., Steady state studies for different oxygen avail...

Studies: Analysis of Escherichia coli strains with linea..., Characterization of mutant strains with defects..., Determination of the impact of specific enzyme ..., The Escherichia coli dynamical response to oxyg..., The Escherichia coli steady state response to o...

Assays: Analysis of by-product formation rates in MG1655, Analysis of gene expression rates at different ..., ArcA phosphorylation at different aerobiosis le..., Characterization of MG1655 and mutant strains u..., Determination of by-product formation and gluco..., Determination of intracellular metabolite conce..., Determination of intracellular redox state by m..., Gene expression analysis of mutants with linear..., Physiological measurements from Sheffield chemo..., Transcriptional profiling of E. coli during ana...

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Created: 26th Oct 2009 at 14:48

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Basic Regulatory Principles of Escherichia coli's Electron Transport Chain for Varying Oxygen Conditions

SUMO

Abstract (Expand)

For adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known … that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.

Editor:

Date Published: 30th Sep 2014

Publication Type: Not specified

PubMed ID: 25268772

Citation:

Created: 7th Oct 2014 at 15:28, Last updated: 8th Dec 2022 at 17:26

A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen

SUMO

Abstract (Expand)

The efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of … energy supply, carbon, and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding. Mathematical modeling has the potential to provide a coherent and quantitative description of the interplay between gene expression, metabolite concentrations, and metabolic fluxes. Escherichia coli undergoes major adaptations in central metabolism when the availability of oxygen changes. Thus, an integrated description of the oxygen response provides a benchmark of our understanding of carbon, energy, and redox metabolism. We present the first comprehensive model of the central metabolism of E. coli that describes steady-state metabolism at different levels of oxygen availability. Variables of the model are metabolite concentrations, gene expression levels, transcription factor activities, metabolic fluxes, and biomass concentration. We analyze the model with respect to the production capabilities of central metabolism of E. coli. In particular, we predict how precursor and biomass concentration are affected by product formation.

Editor:

Date Published: 27th Mar 2014

Publication Type: Not specified

PubMed ID: 24723921

Citation:

Created: 7th Oct 2014 at 15:32, Last updated: 8th Dec 2022 at 17:26

Transcript profiling and inference of Escherichia coli K-12 ArcA activity across the range of physiologically relevant oxygen concentrations

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Matthew Rolfe

Alexander Ter Beek

Alison Graham

Joost Teixeira de Mattos

Robert Poole

Jeff Green

Abstract (Expand)

Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Whilst much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic … conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here for the first time the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundances of cytochrome bd and bo and outer membrane protein W. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself, or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFinfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis, but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. Measurement of the amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.

Authors: , , , Eleanor W Trotter, H M Shahzad Asif, Guido Sanguinetti, , ,

Date Published: 22nd Jan 2011

Publication Type: Not specified

PubMed ID: 21252224

Citation:

Created: 24th Jan 2011 at 08:31, Last updated: 8th Dec 2022 at 17:26