Studies

PGK-GAPDH reactions were studied in vitro at 30 and 70 using yeast or Sso enzymes

SED-ML: https://jjj.bio.vu.nl/models/experiments/kouril2017_fig4b/simulate https://jjj.bio.vu.nl/models/experiments/kouril2017_fig4c/simulate

PGK reaction at 30 C. Yeast PGK was incubated at 30 C, in the presence or absence of the ATP recycling system, and the conversion of 3 PG to BPG was followed. SED-ML simulations Fig. 1A: https://jjj.bio.vu.nl/models/experiments/kouril2017_fig1a/simulate

PGK reaction at 70C. Sulfolobus solfataricus PGK was incubated at 70C in presence and absence of an ATP recycling system. Changes in metabolite concentrations was followed via 31P NMR or enzymatic analyses.

SED-ML: https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3b/simulate https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3c/simulate https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3d/simulate

BPG produced with yeast PGK was incubated at 70 C,upon which BPG rapidly dephosphorylates to 3PG. SED-ML: https://jjj.bio.vu.nl/models/experiments/kouril2017_fig2b/simulate

Genotype: Wildtype (M145E) Medium: Phosphate-limited (F134)

Here you will find guidelines for creating MIAPE compliant proteomics data files as well as examples and links to online tools and resources

Here you will find guidelines for creating MAGE-TAB compliant transcriptomics data files as well as examples and links to online tools and resources.

Determination of essential amino acids for Streptococcus pyogenes M49

Lactic acid bacteria generally use homolactic fermentation for generation of ATP. Here we studied the role of Arginine and Glutamine metabolism on the general physiology of the lactic acid bacteria Streptococcus pyogenes. A deletion mutant of glnA (glutamine synthetase) has been constructed in the S. pyogenes M49 591 background. The glnA mutant strain shows decreased growth in low glutamine and excess glutamate conditions and no growth at all in low glutamine and low glutamate conditions. An arcA ...

The reconstruction of the metabolic networks is done by sequence comparison with already annotated genomes of L. lactis, L. plantarum, B. subtilis and E. coli

Here you will find all pre-liminary data

Pyruvate kinase (PYK, EC 2.7.1.40) is a key step in glycolysis converting phosphoenolpyruvate into pyruvate. The activity of PYK is activator-dependent, with the allosteric activation mostly being due to fructose-1,6-bisphosphate (FBP).

Pyruvate formate-lyase (PFL) is an important enzyme in the metabolic pathway of lactic acid bacteria (LAB) and is held responsible for the regulation of the shift between homolactic acid to mixed acid fermentation. PFL catalysis the reversible reaction of acetyl-CoA and formate into pyruvate and CoA. A glycyl radical, who is regenerated within the reaction, is involved; therefore, PFL works only under strictly anaerobic conditions. For its activation, the C-terminal domain has to bind to the ...

The two lactic acid bacteria L. lactis and S. pyogenes were studied with respect to the concentration of intracellular metabolites involved in glycolysis in time upon a glucose pulse. Models that describe this behavior are also constructed

Lactic acid bacteria generally use homolactic fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three lactic acid bacteria Lactococcus lactis, Enterococcus faecalis and Streptococcus pyogenes. Surprisingly deletion of the ldh genes hardly affected the growth rate in chemically defined medium, however growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various ...

Since over 40 enzymes will be investigated for their mRNA abundance, processing, and degradation kinetics, the less tedious and more accurate Next Generation Sequencing of the entire mRNA repertoire of the cell is employed. To optimise the proportion of useful sequence, while including RNA fragments that are products of of degradation, rRNA is depleted using the eukaryotic Ribominus kit (Ambion). Two biological replicates are treated with Sinefungin and Actinomycin D to inhibit RNA processing and ...

For cells to accurately read out the genomic content, high fidelity during transcription is required. This is mainly established by the accuracy of the active centre of RNA polymerase (RNAP). Based on in vitro experiments with Escherichia coli RNAP it was also suggested that proofreading of transcription via RNA hydrolysis by RNAP may contribute to overall fidelity and processivity. RNAP’s intrinsic cleavage activity is stimulated by the highly conserved Gre factors suggesting that Gre factors ...

The enzyme Trypanothione Synthetase (TryS) is a complex enzyme that catalyses the two step reaction that forms trypanothione from 2 molecules of GSH and 1 molecule of Spd and the use of ATP

A set of isogenic mutant strains was constructed which lack NADH Dehydrogenase I as well as two terminal oxidases, resulting in strains with linear respiratory chain. The different strains hence differ in the terminal oxidase and express either cytochrome bo, cytochrome bdI or cytochrome bdII. The different strains were cultivated in glucose-limited chemostats with defined low levels of oxygen supply. Biomass and by-product formation, gene expression and the phosphorylation state of the important ...

Carbon loss due to instability of gluconeogenic pathway intermediates (BPG, GAP, DHAP) at high temperature in S. solfataricus

Mathematical model of a subset of reactions comprising the three most temperature sensitive intermediates of the gluconeogenic pathway in S. solfataricus

Mutant strains in which one or more of the potential glucose uptake systems was deleted have been analyzed in aerobic and anaerobic batch cultures, as well as aerobic chemostat cultures.

The aim of the study is to assess the global function of RNase Y in RNA processing and degradation in Bacillus subtilis. To this end we constructed a strain allowing controlled depletion of RNase Y and used microarrays to analyze the transcriptome in response to the expression level of RNase Y.

Conversion from KEGG Reactome Information to SBTOOLBOX2 format.

Flux will be measured using the metabolomics platforms based on absolute quantification method (isotope ratio based MS technique) by LC-MS, using heavy-isotope labelled precursors of the metabolites of interest. For example, 15N labelled cysteine, glycine and glutamate will be used to determine rates of synthesis of glutathione. 15N-labelled methionine to measure S-adenosyl methionine (and its decarboxylated form, as well as methionine cycle intermediates). 15N labelled arginine is used as precursor ...

In addition to the highly targeted quantification of metabolites already known to play major roles in oxidative stress, to provide data directly compatible with current models, we will also take an untargeted metabolomics approach. This will enable us to identify other areas of the metabolome influenced by, or influencing, oxidative stress and will allow us to compare changes in each of the stress-inducing stimuli. We have recently pioneered untargeted metabolite profiling of T. brucei using ...

We have already demonstrated that the key metabolites of polyamine biosynthesis (arginine, ornithine, putrescine and spermidine) can be identified using HILIC chromatography coupled to the Orbitrap mass spectrometer, as can glycine, glutamate and cysteine used in glutathione biosynthesis, glutathionyl spermidine and trypanothione itself. Furthermore the key metabolites of the methionine cycle (methionine, S-adenosyl methionine, decarboxylated S-adenosyl methionine, methylthioadenosine) can all ...

Annotation retrieval

Creation of the KEGG based Reactome

Parasites will be harvested at different growth phases and the total amount of the proteins will be followed by western blot. The absolut concentration will be obtained by comparison with a know amount of the recombinant untagged protein. The thiol redox state of the proteins will be followed by modification of the free cys with methoxy-ethyl-maleinimide poly(ethylenglycol) (Meo-PEG-mal).

The enzymes involved in the trypanothione metabolism will be studied in a uniform assay medium that mimics the intracellular milieu of the parasite.

Key enzymes of critical points in the pathways will be targeted for disruption by the generation of RNAi cell lines and lines which drive tetracycline-regulatable ectopic over expression of either wild type enzyme or, if appropriate, dominant-negative or mis-targeted mutants of these. In all cases perturbed lines will be analysed with respect to the mRNA, protein or enzymatic activities of other components of the subsystem, this being directed iteratively by the predictions from systems modelling. ...

We developed a new metabolomics protocol, which involved a comparison of different harvesting techniques, quenching solutions and extraction methods.

The Lactate dehydrogenases (LDH) are key metabolic enzymes in lactic acid bacteria (LAB). The LDH ( E.C. 1.1.1.27) catalyzes the reaction of pyruvate and NADH into lactate and NAD+.We have carried out an experimental and computational study of the effects of fructose-1,6-bisphosphate (FBP), phosphate (Pi) and ionic strength (NaCl concentration) on 3 LDHs from 3 LABs studied at pH 6 and pH 7.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see (1,2,3)). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard ...

The output of the initial model of redox metabolism will be compared to experimental flux and metabolite data. Deviations between model and experiment will be prioritized together with WP2. We will apply Metabolic Control Analysis (Fell 1992 PMID: 1530563) to diagnose which enzymes control the deviating metabolite concentrations and/or rates. When the agreement between model and experiment is sufficient we will first link it to the existing model of trypanosome glycolysis and repeat the same ...

Our current gene-expression model (Haanstra et al. 2008 PMID: 19008351) will be parameterized for the different genes of interest. The framework of this gene expression model has been used to include mRNA half life data into the model of glycolysis For the enzymes of redox metabolism we will use newly measured rates of transcription, RNA precursor degradation, mRNA degradation, concentrations of mature mRNAs and proteins, enzyme turnover, Vmax values and metabolic fluxes (WP3&5). Regulation ...

We are in the process of construct an ODE model of the trypanothione pathway. As input we will use newly determined and existing kinetic data and measured metabolite concentrations at the boundaries (from WP3&6). Recently the glycolysis model was extended with the pentose phosphate pathway. This pathway will yield the NAPDH that maintains trypanothione in a reduced state. For some complex enzymes (i.e trypanothione synthase) we are intensively discussing the kinetic data obtained on the ...

These templates can be used for a selection of metabolomics data types. There is a MASTER template for general use and adaptation as well as several more specific templates for particular types of experiment (e.g. HPLC), or specific assay types (e.g glucose pulse)

Multi experimental approach to define the gene expression remodelling under potassium starvation conditions.

How does the Volume, internal pH, membrane potential and intracellular cation content change in a time dependent scale during potassium limitation.

How do the fluxes of rubidium (potassium) change during potassium starvation.

What is the proteome of starved cells. Main characteristics?

Are there one or several stationary states for physiological parameters dependent on external KCl? For which range of external potassium the cell is able to maintain a constant pH, potassium content, membrane potential and volume?

How does the current mediated by Trk1,2 depend on external and internal ion concentrations? How is the membrane potential shifted by the concentrations of various ions, especially ammonium?

Does the general proteome of yeast cells change in a strain lacking Trk1,2 transporter? Under which conditions?

Studies on the effect of deletion of genes encoding 14-3-3 proteins on the transcriptional response of yeast cells to potassium starvation.

Studies on genetic interactions between genes encoding 14-3-3 proteins and genes encoding transporters to elucidate which transporter(s) are regulated by 14-3-3 proteins.

Bioinformatic studies to elucidate the role of 14-3-3 proteins in cation homeostasis.

How does the flux of potassium, hydrogen change during potassium uptake? Which ions are involved in maintaining the required charge balance?

How does the transport of potassium and hydrogen ions effect the concentrations in the near surrounding of the cell.

How does the current mediated by Trk1,2 depend on external and internal ion concentrations? How is the membrane potential shifted by the concentrations of various ions, especially ammonium?

At external potassium concentrations in the range of 10uM to 1mM Trk is major cellular uptake system for potassium. This system responds to the activiy of the proton pump. Transmembrane fluxes of protons and potassium cations are analysed in a signal-response relationship.

This study aims to establish the optimum conditions and assay methods for measuring ATP levels

To see changes in ATP levels in cells with induced ABC transporters. Cells with Pdr12 pump by 10 mM benzoic acid are used.

ATP levels of cells stressed with higher concentrations of benzoic acid (30 mM and 50 mM).

Effect of benzoate treatment (high concentrations) on ATP levels and Pdr12 expression after pretreatment of cells with low concentrations of benzoic acid.

Cell survival was determined under different benzoic acid concentrations

Mathematical modelling of the dynamic shift experiments and the effect of pH upon gene regulation.

B. subtilis was grown in minimal media in a chemostat at different growth rates (µ= max, µ=0.1, µ=0.4) and in the presence of 1.2M NaCl (µ=0.1) with or without glycinebetaine. Transcriptome, proteome and metabolome were investigated.

Here we develop a set of new tools for S. pneumoniae and as a case study we show that S. pneumoniaea SMC is recruited to oriC by ParB and promotes chromosome segregation.

Goals:

1. Understanding the regulatory principles of Escherichia coli’s electron transport chain (ETC) for varying oxygen conditions in glucose-limited continuous cultures (especially regulatory loops via the transcription factors FNR and ArcA).
2. Explaining the observed phenomena in the measurement data.
3. Predicting unmeasured variables especially of the gene expression regulatory loops.

Means:

1. Experiments (chemostat experiments within the aerobiosis scale).
2. Kinetic modelling (especially ...

Selected strains from the collection of GFP-tagged S. cerevisiae strains are cultivated at different extracellular caion concentrations and the localization of the GFP-tagged protein will be studied by confocal microscopy. The effect of deletion of the BMH1 gene, encoding the major 14-3-3 isoform, will be analyzed.

3 chemostat experiments:

each in 4 biological replicates incl. 1 fed with labelled glucose T = 37°C pH = 7.1 V_R = 300 mL (dasgip parallel bioreactor system) V_G = 9 sL/h (0.5 vvm) M9 Minimal medium + 3,4-dihydroxybenzoate (chelating agent) + 1g/L Glucose n = 1000 rpm

3 conditions:

"reference" without additional sodium chloride as control "stress" supplemented with 1.2M sodium chloride "osmoprotection" supplemented with 1.2M sodium chloride and 1mM glycine betaine (osmoprotectant)

Provided with genomic data over different pH values we have the opportunity to study the similarity of gene expression profiles and cluster groups of very simlar gene expression profiles. Via PCA we can furthermore study dynamic similarity and compe genes that are possible co-regulators or anti-regulators in the clostridial metabolism.

The main aim of this experiment is to actively grow B.subtilis in presense of glucose until high optical density in an aerobic fermentor and then, at a definite point of the growth, the glucose supply is shut down which leads to complete glucose exhaustion in the media. Simultaneusly samples for transcriptomics, intra and extracellular metabolomics, intra and extracellular proteomics are harvested through out the experiment.

test test

Experiments using shake flask cultures to measure dynamics associated with sigB response.

To model the ENA1 transcriptional regulation a model has to be established. First this will be just a graphical representation, it shall then be extended to a boolean model and shall at one point be converted to a kinetic model.

Mutant strains which carry deletions of important metabolic enzymes, as well as mutant strains with altered regulation, need to adapt by changing fluxes or gene expression to compensate with the absence/differed concentration of these key enzymes. This may give new insights in the regulation of these pathways/enzymes.

The pilot experiment has been set up in order to develop uniform SOPs for the Sulfosys consortium. It comprises proteomics, transcriptomics, metabolomics as well as enzymatic essays. Cells for all the members of the consortium have been obtained from the same batch fermentations according to fermentation SOP of Sulfosys. The pilot resulted in creating a procedures for all the techniques used in consortium.

Cells are starved. Potassium and subsequently glucose are added to the medium. Proton and potassium fluxes across the plasma membrane are recorded before and after these events for WT and mutants lacking specific transporter proteins.

In this kind of studies sigmaB stress response is induced by the addition of artificial inducers of sigmaB. For example simgaB is downstream of a Pspac promoter and induced by the addition of IPTG. A ctc::lacZ reporter gene is used to monitor sigmaB activity.

The steady state anaerobic culture (D = 0.1 h-1) was pertrubed by sudden increase of the extracellular glucose up to 1 g/L and both extra- and intracellular transient metabolite concentrations were measured

The aim of the study is to test the interplay of the effects of different transcriptional regulators on gene expression during different environmental responses in B. subtilis. For a selected set of transcriptional regulators native proteins are fusion-labelled. The intensity of expression of each reporter protein is measured in the absence of several transcriptional regulators that compete for the RNA polymerase.

Steady state fluxes in yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1

Steady state concentrations of metabolites in yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1

The main aim of this experiment is to actively grow B.subtilis in presense of glucose until high optical density in an aerobic fermentor and then, at a definite point of the growth, the glucose supply is shut down which leads to complete glucose exhaustion in the media. Simultaneusly samples for transcriptomics, intra and extracellular metabolomics, intra and extracellular proteomics are harvested through out the experiment.

Growth of B.subtilis in shakeflask at 57°C; 16°C, 37°C(Control) and 1,2M NaCl in SMM.

BMM EtOH, 16, 57 SMM NaCl