BaCell-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 ...

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.

The objective of this project is an integrated understanding the metabolic, proteomic and genetic network that controls the transition from growth to glucose starvation. This transition is a fundamental ecophysiological response that serves as a scientific model for environmental signal integration and is pivotal for industrial fermentations of Bacillus that occur predominantly under nutrient starvation.

Keywords: Glucose starvation, Transcriptomics, Proteomics, Metabolomics,Bacillus subtilis,

Bacillus subtilis was subjected to various stress conditions like high temperature(57°C), low temperature(16°C), high osmalarity(1.2M NaCl). The above mentioned stress conditions are again split into two different types as 'continuous stress condition' and 'sudden shock'. All the conditions were then done in biological triplicates. Transcriptome for these samples was then analysed with Nimblegen Tiling array.

The aim of this project is to develop a detailed kinetic model of the CcpA-dependent regulatory network, the key regulon of flux regulation in B. subtilis. Thereby involved are more than 300 genes e.g. catabolism, overflow metabolism, the TCA cycle and amino acid anabolism which are regulated via carbon catabolite regulation (CCR)

High salinity chemostat cultivation, multiomics sampling (proteome, transcriptome, metabolome, fluxome) and modelling of carbon core metabolism of Bacillus subtilis 168.

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.

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.

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)

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.

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

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 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.

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

Measurement of intra- and extra-cellular metabolome.

We use BSA115 strain which lacks RsbU and RsbW proteins. Therefore, there is limited post-transcriptional regulation of sigmaB activity. SigmaB itself is placed downstream of Pspac, inducible by IPTG. The lacZ reporter gene is downstream of Pctc promoter. IPTG concentrations of 0.1, 0.2 and 1 mM are added in mid-exponential phase at an OD of appr. 0.3. The whole experiment runs for about eight hours.

Theoretical analysis of hypothetical sigma factor competition. Based on the model 'transcription factor competition' possible dynamics of sigma factor competition are simulated and analysed using Lineweaver-Burk representations.

B. subtilis was grown in SMM media with glucose as carbon source and the samples for RNA were harvested OD578nm- 1.0). The stress conditions that were applied over here are growthat 57°C, 16°C, 1.2M Nacl and 37°C(control). All the samples were analysed for transcriptome as biological triplicates.

B. subtilis was grown in M9 media with glucose as carbon source and the samples were harvested during exponential phase (OD600nm- 0.4), early stationary phase(OD600nm- 1.3), late stationary phase(OD600nm- 1.0). All the samples were analysed for transcriptome as biological triplicates.

Theoretical analysis of hypothetical sigma factor competition. Based on the model 'transcription factor competition' possible dynamics of sigma factor competition are simulated and analysed using Lineweaver-Burk representations.

We use BSA115 strain which lacks RsbU and RsbW proteins. Therefore, there is limited post-transcriptional regulation of sigmaB activity.

There occurs an unexpected drop in the beta-Gal activity after sigB induction. This modelling effort aims to clarify the reasons.

For analyzing the binding of CcpA-HPrSer46P-complexes to various cre-elements, Surface Plasmon Resonance was used. All operations were carried out on a Biacore X instrument (Biacore, Uppsala, Sweden). Biotinylated cre DNA was coupled on a Neutravidin coated chip in flowcell two, a biotinylated reference DNA in flowcell one. For visualizing only the interactions of the CcpA-HPrSer46P-complex with the cre elements, CcpA was saturated with 50µM HPrSer46P. Titrations were carried out with 5-100nM ...

Transcriptome analysis for the samples harvested from Chemostat cultivated samples.

• Comparison of metabolic flux distribution in carbon core metabolism (EMP, PPP, TCA) of Bacillus subtilis under 3 different conditions: "salt-free" reference, "stress" chemostat, "osmoprotected" chemostat.
• Model created using OpenFLUX and Microsoft Excel
• Model computed using MatLAB

Absolute quantification of proteins using heavy labeled QconCAT as an internal standard and quantifying the native proteins in the complex sample via scheduled Multiple Reaction Monitoring(MRM) .

extracellular metabolite concentrations measured by 1H-NMR

Excel sheet contains:

• flux distribution solution from best iteration cluster
• quality of the fit (experimental MIDs vs. simulated MIDs)
• Sensitivity analysis for 95% flux parameter confidence interval using a Monte-Carlo approach

Excel sheet contains:

• flux distribution solution from best iteration cluster
• quality of the fit (experimental MIDs vs. simulated MIDs)
• Sensitivity analysis for 95% flux parameter confidence interval using a Monte-Carlo approach

Excel sheet contains:

• flux distribution solution from best iteration cluster
• quality of the fit (experimental MIDs vs. simulated MIDs)
• Sensitivity analysis for 95% flux parameter confidence interval using a Monte-Carlo approach

RNA processing and degradation is initiated by endonucleolytic cleavage of the target RNAs. In many bacteria, this activity is performed by RNase E which is not present in Bacillus subtilis and other Gram-positive bacteria. Recently, the essential endoribonuclease RNase Y has been discovered in B. subtilis. This RNase is involved in the degradation of bulk mRNA suggesting a major role in mRNA metabolism. However, only a few targets of RNase Y have been identified so far. In order to assess the ...

batch fermatation - The transition from growing to non-growing Bacillus subtilis cells

batch fermatation - The transition from growing to non-growing Bacillus subtilis cells

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. Here you'll find cell sizes for every sample.

B. subtilis was grown in minimal media in a chemostat at growth rate µ=0.1, with 1.2M NaCl and glycine betaine. Relative quantification for the proteome was done using metabolic labeling.

B. subtilis was grown in minimal media in a chemostat at growth rate µ=0.1, with 1.2M NaCl, without glycine betaine. Relative quantification for the proteome was done using metabolic labeling.

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. Here you'll find cell titers for every sample.

Array hybridisation was done at Roche Nimblegen Inc. It cannot be displayed over here.

SysMo2: Intra- and extracellular metabolome data of the chemostat experiments: nitrogen limitation, nitrogen limitation+ NaCl, nitrogen limitation + glucose

Data sheet generated in Greifswald to measure the response of sigB activity in response to different media composition.

Strain BSA115 is grown until appr. OD 0.25 then expression of sigB is induced by the addition of IPTG. The extend of stress response is measured by the expression of lacZ via beta-Gal assay. The experiment lasts for appr. 400 min.

The stressosome is composed of three proteins that assemble in the form of an icosahedron. Icosahedra can be modelled in different ways with different abstraction levels regarding the original stressosome structure. The pdf-figure introduces geometric modelling of the stressosome using origami and particle dynamics simulations.

The pdf-file shows simulations of a hypothetical model of sigma factor competition. It simulates the dynamics that we can expect from the experiments and prepares for the analysis of the experimental data. Analysis of sigma factor competition is based on a Lineweaver-Burk representation of RNApolymerase and competing sigma factors.

In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a ...

Table describing the catabolite repression of β-xylosidase by different carbon sources(glucose, sorbitol, fructose, maltose, glycerol. mannitol) in various mutants of CcpA cofactors (HprK, crh)

The zip file contains two executable Matlab functions.

File named 'fnct_gen_tfcompmod.m' generates a Simbiology model based on the following interactions: R + X <-> RX -> R + X + Px R + Y <-> RY -> R + Y + Py R + Z <-> RZ -> R + Z + Pz Y + Pz -> Pz Px -> Py -> Pz ->

We assume much higher reaction speeds of sigma factor RNApol binding/unbinding compared to protein expression. Protein expression can therefore be represented by Michaelis-Menten like kinetic ...

only lacZ synthesis reduced by inhibitor in BSA115

The model file represents the expression of beta-gal from a sigB dependent promoter after sigb production was stimulated by IPTG. The model is based on an assumption that a hypothetical protein degrates the sigb factor.

This is a JWS model of the successful model for data representation. It realises regulation by a hypothetical sigB dependent protein that degrades beta-Gal.

The model represents a hypothetical situation in which an anti-sigmafactor reduces sigB efficacy.

The zip folder contains files that allow simulation of stressosome dynamics. The models are based on a cellular automaton approach. Each protein of RsbR and RsbS is located in the crystal structure of the stressosome. The proteins can be phosphorylated or not and these states determine the future of neighbouring proteins. To simulate the model open the file 'liebal_stressosome-model_12_workflow-matlab.m' in Matlab. It is written in the cell-model, put the cursor into a cell that you wish to ...

The zip file contains model files and an experiment file. Unpack it in a directory and navigate with matlab to there. Use the 'matlab_execution_guide.m' for simulation and visualisation of the model. This file is written in matlab cell mode, so it is not a stand alone function.

Three models have been developed to test their capacity to reproduce the experimental data from Study: 'Controlled sigmaB induction in shake flask' with Assay: 'IPTG induction of sigmaB in BSA115'. One model assumes a ...

The zip-folder contains files for execution in matlab that allow for the simulation of stressosome dynamics and reproduction of published data on the stressosome. The important file for execution is 'liebal_stressosome-model_12_workflow-matlab.m'.

A model of E. coli central carbon core metabolism, used as starting point for B. subtilis modelling. It is developed by Chassagnole et al. doi:10.1002/bit.10288.

The model can simulate the the dynamics of sigB dependent transcription at the transition to starvation. It is was developed along the comic in 'sigB-activation-comic_vol1'. Parameters were partly taken from Delumeau et al., 2002, J. Bact. and Igoshin et al., 2007, JMB. Parameter estimation was performed using experimental data from '0804_shake-flask'. Use the .m-file with matlab as: % reading initial conditions from the file: inic = sigb_model_liebal;

% performing the simulation: [t,y] = ...

Isolation of total RNA from Bacillus Subtilis using phenol-chloroform extraction method by maintaining cryogenec conditions initailly to prevent RNA degradation. Quality of the obtained RNA is then tested with Agilent Bioanalyser before proceeding for gene expression analysis.

SOP for extracellular metabolite measurment

SOP for ß-Galactosidase assay.

SOP for shake flask cultivation of B.Subtilis in Bacell-Sysmo

The reverse transcriptase synthesizes DNA, which complements the mRNA template (complementary DNA, cDNA). Cy3/Cy5-dCTP are incorporated into cDNA during Reverse transcription. The obtained Cy3/Cy5 cDNA are then competitively hybridised onto Agilent microarray slide and subsequently scanned.

Cells were harvested from culture keeping the cells cold to quench the physiological condition of RNA and the cells were mechanically disrupted. RNA was isolated from the cells by conventional acid-phenol method and the quality was checked by Agilent bioanalyser.

In the talk I show that an increase in the concentration of one of two channelling enzyme has no effect on the flux. In contrast a decrease in the concentration of one of two channelling enzymes has the same effect like a concentration decrease of both enzymes.

Presentation given by Colin Harwood presenting results about ITC experiments to confirm interactions. Preliminary crystallisation experiments commenced on suitable candidates.

The talk shows that proline biosynthesis and accumulation is connected to glycine betaine availability. Glycine betaine complements proline function.

The talk shows that position 153 in various glutamate-kinases regulates proline feed-back regulation. This difference is shown for ProB being proline sensitivie and ProJ being proline independent.

Michael Kohlstedt presented flux data as integrated output of cellular components. Moreover, proteomics and metabolomics data reveal significant changes in intracellular protein and metabolite level and osmoprotection (supplementing glycine betaine) creates a novel metabolic state.

Presentation by Thomas Millat of a tool for automatized analysis of flow cytometry data. It allows characterisation of different growth modalities, statistical measures of mean and variance and estimation of population overlap all along with appropriate visualization.

The image file represents the activation of sigB in response to glucose starvation. It was drawn alongside the model 'sigb-response_starvation_shakeflask'. Biomass catalyses the conversion of glucose to biomass, while a low glucose concentration will negatively affect cell viability (inverted shown in the figure, where Glc inhibits cell death). W (RsbW) can form dimers with sigB (B) and dimers and trimers with V (RsbV)(WV, WV2) and is able to phosphorylate V to VP. A high glucose concentration ...

Annual Conference of Bacillus subtilis community.

BaCell consotium in Groningen.