Assays

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.

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

intracellular metabolite measured by GC/MS and LC/MS

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.

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.

The stressosome is an important sensor of environmental stresses in B. subtilis. It is formed by three protein types that form an icosahedral geometric protein complex. There are uncertanties how protein interactions take place, what the effects on the response behaviour of activation and inhibition of phosphorylation among proteins is, and what kind of proximal signal activates the stressosome in the first place. To answer these questions a computational modelling approach was developed. This ...

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.

• 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