Multiomics study of Bacillus subtilis under osmotic stress

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 ...

BaCell-SysMO 2 Modelling carbon core metabolism in Bacillus subtilis – Exploring the contribution of protein complexes in core carbon and nitrogen metabolism.

Bacillus subtilis is a prime model organism for systems biology approaches because it is one of the most advanced models for functional genomics. Furthermore, comprehensive information on cell and molecular biology, physiology and genetics is available and the European Bacillus community (BACELL) has a well-established reputation for applying ...

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)

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

intracellular metabolite measured by GC/MS and LC/MS

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

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.

intracellular metabolites measured by LC/MS

extracellular metabolites concentrations [mM] measured by 1H-NMR i

intracellular metabolite measured by GC/MS

SOP for extracellular metabolite measurment

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.