Modelling carbon core metabolism in Bacillus subtilis - Exploring the contribution of protein complexes in core carbon and nitrogen metabolism

Fires can be extinguished in two ways. One uses pumps with pipes that fill up a ditch (the 'pool') that leads to where the fire is, and then another pump taking out the water squirting it onto the fire. The other one is a line of people where the first fills each bucket in a nearby river and then hands the full bucket to the person next in line, who hands it to the next-next, etcetera, i.e. the 'bucket-brigade' mechanism. The chemistry of how living organisms extract energy and carbon out of their food is organized in terms of pathways. This organization could use either the pool mechanism or the bucket brigade mechanism. In most of the text books on this topic it is assumed that it is the pool mechanism that operates, but this is mainly because this is simpler to understand and more readily examined experimentally. Accordingly, mathematical models of the chemistry of living cells tend to assume that pool mechanisms are operative. The biological 'machines' that carry out the individual steps/chemical reactions in the pathways are called enzymes. In recent work in an important bacterium, it has been discovered that some of these enzymes embrace each other, i.e. form complexes. This proposal aims at defining and understanding the impact of metabolic and regulatory multienzyme complexes on the central carbon and nitrogen metabolism. It will do this in Bacillus subtilis, a highly tractable model organism for systems biology approaches. It will examine whether any observed impact is through the facilitation of the bucket-brigade mechanism (also called metabolic channelling). It will do this through a close integration of modelling and experimentation. The outcome of this project could greatly change our understanding of the chemistry of Life. It could also lead to new ways of interfering with that chemistry, for instance by breaking up or promoting the enzyme-enzyme love affairs. Implications could be in the domains of the production of food and chemicals by microorganisms (beer, bread, wine, gasohol) and health management, but would require follow-up projects.

Based on the finding in the first funding period of SYSMO that glycolytic enzymes of B. subtilis form complexes, we wish to define the role of enzyme complexes in the physiology of B. subtilis at the Systems level. To this end, we will first quantify the concentrations of relevant cellular components (proteins, mRNAs, metabolites). Asking whether enzyme concentrations are identical or otherwise stoichiometric to each other and asking for metabolic over- and undershoots around enzymes, we will identify candidates for enzyme-enzyme complexes. In parallel, we will identify protein complexes involved in glycolysis, in tricarboxylic acid cycle, in gluconeogenesis and in anabolic and stress-induced proline synthesis, by implementing our established complex isolation methodology. After an initial validation of these potential complexes by bacterial two-hybrid screening, we will compare these to the above mentioned candidates and come to a combined list of more likely candidates. Meanwhile integral kinetic models of the pathways will be built (topological, then blueprint kinetic), and populated with the measured enzyme concentrations and literature kinetic data. The metabolic fluxes will be determined during balanced growth. By comparing model prediction with experimental results for fluxes and metabolite concentrations, first functional indications for metabolite channelling or alteration of enzyme kinetics upon complexation, will be identified. The promising candidate complexes will then be studied in more detail: We will provide evidence for the in vivo interaction of the partners in the complexes in single cells of B. subtilis using fusions to fluorescent proteins and time lapse and confocal microscopy. Moreover, we will determine kinetic parameters of the interactions to obtain data needed for refined modelling and to select the most relevant candidate complexes for detailed analysis. For these key complexes, we will study the dynamic behaviour.

From the research planned by our consortium we expect to obtain for the first time ever a clear picture of the functional role of protein complexes in bacterial physiology. The outcomes will represent a major advance in our understanding of the metabolism of B. subtilis, an organism of high biotechnological relevance. Moreover, we will contribute to the general understanding of metabolic compartments in bacteria, leading to new strategies for metabolic modeling that take the existence and importance of such complexes into account. Finally, with our research program, which involves extensive collaboration between several European laboratories, our consortium will strengthen the European research community and the collaborations within this community. Last, but not least, there will certainly be important results to be published that will increase the visibility of European science in general, and European microbial Systems Biology in particular.