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

In this proposal, we aim to combine data obtained by direct experimentation with mathematical models that seek to predict the behaviour of central carbon metabolism in a model bacterium. This interplay between biologists and mathematicians, using a systems level approach, provides a potentially powerful tool for characterising biological processes. The system that we seek to understand is that of central carbon metabolism - the means by which nutrients are converted into energy - in the model Gram+ bacterium called Bacillus subtilis. B. subtilis has been particularly well studied and thus acts as a highly amenable model organism for studies of bacterial physiology. In our consortium, which brings together biologists from complementary disciplines and mathematicians, we will focus on the way in which carbon is assimilated by this organism and the interplay between carbon and nitrogen metabolism. In recent years it has become clear that many key aspects of physiology are regulated not by discrete enzymes, but by multi-component complexes. Central to this research proposal is the way in which many of the key enzymes in carbon/nitrogen assimilation may function as complexes. We seek to identify and validate these complexes in metabolism. These macromolecular complexes are likely to confer special properties to the enzymatic reactions that are catalysed by these essential proteins. For instance, maintaining two enzymes that catalyse consecutive steps in the biochemical pathway of carbon/nitrogen metabolism in a tightly-associated complex may increase the rate at which substrate A is converted to product C via intermediate B through the process of substrate channeling. Although it may seem intuitive that this ought to be the case, it has not been demonstrated unequivocally. Moreover, these essential protein:protein complexes may change composition depending on the energy status of the cell, a possibility that has yet to be explored. Consequently, by a series of experiments designed to identify key complexes, we will assess the impact on the life cycle of the organism by targeted disruption by mutagenesis of interface regions. By feeding strands of data from a variety of sources into a computer model that aims to describe the metabolic pathway on a mathematical basis, we can use the model to form a hypothesis about the likely effect of changes to the system, and then go back to the laboratory to test these predictions. Central to our aims is the description of key biochemical parameters that define the interactions between proteins for the completion of the computer model in the most robust form, and also to determine the 3-dimensional structures of key complexes so that we can place a cellular phenomenon on an atomic scale.

This proposal aims to use a systems biology approach to define and to understand, through integrated modelling and experimentation, the impact of metabolic and regulatory multienzyme complexes on the central carbon and nitrogen metabolism of a highly tractable, low-GC Gram+ model organism, Bacillus subtilis, the focus of our consortium. This bacterium has a proteome of ~4,100 proteins, about half of which have yet to have a function assigned. While transcriptomics and proteomics can reveal which proteins are expressed in the cell under any given condition, they are not able to reveal their interactions with each other, and with other cellular components. Such interactions are likely to have important system-wide implications with respect to cellular physiology. It is the interaction of a key subset of these proteins and how these complexes may change temporally, that we seek to understand, for which a systems approach is an absolute requirement. So far, it has been possible to model the metabolism of B. subtilis with the inherent assumption that both metabolites and enzymes are more or less evenly distributed in the cell. However, the existence of enzyme complexes would have a dramatic impact on the kinetics of the reactions (second order kinetics vs. third order kinetics) as well as on the relevant local concentrations of metabolites. This field of research is so far unexplored and urgently requires new experimental approaches to address the important issue of (micro)compartmentalisation. In this project, we will utilise quantitative approaches for the detection of protein and metabolite levels and novel mass spectrometry methods for the detection of protein-protein interactions. The most promising candidate complexes will be targeted for detailed analysis, including structure determination by X-ray crystallography, and/or for larger complexes, cryo-electron microscopy and the derivation of thermodynamic constants by biophysical techniques, such as ITC/SPR.

Bacteria are the most abundant and diverse organisms on Earth, essential to the ecosystems that maintain our planet. They have developed relationships with plants and animals that are, for the most part beneficial, with only a small minority causing disease. In addition, bacteria are exploited by the 'white' biotechnology sector, an exploitation that is likely to be expanded as we seek new ways to reduce reliance on environmentally-damaging chemicals or to increase the production of 'bio-fuels'. Thus we must understand the fundamental properties of bacteria for the long-term benefit of the planet. Both Harwood and Lewis labs host ~3 intern visits per year from post-graduate students, usually from Europe (including other partners in BACELL SysMO), China and Australia. Consequently, these young scientists receive training and expertise from the research staff in our laboratories and in the Institute in which we are embedded. We anticipate further visits will take place during the current project for the effective exchange of ideas and expertise. Press releases are made to coincide with major publications. The most recent, in October 2008, accompanied a paper in Science with interviews in printed media and commentaries in more scientific media forums. The Lewis lab has featured regularly in newsletters from the Diamond synchrotron, and formed part of the highlights presented at the AAAS in 2009. The Lewis lab will also participate in the 350th anniversary of the Royal Society in June 2010, at which many internationally-renown scientists, politicians and other policy makers will be present. We will be the only structural biology lab at these celebratory events, representing an opportunity to showcase our science, much of which is BBSRC-funded. The work in the Harwood lab on a patient-side bacterial detection system has figured in the press and is being featured in the EPSRC Impact! Art event, a mixed-media exhibition of original art works exploring the relationship between science and society, and the impact that engineering and the physical sciences have on our world. The month-long exhibition at the Royal College of Art will be followed by a travelling exhibit. Various European companies that form the Bacillus Industrial Platform (BACIP) use Bacillus for a variety of biotechnological and biomedical applications, such as the production of key biologicals, compounds that are difficult to produce chemically. Hence the European 'white' biotechnology sector has a keen interest in the exploitation of bacteria in general, and B. subtilis in particular, for bioproduction. BACIP supports the annual European Bacillus conference, the BACELL website and publicity material (www.bacell.eu) while Fonds der Chemischen Industrie supports the B. subtilis Wiki (http://subtiwiki.uni-goettingen.de/wiki/). The scientists employed on this project, and the students with whom they will engage, will receive training in the identification and characterisation of protein complexes and in systems biology, two areas of strategic importance for the BBSRC. They will also receive opportunities for public engagement, with scientists and non-scientists (through University open days and the Royal Society celebrations), and develop project management skills. Their numeracy skills will also be improved by our links to mathematicians. These benefits will take place during the life-time of the project. The multi-media that we develop for the Royal Society celebrations will be made available on the internet. There are no immediate plans for commercial exploitation at this stage, but the results we will obtain will inform the 'white' biotechnology sector, and provide essential information for the burgeoning field of synthetic biology, where knowledge of substrate channelling and the interplay of enzymes in multi-component complexes remain sparse. The 3D structures that we will solve will be deposited in publically accessible databases (e.g.the PDB).