SysMo: Ion and solute homeostasis in enteric bacteria: an integrated view generated from the interface of modelling and biological experimentation.

The growth and survival of bacterial cells is dependent upon their ability to maintain a relatively constant cytoplasm where the biochemical processes of producing proteins takes place. The major elements of the cytoplasm that must be maintained relatively constant are the ionic composition, simple ions, such as potassium, sodium, chloride, magnesium, iron and protons (pH). In general, a high potassium concentration, low sodium, chloride and proton concentrations are desirable for the normal functioning of enzymes that generate the molecules needed for growth and survival. The cytoplasmic pools of magnesium and iron are maintained constant by complex regulatory networks that will not be considered directly in the current project. When the cell cannot maintain its cytoplasm in the desired state, inhibition of growth occurs and, eventually, bacterial death results. This observation has two immediate consequences. Firstly, many large scale commercial processes for the production of antibiotics and novel therapeutic proteins rely on bacterial growth - understanding how to achieve the best growth is one the major objectives for the pharmaceutical industry. Secondly, the growth of bacteria in the body is normally prevented by the immune system, which can be aided by the use of antibiotics. In food processing and in the environment, bacterial growth should is limited by trying to interfere with the composition of the cytoplasm. New insights arising from the studies proposed here will help us to understand how we can optimise the inhibition and killing of bacterial cells. The project builds upon work in the partner laboratories that have sought to develop an understanding of the mechanisms that regulate potassium accumulation by bacteria, the control over the cytoplasmic pH and the adaptation to high salt conditions. All the partners have achieved excellence in their biological research and in this programme they are now joined by chemists, mathematicians, computer scientists and physicists who will to build models that predict the behaviour of cells. Modelling can describe our current understanding of a biological process and if the accuracy of the model is high, which is dependent on clear communication between the physical scientists and the biologists, the model will predict the outcome of experiments that have not yet been performed. The researchers can then design experiments that test the model and then allow the model to be refined. Ultimate success in this strategy is defioned by being able to predict accurately the outcome of specific experiments and the bacterial response to specific changes in the environment. This information can then be used to try to inform protocols that either improve or reduce bacterial growth and survival.

This programme brings together four groups of biologists and three groups of mathematicians in four countries, and builds upon existing collaborations to develop and test models for ionic homeostasis and its role in regulating cell behaviour. WP1: Analysis of signal perception, transmission and integration of the Kdp system for K+ transport into E. coli: (funded by DFG) WP2: Glutathione-based detoxification of chemicals is directly linked to changes in cytoplasmic pH via modulation of the KefC class of K+ channels. The proposed studies will build and test a model for the regulation of the channel based upon competition between different glutathione ligands and will integrate changes in pH,with cell survival. WP3: we will investigate the hypothesis that cytoplasmic crowding transient networks of 'electrolyte pathways and pools'. Using fluorescence correlation spectroscopy, the mobility of fluorescently-labeled proteins as a function of osmotic stress will be probed to determine the extent to which intracellular transport becomes limiting for vital cellular processes. Fluorescence measurements will be correlated with the activity of cellular systems using the osmoregulatory glycine betaine transporter OpuA as a reporter of changes in intracellular ionic strength. WP4: DNA topology and transcription factor abundance and activity are two major determinants of gene expression. In vivo ChIP-Chip techniques will be used to measure the global patterns of gene expression, protein stability and protein synthesis in response to variations in cytoplasmic constitution that can arise naturally in response to activation of the transporters and channels analysed in WP1-3. WP5: we will generate a predictive model for the response of the cell to changes in ionic homeostasis, built from existing datasets and then be combined with accurate measurements of cellular ionic composition (total ion concentrations), internal pH, the protein mobility measurements from WP1-4.