Solute Stress Mechanisms and Responses

Pseudomonas putida (P. putida for short) is a bacterium that was originally isolated from soil, a stressful and ever-changing environment; as such it has an inherent capacity to be able to respond to changes in its environment. This bacterium is non-pathogenic, and because of its ability to produce a wide variety of compounds useful to mankind, and to degrade a variety of pollutants that are dangerous to environmental and human health, it has been the object of intensive scientific study. There is growing public and political pressure to reduce reliance on fossil fuels such as oil, coal and gas, to develop new fuels that do not cause climate change, and to build a sustainable, non-polluting industrial and biotechnological base. The overall long-term goal of the project is to develop a 'systems understanding' of P. putida, i.e. an understanding of its activities at the multiple levels (gene, protein and metabolites), and how these are integrated and controlled. This understanding will provide a quantum increase in performance of this microorganism in diverse biotechnological applications through knowledge-based interventions. These applications will include the production of enzymes for diverse uses: industrial products, food, body-care products, medical treatments and diagnostics, production of biopolymers (e.g. biodegradable plastics), plant protection and growth promotion, and bioremediation of polluted environments. Many of these processes occur under conditions that are sub-optimal for the function of P. putida, for example it is likely to be stressed by high concentrations of solutes (substances that dissolve in the watery environment of this bacterium). Our primary goal is to understand how P. putida is affected by different classes of solute and in turn how it responds, with a view to optimizing its performance. Solutes fall into several different classes: charged (e.g. common salt), uncharged (e.g. glucose), chaotropic (those that cause chaos in biological molecules and membranes, e.g. ethanol and phenol), kosmotropic (order-forming, e.g. polyethylene glycol), hydrophobic (water-hating, e.g. benzene), and many more. We will added these stressful solutes at inhibitory concentrations, and in collaboration with our scientific partners in other European countries investigate how the cell responds by examining gene expression, proteins produced, and various other stress-protection measures. One particular question that we will be addressing is whether and to what extent different classes of solute result in specific responses, and in turn whether there are general solute-stress response mechanisms. These studies will result in mathematically-designed models, created from our biological (experimental) data, that function via computer programs, and that will be used to predict how the cell will behave under different conditions, and we aim to put these models to the test. Ultimately these models will help us to predict how bacterial function can be more efficient under biotechnologically-important conditions. Many biotechnological processes involve a mixture of solutes, and we have observed that the stressful effects of different classes of solutes impact on each other. This is sometimes beneficial in that one solute offsets the deleterious effects of another (e.g. kosmotropes and chatotropes), but the mixtures can sometimes be antagonistic. Similarly, the physical environment (e.g. temperature and pressure) will alter the effect that solutes have on bacteria, and how they respond. We have designed a variety of experiments that will test these issues by examining the effects of mixtures on the growth rate of P. putida and its response mechanisms as outlined above.This research, as well as the model that will be produced, has the potential to improve all types of biotechnological and industrial processes in which bacterial cells are invloved, and represents the first research of its kind in this area.

We aim to develop the knowledge base, material and computational resources to establish the soil bacterium Pseudomonas putida as the vehicle for implementing biological activities into a range of industrially-related processes. All biotechnological reactions occur in the presence of solutes and dissolved organic solvents, many of which prevent optimal performance; yet there is currently no rational basis for predicting their metabolic impact either singly or in mixtures. The overall goal of the Essex/Queen's partnership is to understand how P. putida is affected by different classes of solute (polar, ionic, chaotropic, kosmotropic, organic, inorganic, hydrophobic), and how it responds at multiple levels (transcriptome, proteome, stress protectants, membrane lipids, metabolome) in order to optimise P. putida performance as a cell factory. We will employ a range of stressors/ stressor mixtures using complementary shake-flask and chemostat experiments and investigate the extent to which solute activities can have protective or antagonistic effects on the cell. Similarly, we will determine how factors that reduce entropy (high pressure, low temperature, specific solutes) alter the effects of solutes that increase (chaotrope) or decrease (kosmotrope) entropy, and the corresponding cellular responses. All relevant 'omics and other data generated will be used by partners to model responses of the cellular system; this model in turn will feed back into our experimental design. We will test the potency of the P. putida model using mutant strains, cellular protectants, untested stressors, and other techniques. This research and the resulting P. putida model will be used to improve all types of microbially driven industrial processes, e.g. by rational use of solutes/ environmental conditions, manipulation of phenotypic plasticity, and/ or rational design of mutant strains, and represents the first research of its kind in this area.