Solute Stress Mechanisms and Responses

恶臭假单胞菌（简称P. putida）是一种最初从土壤中分离出来的细菌，土壤是一个充满压力和不断变化的环境;因此，它具有能够对其环境变化做出反应的内在能力。这种细菌是非致病性的，并且由于其能够产生对人类有用的多种化合物，并且能够降解对环境和人类健康有害的多种污染物，因此它一直是深入科学研究的对象。公众和政治压力越来越大，要求减少对石油、煤炭和天然气等化石燃料的依赖，开发不会造成气候变化的新燃料，并建立可持续、无污染的工业和生物技术基础。该项目的总体长期目标是发展对恶臭假单胞菌的“系统理解”，即了解其在多个层面（基因、蛋白质和代谢物）的活动，以及如何整合和控制这些活动。这种理解将通过以知识为基础的干预措施，在不同的生物技术应用中提供这种微生物的性能的量子增加。这些应用将包括生产各种用途的酶：工业产品、食品、身体护理产品、医疗和诊断、生产生物聚合物（例如生物降解塑料）、植物保护和促进生长以及污染环境的生物修复。许多这些过程发生在恶臭假单胞菌功能的次优条件下，例如，它可能受到高浓度溶质（溶解在该细菌的水环境中的物质）的胁迫。我们的主要目标是了解恶臭假单胞菌如何受到不同种类溶质的影响，以及它如何响应，以优化其性能。溶质分为几个不同的类别：带电（如食盐），不带电（如葡萄糖），离液（那些导致生物分子和膜混乱，如乙醇和苯酚），亲液（有序形成，如聚乙二醇），疏水（憎水，如苯），等等。我们将在抑制浓度下添加这些应激溶质，并与我们在其他欧洲国家的科学合作伙伴合作，通过检查基因表达、产生的蛋白质和各种其他应激保护措施来研究细胞的反应。我们将讨论的一个特殊问题是，不同种类的溶质是否以及在多大程度上导致特定的反应，以及是否存在一般的溶质应激反应机制。这些研究将导致从我们的生物（实验）数据创建的自动设计的模型，通过计算机程序发挥作用，并将用于预测细胞在不同条件下的行为，我们的目标是测试这些模型。最终，这些模型将帮助我们预测细菌功能如何在生物技术重要条件下更有效。许多生物技术过程涉及溶质的混合物，并且我们已经观察到不同种类的溶质的应激效应相互影响。这有时是有益的，因为一种溶质抵消了另一种溶质（例如亲液剂和促变色剂）的有害影响，但混合物有时可能是拮抗性的。同样，物理环境（例如温度和压力）会改变溶质对细菌的影响以及它们的反应。我们已经设计了各种实验，通过检查混合物对恶臭假单胞菌生长速率的影响及其上述反应机制来测试这些问题。这项研究以及将产生的模型，有可能改善涉及细菌细胞的所有类型的生物技术和工业过程，并代表了该领域的第一个同类研究。

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.