Does Composition of the Exopolysaccharide Matrix of Pseudomonas Putida Control Biofilm Architecture and Fitness In Low-water-content Environments?

智力优势：土壤和其他不饱和环境中的细菌，如肺或植物的空气表面，通常以细胞聚集体（生物膜）的形式生活在部分由它们自己制造的细胞外多糖（EPS）组成的基质中。尽管人们普遍认为，包括水生生物膜群落在内的许多环境中的细菌都生活在EPS基质中，但对EPS层的一般功能甚至基质中特定多糖成分的功能知之甚少。一种经常被讨论但相对较少研究的可能性是，EPS包膜可以保护细菌免受干燥，因此在低含水量栖息地中起着适合的作用。土壤微生物介导了许多重要的陆地生态系统过程，包括全球生物地球化学循环、有机污染物的降解以及与植物的有益和有害相互作用，但我们对水的有效性如何影响生物膜的发育和代谢活动以及群落成员的生存仍然知之甚少。该项目的目标是了解细菌在土壤中定植的过程，以及环境如何影响细菌的适应性，包括将环境线索整合到参与适应这些压力的调节网络中的机制。作为这个项目框架的中心假设是，水对细菌的可用性是影响细菌生理、生长和生存的主要力量。将被测试的假设是，EPS基质组成的调节是一个由脱水压力驱动的活跃过程，特定的EPS成分保持大量的水，从而创造一个微环境，减缓生物膜干燥的速度，通过增加代谢调节的时间来增加细菌的存活率。此外，胁迫介导的EPS基质调节改变了生物膜的发育过程和结构，最终影响了生物膜的生物物理特性和群落成员的代谢能力。该项目的目标是：1、鉴定参与细胞外多糖生物合成的基因，评估其调控并灭活它们；2、评估细胞外多糖是否创造了一个更水合的微环境，保护生物膜细胞免受干燥胁迫；3 .评估低含水量生境中EPS对生物膜发育模式和结构的影响。更广泛的影响：这项研究的一个重要组成部分是填补了我们对陆地生态系统中生物膜生物学的理解上的一个主要空白，这一空白通常依赖于从实验系统中获得的信息，而这些信息往往不能反映土壤条件。这个项目将提供多种机会，使研究训练与教学相结合，并促进以探究为基础的学习策略。学生将面临的挑战是确定这个项目与调控等级和信号转导途径的联系，以及这些生长形式对陆地生态系统过程的更广泛影响。信息将通过出版物和会议上的介绍以及一个网站来传播，该网站将包含未以出版物形式提供的不饱和生物膜的发展和特性的图像和描述。此外，实验结果和实验方法将被纳入微生物学本科课程，并纳入PI目前正在开发的新讲座和实验课程。

Intellectual Merit: Bacteria in soil and other unsaturated habitats, such as lungs or aerial surfaces of plants, generally live as aggregates of cells (biofilms) within a matrix comprised, in part, of extracellular polysaccharides (EPS) of their own making. Although there is general agreement that bacteria in many environments, including aquatic biofilm communities, live within an EPS matrix, relatively little is known about the function of the EPS layer in general or even specific polysaccharide components of the matrix. One possibility that has been often discussed but has been the subject of relatively few studies is that an EPS envelope may protect bacteria from drying, thus functioning as a fitness trait in low water content habitats. Soil microorganisms mediate many critical terrestrial ecosystem processes, including global biogeochemical cycles, the degradation of organic pollutants, and beneficial and detrimental interactions with plants, yet we still have a poor understanding of how water availability influences biofilm development and metabolic activities and the survival of community members. The goal of this project is to understand the processes involved in bacterial colonization of soil and how the environment influences bacterial fitness, including the mechanisms by which environmental cues are integrated into the regulatory networks involved in adaptation to those stresses. The central hypothesis that serves as a framework for this project is that the availability of water to bacteria is a major force influencing bacterial physiology, growth, and survival. The hypotheses that will be tested are that modulation of the composition of the EPS matrix is an active process that is driven by dehydration stress and that specific EPS constituents hold substantial amounts of water thereby creating a microenvironment that slows the rate of biofilm drying, which increases bacterial survival by increasing the time for metabolic adjustment. Furthermore, stress-mediated modulation of the EPS matrix alters biofilm developmental processes and architecture, which ultimately influences the biophysical properties of the biofilm and the metabolic capabilities of community members. The objectives of the project are to: 1, identify genes involved in extracellular polysaccharide biosynthesis, assess their regulation and inactivate them; 2, assess whether extracellular polysaccharides create a more hydrated microenvironment that protects biofilm cells from desiccation stress; and 3, assess the role of EPS on biofilm developmental patterns and architecture in low water content habitats.Broader impacts: An important component of this research is to fill a major gap in our understanding of biofilm biology in terrestrial ecosystems, which has had to typically rely on information obtained from experimental systems that frequently don't reflect soil conditions. This project will provide multiple opportunities for research training coupled to instruction and to promote inquiry based learning strategies. Students will be challenged to identify linkages of this project to regulatory hierarchies and signal transduction pathways and to the broader implications of these growth forms on terrestrial ecosystem processes. Information will be disseminated through publications and presentations at meetings and through a website that will contain images and descriptions of unsaturated biofilm development and properties that are not available in publication format. Furthermore, the results and experimental methodologies will be incorporated into the undergraduate microbiology curriculum into new lecture and laboratory courses the PI is currently developing.