Temporal and spatial expression of regulators affecting Escherichia coli biofilm

描述(由申请人提供)：该项目的长期目标是确定有效预防和治疗生物膜相关感染的目标。我们选择了一种方法，在这种方法中，我们对生物膜形成背后的细菌生理的了解的增加将导致提高操纵细菌的能力。以肠道细菌大肠杆菌为模式生物，对影响生物膜相关细胞表面细胞器的部分转录调控网络进行了综述。这项建议侧重于这一网络的一部分，包括鞭毛状和全球调节复合体FlhD/FlhC和几个双组分系统，如EnvZ/OmpR和RcsCDB。与醋酸盐代谢有关的代谢信号分子也包括在内。通过这些分子的信号通路在上游通路中是平行的，在下游通路中是相交的。因此，多种环境信号被整合到一个最终结果--生物膜的形成。 目前的大多数研究都是在一个时间点上研究整个生物膜的基因表达。这并没有承认生物膜经历了一个发展过程，即使是完全发育成熟的生物膜也由几个不同的生态位组成。我们提出的研究旨在确定生物膜形成过程中基因表达的时间和空间分布。我们预计，这将是我们对生物膜形成的环境控制的理解的重大突破。作为翻译方面，拟议的研究结果将有助于开发新型接触活性化合物，这些化合物将被集成到基于有机硅的抗菌涂层和表面中，该项目由南加州大学纳米科学与工程中心的合作者完成。生物膜形成所需的基因和在生物膜发育早期表达的基因将对这一有趣的应用特别感兴趣。 具体目标的基础是一组定量的生物膜数据，这些数据是在多种环境条件组合下用许多大肠杆菌突变体获得的。利用flhd、rcsb、ompR突变株和醋酸酯代谢酶基因之间生物膜相关生物量差异最大的环境条件，将使用固定生物膜和扫描电子显微镜以及连续生物膜和共聚焦显微镜对生物膜的形成进行定性和定量评估。这将构成具体的目标一。将包括突变的组合，以建立调控效应之间的等级关系。在第二个特定目标中，来自上述一些基因的启动子将与编码荧光蛋白的基因融合，并将在生物膜中研究启动子的时间和空间表达。这将在野生型细菌和一些突变体中完成。通过对共聚焦显微镜图像中的生物膜进行量化，定性数据将与环境筛选实验相关。

DESCRIPTION (provided by applicant): The long-term goal of the project is to identify targets for the effective prevention and treatment of biofilm-associated infection. We have selected an approach where an increase in our understanding of the bacterial physiology that underlies biofilm formation will lead to an improved ability to manipulate the bacteria. Using the enteric bacterium Escherichia coli as a model organism, a partial network of transcriptional regulation affecting biofilm-associated cell surface organelles was previously summarized. This proposal focuses on a portion of this network, including the flagellar and global regulator complex FlhD/FlhC and several two-component systems, such as EnvZ/OmpR and RcsCDB. Metabolic signaling molecules relating to acetate metabolism are included. The signaling pathways through these molecules are parallel in the upstream pathways and intersecting in the downstream ones. As a consequence, multiple environmental signals are integrated to one final outcome, the formation of biofilms. Most current studies investigate gene expression of the entire biofilm at one time point. This does not acknowledge that biofilms undergo a developmental process and that even the fully developed, mature biofilm consists of several distinct ecological niches. Our proposed research aims at determining the temporal and spatial distribution of gene expression during biofilm formation. We anticipate that this will be a major breakthrough in our understanding of the environmental control of biofilm formation. As a translational aspect, results from the proposed research will aid in the development of novel contact-active compounds that will be integrated into silicone based antimicrobial coatings and surfaces, a project that is done by collaborators at the Center for Nanoscale Science and Engineering at NDSU. Genes that are required for biofilm formation and expressed early in biofilm development will be of particular interest for this intriguing application. The basis for the Specific Aims is a data set of quantitative biofilm data that was obtained with many E. coli mutants under multiple combinations of environmental conditions. Using the environmental conditions that gave the largest differences in biofilm associated biomass between mutants in flhD, rcsB, ompR, and genes encoding enzymes of acetate metabolism, qualitative and quantitative assessment of biofilm formation will be performed with stationary biofilms and scanning electron microscopy, as well as continuous biofilms and confocal microscopy. This will constitute Specific Aim I. Combinations of mutations will be included to establish hierarchies among regulatory effects. In a second Specific Aim, promoters from some of the above genes will be fused to genes encoding fluorescence proteins and the temporal and spatial expression from the promoters will be investigated within the biofilms. This will be done in wild type bacteria and a selection of the mutants. The qualitative data will be related back to the environmental screening experiment by quantifying biofilms from the confocal microscopy images.