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靶点。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学的理解的增加将导致操纵细菌的能力的提高。利用肠道细菌大肠杆菌作为模式生物，先前总结了影响生物膜相关细胞表面细胞器的部分转录调控网络。该提案侧重于该网络的一部分，包括鞭毛和全局调控复合物FlhD/FlhC和几个双组分系统，如EnvZ/OmpR和RcsCDB。包括与醋酸代谢有关的代谢信号分子。通过这些分子的信号通路在上游是平行的，在下游是交叉的。因此，多种环境信号被整合成一个最终结果，即生物膜的形成。

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. PUBLIC HEALTH RELEVANCE: Bacterial biofilms are aggregates of bacteria that embed themselves in an extracellular polymeric matrix and form on biologic or non-biologic surfaces. While more than 80% of all bacterial infections involve the formation of biofilms, the majority of the research to date has been performed on free swimming, planktonic bacteria. However, the physiology of biofilm- associated bacteria is much different from planktonic ones, which causes phenotypes such as the unusually high resistance to antibiotics and the host immune system. Understanding the underlying physiology of biofilm formation will permit the development of novel intervention techniques, inhibiting the formation of biofilms.