Plant Biofilm Inhibitors to Discover Biofilm Genes

描述(由申请人提供)：为了表征不影响细菌生长的无毒、有效的抗生物被膜剂，提出了一项研究计划，以调查自然产生的海洋呋喃酮和其他植物衍生化合物抑制大肠杆菌生物被膜形成的能力。此外，将利用时间DNA微阵列研究、基因敲除和已发现的生物膜调节蛋白的核磁共振结构测定来研究生物膜形成的遗传基础。该项目将通过使用DNA微阵列的两种互补方法来发现大肠杆菌生物膜形成所需的基因：直接鉴定生物膜基因和通过用植物化合物抑制生物膜。此外，这个项目将确定植物是如何衍生出来的 生物膜抑制剂影响生物膜，并将发现其他植物衍生化合物抑制生物膜。已发现的生物被膜基因将成为其他类别化合物的丰富靶标，这些化合物可能被用于防止工业和医药中的生物被膜。 该项目的具体目标是： 1.从红藻Delisea Pulchra合成天然呋喃酮类化合物，并用共聚焦显微镜和96孔快速分析法评估这些呋喃酮类化合物和其他植物衍生化合物防止生物膜形成的能力。 2.使用DNA微阵列评估植物来源的抑制剂对大肠杆菌基因表达的影响(确定哪些基因受到影响，而抑制剂不影响生长) 3.利用dna芯片发现生物被膜基因。 4.通过抑制与呋喃酮的通讯，利用dna微阵列发现细胞间的信号基因。 5.研究基因芯片阐明的基因在生物膜形成和形成方面的重要性。 利用绿色荧光蛋白标记的敲除突变体和标记生物膜调节蛋白启动子的体系结构 6.利用核磁共振技术确定重要生物膜调节蛋白的结构和功能。 7.推导了基于遗传信息(哪些基因)的细菌生物膜形成模型。 诱导/抑制)和蛋白质信息识别(通过核磁共振)

DESCRIPTION (provided by applicant): To characterize non-toxic, effective anti-biofilm agents that do not affect bacterial growth, a research program is proposed to investigate the ability of naturally-occurring marine furanones and other plant-derived compounds to inhibit the formation of E. coli biofilms. In addition, the genetic basis of biofilm formation will be investigated using temporal DNA microarray studies, knock-outs, and NMR structure determinations of the discovered biofilm regulatory proteins. This project will discover the genes required for E. coli biofilm formation by two complementary approaches using DNA microarrays: direct identification of biofilm genes and by inhibiting biofilms with plant compounds. In addition, this project will identify how the plant-derived biofilm inhibitors influence biofilms, and will discover other plant-derived compounds which inhibit biofilms. The biofilm genes that are found will be rich targets for other classes of compounds that may be used to prevent biofilms in industry and medicine. The specific objectives of this project are: 1. synthesize natural furanones from the red alga Delisea pulchra and evaluate these furanones and other plant-derived compounds for their ability to prevent biofilm formation using confocal microscopy and 96-well rapid assays 2. evaluate the impact of the plant-derived inhibitors on the gene expression of E. coli using DNA microarrays (identify which genes are affected while the inhibitor does not affect growth) 3. discover biofilm genes using DNA microarrays for E. coli (submerged biofilm) 4. discover cell-to-cell signaling genes using DNA microarrays by inhibiting communication with furanones 5. study the importance of the genes elucidated with the gene chips in terms of biofilm formation and architecture using knock-out mutants that are labeled with the green fluorescence protein and by tagging the promoters of biofilm regulatory proteins 6. determine the structure and thereby the function of the important biofilm regulatory proteins using NMR 7. deduce a model for bacterial biofilm formation based on the genetic information (which genes induced/repressed) and protein information discerned (via NMR)