Pseudomonas aeruginosa sensing and response

描述(申请人提供)：这项申请是为了探索在条件致病菌铜绿假单胞菌中发现的趋化性基因簇II在附着和生物膜形成中的功能。铜绿假单胞菌有多组趋化基因簇排列。鞭毛介导的趋化作用绝对需要I/V簇中的基因。第IV簇中的那些是由毛发介导的运动和反应所必需的。越来越多的证据表明，细菌中的一些趋化性基因可能不主要参与趋化性或运动性。铜绿假单胞菌第二簇基因似乎就是这种情况。类CHE和MCP基因参与了细胞表面的附着和生物膜的形成。它们似乎在鞭毛介导的趋化作用中最多只起到很小的作用。簇II突变体在菌毛介导的抽动运动或在固体表面蜂拥而至方面没有缺陷。这项拟议工作的长期目标是确定推测的簇II信号转导复合体引导细胞附着到表面并启动生物膜形成的机制。在特定的目标中，铜绿假单胞菌基因芯片将被用来识别被类CHE蛋白II转录激活或抑制的基因集。其他实验将探索在生长的静止阶段，簇II信号复合体在直接调节细胞表面分子机活动方面发挥主要作用的可能性。还将研究簇II蛋白通过直接物理接触调节其他蛋白活性的可能性。在特定的目的中，2个被鉴定为受簇II信号转导调控的基因或编码其活性受簇II蛋白调控的蛋白的基因将被突变失活。将对突变体的附着和生物膜形成表型进行分析。将使用互补的生理学方法来探索编码蛋白质的活性如何影响生物膜的形成。簇II基因在生长的稳定期以依赖于细胞密度的方式被诱导。具体目标3将探索这样的假设，即簇II基因和某些emcp基因受控制铜绿假单胞菌从指数到稳定期生长转变的全球调控因子的协调调控。簇II蛋白相互作用形成信号转导复合体的假设将在特定的目的中得到验证4.将进行磷酸转移试验，并将确定簇II蛋白对其他绿色荧光标记簇II蛋白的亚细胞定位的影响。铜绿假单胞菌在囊性纤维化患者的肺部以生物膜的形式生长，在医疗器械留置引起的感染中以生物膜的形式生长。这项拟议的工作将揭示原核生物生物膜发育所需的蛋白质和基因。

DESCRIPTION (provided by applicant): This application is to explore the function in attachment and biofilm formation of cluster II chemotaxis-like genes found in the opportunistic human pathogen Pseudomonas aeruginosa. P. aeruginosa has multiple sets of chemotaxis-like genes arranged in clusters. Genes in clusters I/V are absolutely required for flagella-mediated chemotaxis. Those in cluster IV are required for pilus-mediated motility and response. Accumulating evidence suggests that some chemotaxis-like genes from bacteria may not be primarily involved in chemotaxis or motility. Such appears to be the case for P. aeruginosa cluster II genes. Cluster II che-like and mcp-like genes are involved in attachment of cells to surfaces and biofilm formation. They appear to play, at most, a minor role in flagella-mediated chemotaxis. Cluster II mutants are not defective in pilus-mediated twitching motility or in swarming across solid surfaces. The long-term objective of the proposed work is to determine the mechanism by which a presumed cluster II signal transduction complex directs cells to attach to surfaces and initiate biofilm formation. In specific aim 1 P. aeruginosa gene chips will be used to identify sets of genes that are transcriptionally activated or repressed by cluster II che-like proteins. Other experiments will explore the possibility that a cluster II signaling complex has a major role in directly modulating the activity of a cell surface molecular machine during the stationary phase of growth. The possibility that cluster II proteins modulate the activities of other proteins through direct physical contact will also be examined. In specific aim 2 genes identified as being regulated by cluster II signal transduction or genes encoding proteins whose activities are modulated by cluster II proteins, will be inactivated by mutation. The attachment and biofilm formation phenotypes of the mutants will be analyzed. Complementary physiological approaches will be used to explore how the activities of the encoded proteins may affect biofilm formation. Cluster II genes are induced in the stationary phase of growth in a cell-density dependant manner. Specific aim 3 will explore the hypothesis that cluster II genes and somemcp genes are coordinately regulated by global regulators that control the exponential-to-stationary phase growth transition in P. aeruginosa. The hypothesis that cluster II proteins interact to form a signal transduction complex will be tested in specific aim 4. Phospho-transfer assays will be carried out and effects of cluster II proteins on the subcellular localization of other green fluorescent tagged cluster II proteins will be determined. Pseudomonas aeruginosa grows as a biofilm in the lungs of cystic fibrosis patients and it grows as a biofilm in infections caused by indwelling medical devices. The proposed work will reveal proteins and genes that are required for biofilm development by prokaryotes.