Pseudomonas aeruginosa sensing and response

描述（由申请人提供）：本申请旨在探索在机会性人类病原体铜绿假单胞菌中发现的簇II趋化性样基因在附着和生物膜形成中的功能。铜绿假单胞菌具有多组成簇排列的趋化性样基因。簇I/V中的基因是鞭毛介导的趋化性所必需的。簇IV中的那些是菌毛介导的运动和反应所需的。越来越多的证据表明，一些趋化性类似基因的细菌可能并不主要参与趋化性或运动。这似乎是铜绿假单胞菌簇II基因的情况。簇II che样和mcp样基因参与细胞与表面的附着和生物膜的形成。他们似乎发挥，最多，在鞭毛介导的趋化性的一个小角色。簇II突变体是没有缺陷的菌毛介导的抽搐运动或在整个固体表面群集。拟议的工作的长期目标是确定的机制，其中一个假定的集群II信号转导复合物指导细胞附着到表面，并启动生物膜的形成。在具体目标1中，铜绿假单胞菌基因芯片将用于鉴定被簇II che样蛋白转录激活或抑制的基因组。其他实验将探讨的可能性，簇II信号复合物有一个主要的作用，直接调节细胞表面的分子机器的活动，在稳定期的增长。簇II蛋白质通过直接物理接触调节其他蛋白质活性的可能性也将被检查。在特定目的中，将通过突变使被鉴定为受簇II信号转导调节的基因或编码其活性受簇II蛋白调节的蛋白的基因失活。将分析突变体的附着和生物膜形成表型。互补的生理学方法将用于探索编码蛋白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细胞密度依赖性方式被诱导。具体目标3将探索以下假设：簇II基因和somemcp基因由控制铜绿假单胞菌中指数生长期到稳定期生长转变的全局调节因子协调调节。簇II蛋白相互作用形成信号转导复合物的假设将在具体目标4中进行测试。将进行磷酸转移测定，并确定簇II蛋白对其他绿色荧光标记的簇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中作为生物膜生长，并且在由留置医疗器械引起的感染中作为生物膜生长。这项工作将揭示原核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.