Pseudomonas aeruginosa sensing and response

描述（由申请人提供）：本申请旨在探索机会性人类病原体铜绿假单胞菌中发现的簇II趋化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簇状趋化样基因。簇I/V中的基因对于鞭毛介导的趋化性是绝对必需的。IV类细胞是由菌毛介导的运动和反应所必需的。越来越多的证据表明，来自细菌的一些趋化性基因可能主要不参与趋化性或运动性。这似乎是铜绿假单胞菌簇II基因的情况。类簇基因和类簇基因参与细胞附着于表面和生物膜的形成。它们似乎最多在鞭毛介导的趋化性中起次要作用。簇II突变体在菌毛介导的抽搐运动或在固体表面上群集方面没有缺陷。这项工作的长期目标是确定一个假定的簇II信号转导复合物指导细胞附着在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用于鉴定被簇II类che蛋白转录激活或抑制的基因组。其他实验将探索簇II信号复合物在生长固定阶段直接调节细胞表面分子机器的活性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。在特定的目标中，被鉴定为受簇II信号转导调节的2个基因或编码活性受簇II蛋白调节的蛋白质的基因将因突变而失活。将分析突变体的附着和生物膜形成表型。补充的生理方法将用于探索如何编码蛋白质的活动可能影响生物膜的形成。簇II基因在生长的固定阶段以细胞密度依赖的方式被诱导。具体目标3将探讨假设集群II基因和一些emcp基因是由控制P. aeruginosa的指数阶段到平稳阶段的生长转变的全球调节协调调节。簇II蛋白相互作用形成信号转导复合物的假设将在特定目标4中进行验证。将进行磷酸化转移试验，并确定簇II蛋白对其他绿色荧光标记簇II蛋白亚细胞定位的影响。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，在留置医疗器械引起的感染中也以生物膜的形式生长。这项工作将揭示原核生物发育生物膜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.