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Genetic analysis of metyl-accepting chemotaxis protein family in Pseudomonas aeruginosa.

铜绿假单胞菌甲基接受趋化蛋白家族的遗传分析。

基本信息

批准号：
12660079
负责人：
KATO Junichi
金额：
$ 0.58万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

The methyl-accepting chemotaxis proteins (MCPs) from phylogenetically diverse bacteria have been shown to possess the highly conserved domain (HCD) which is likely to be important for intracellular chemotactic signalling. Based on the conserved 44-amino acid sequence, computer analysis of the P. aeruginosa PAO1 genome sequence predicted that PAO1 possesses 26 open reading frames (ORFs) which likely encode proteins containing HCD. Twenty six ORFs were individually amplified by PCR and cloned into pGEM-Teasy. Individual genes were then disrupted by inserting a kan cassette into the wild-type genes in the PAO1 genome, and the phenotype of kanamycin resistant mutants and PAO1 were analyzed. Chemotaxis assays revealed that pctA, pctB, and pctC were involved in chemotctic responses to ammo acids. The ctpH was required for exhibiting phopshate taxis at high concentrations of inorganic phosphate (P_i), while the gene product of ctpL served as the major chemoreceptor for P_1 at low concentration. The mcp96-3 disruptant failed to respond to O_2, suggesting that MCP96-3 is involved in the detection of C_2. We found that PAO1 was attracted by 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenoxyacetic acid, and phenoxyacetic acid and repelled by volatile chlorinated aliphatic compounds including trichloroethylene, tetrachloroethylene, trichloroethane, and chloroform. All of the disruptants exhibited normal chemotaxis toward these compounds. These results suggest that each of these compounds is detected by more than two MCPs. PAO1 did not grow on LB broth in the presence of 10% (v/v) toluene. The pilJ mutant showed the sparse growth on LB containing toluene, however, this disruptant emulsified toluene. The pilJ mutant was likely to produce significant amount of biosurfactant, rhamnolipid in the culture condition. The supernatant of the pilJ mutant showed five times more elastase activity detected with PAO1. These results suggest the pilJ gene is involved in quorum sensing.

来自系统发育多样化细菌的甲基接受趋化蛋白（MCPs）已被证明具有高度保守结构域（HCD），这可能对细胞内趋化信号传导很重要。基于保守的44个氨基酸序列，计算机分析了P. aeruginosa PAO1基因组序列，预测PAO1具有26个开放阅读框（orf），可能编码含有HCD的蛋白质。通过PCR扩增出26个orf，并克隆到pGEM-Teasy中。然后通过在PAO1基因组的野生型基因中插入kan盒来破坏单个基因，并分析卡那霉素抗性突变体和PAO1的表型。趋化性实验显示pctA、pctB和pctC参与了对氨基酸的趋化反应。在高浓度无机磷酸盐（P_i）下，ctpH是表现出磷酸盐趋近性所必需的，而在低浓度下，ctpL基因产物是P_1的主要化学受体。mcp96-3干扰物对O_2没有反应，提示mcp96-3参与了C_2的检测。我们发现PAO1被2,4-二氯苯氧乙酸、2,4,6-三氯苯氧乙酸和苯氧乙酸吸引，被挥发性氯代脂肪化合物（包括三氯乙烯、四氯乙烯、三氯乙烷和氯仿）排斥。所有的干扰物对这些化合物都表现出正常的趋化性。这些结果表明，这些化合物中的每一种都可以被两个以上的mcp检测到。在10% （v/v）甲苯的条件下，PAO1在LB肉汤中不生长。pilJ突变体在含有甲苯的LB上生长稀疏，而这种干扰物使甲苯乳化。在培养条件下，pilJ突变体可能产生大量的生物表面活性剂鼠李糖脂。pilJ突变体的上清液显示用PAO1检测的弹性酶活性高出5倍。这些结果表明pilJ基因参与群体感应。