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

来自不同系统发育的细菌的甲基接受趋化蛋白(MCPs)被证明具有高度保守的结构域(HCD)，这可能是细胞内趋化信号的重要组成部分。根据保守的44个氨基酸序列，计算机分析铜绿假单胞菌PAO1基因组序列，预测PAO1有26个开放阅读框(ORF)，可能编码含有HCD的蛋白质。通过聚合酶链式反应分别扩增出26个ORF，并将其克隆到pGEM-Tasy中。然后在PAO1基因组的野生型基因中插入一个Kan盒，打乱单个基因，并分析了卡那霉素抗性突变体和PAO1的表型。趋化实验表明PCTA、PCtB和PCTC参与了氨基酸的趋化反应。在高浓度的无机磷(P_I)中，CTPH是显示趋磷性所必需的，而在低浓度时，ctpL的基因产物是P_1的主要化学受体。2，4-二氯苯氧乙酸、2，4，6-三氯苯氧乙酸和苯氧乙酸对PAO1有吸引作用，三氯乙烯、四氯乙烯、三氯乙烷和氯仿等挥发性氯代脂肪族化合物对PAO1有排斥作用。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被两个以上的MCP检测到。在10%(v/v)的甲苯存在下，PAO1不能在LB液中生长。PilJ突变体在含有甲苯的乳酸菌上生长稀疏，但这种干扰物乳化了甲苯。该突变株在培养条件下可能产生大量的生物表面活性物质鼠李糖脂。用PAO1检测，该突变株的培养上清液中的弹性蛋白酶活性比PAO1高5倍。这些结果表明，PilJ基因参与了群体感应。

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.