Studies on molecular mechanism of chemotaxis toward inorganic phosphate in baeteria.

细菌对无机磷酸盐趋化的分子机制研究。

• 批准号: 10660089
• 负责人: KATO Junichi
• 金额: $ 2.24万
• 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: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660089/
• 关键词: environmental response system; phopshate taxis; phosphate starvation; chemotaxis signal transduction; Pseudomonas aeruginosa; Enterobacter cloacae; Enterobacter cloacae; リン酸飢餓応答; 走化性トランスデューサー; リン酸特異的膜輸送系; Enterbacter cloacae; Psendomonas aerugi

Two chemotactic transducers for inorganic phosphate (PィイD2iィエD2) designated CtpH and CtpL, have been identified in Pseudomonas aeruginosa. The corresponding genes (ctpH and ctpL) were inactivated by inserting kanamycin and tetracycline resistance gene cassettes into the wild-type genes in the P. aeruginosa PAO1 genome. The computer-assisted capillary assays showed that the ctpH single mutant failed to exhibit PィイD2iィエD2 taxis when the concentration of PィイD2iィエD2 in the capillary was higher than 5 mM. Conversely, the ctpL single mutant could not respond to PィイD2iィエD2 at the concentration of 0.01 mM. Like PAO1, these single mutants were induced by PィイD2iィエD2 limitation for PィイD2iィエD2 taxis. The ctpH ctpL double mutant was defective in PィイD2iィエD2 taxis at any concentration ranging from 0.01 to 10 mM. To investigate regulation of PィイD2iィエD2 taxis, the ctpH and ctpL genes were also disrupted individually in the P aeruginosa phoU and phoB single mutants. The ctpH phoU and crpH phoB double mu … More tants were defective in PィイD2iィエD2 taxis, regardless of whether the cells were starved for PィイD2iィエD2' The crpL phoU and ctpL phoB double mutants exhibited PィイD2iィエD2 taxis at PィイD2iィエD2 concentrations higher than 5 mM. In addition, the ctpL phoU double mutant was constitutive for PィイD2iィエD2 taxis, whereas the ctpL phoB double mutant was induced by PィイD2iィエD2 limitation for PィイD2iィエD2 taxis. The region upstream of ctpL, but not ctpH, contained a putative pho box sequence. Expression of ctpL: :lacZ was induced by PィイD2iィエD2 limitation in PAO1, while it was constitutive in the phoU mutant. In contrast, the phoB mutant showed only background levels of atpL: : lacZ expression. These results showed that ctpL is involved in the pho regulon genes in P aeruginosa and, therefore, could explain that the ctpH phoB mutant was defective in PィイD2iィエD2 taxis. Furthermore, the fact that the ctpH phoU mutant failed to exhibit PィイD2iィエD2 taxis despite inducible expression of ctpL suggested that the PィイD2iィエD2 detection by CtpL requires PhoU. On the other hand, like PAO1 , the phoB and phoU single mutants were constitutive for expression of ctpH: :lacZ. Thus, the evidence that the ctpL pho U mutant but not the ctpL phoB mutant and PAO1 , was constitutive for PィイD2iィエD2 taxis raised the possibility that PhoU exerts a negative control on the PィイD2iィエD2 detection by CtpH at posttranscriptional level.A chemotaxis-defective mutants of Enterobacter cloacae IFO3320, designated EC1, was isolated after N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Computer-assisted capillary assays revealed that they failed to show chemotactic responses to peptone and PィイD2iィエD2 Cloning and sequence analysis showed that both of EC1 are a cheR mutant. These results suggest that PィイD2iィエD2 taxis by E. cloacae is dependent on methyl-accepting chemotaxis proteins (MCPs). EC1 was further mutagenized with NTG to isolate cheR pstS and cheR pstA double mutants, designated ECAP1 and ECAP2, respectively. pECT01.1 containing the E. cloacae cheR gene restored the ability of both double mutants to exhibit chemotactic responses toward peptone but not toward PィイD2iィエD2' These results confirm that both of CheR and Pst are simultaneously required for PィイD2iィエD2 taxis in E. cloacae. Less

在铜绿假单胞菌中发现了两种无机磷酸盐（P φ φ D2i φ φ D2）的趋化传感器CtpH和CtpL。通过将卡那霉素和四环素耐药基因盒插入铜绿假单胞菌PAO1基因组的野生型基因中，灭活相应的ctpH和ctpL基因。计算机辅助毛细管化验表明,ctpH单一突变未能表现出PィイD2iィエD2出租车当P的浓度ィイD2iィエD2毛细管高于5毫米。相反,ctpL单一突变不能应对PィイD2iィエD2在0.01毫米的浓度。像PAO1,这些单突变体诱导PィイD2iィエD2限制PィイD2iィエD2出租车。的ctpH ctpL双突变体缺陷在PィイD2iィエD2出租车在任何浓度从0.01到10毫米。调查监管PィイD2iィエD2出租车,ctpH和ctpL基因也破坏了分别在P绿脓杆菌phoU和phoB单一突变体。的ctpH phoU和crpH phoBμ的两倍 ... 更经常被有缺陷的PィイD2iィエD2出租车,无论这些细胞被渴望PィイD2iィエD2”的crpL phoU和ctpL phoB双突变体表现出PィイD2iィエD2出租车在PィイD2iィエD2浓度高于5毫米。此外,在ctpL phoU双突变体本构为PィイD2iィエD2出租车,而ctpL phoB双突变体诱导了PィイD2iィエD2限制PィイD2iィエD2出租车。ctpL上游的区域，而不是ctpH，包含一个假定的phobox序列。ctpL::lacZ的表达在PAO1中受P γ γ γ γ的限制，而在phoU突变体中则是组成型表达。相比之下，phoB突变体只显示背景水平的atpL:: lacZ表达。这些结果表明ctpL参与越南河粉调节子在P绿脓杆菌基因,因此,可以解释ctpH phoB突变缺陷在PィイD2iィエD2出租车。此外，ctpH phoU突变体尽管诱导表达了ctpL，但未能表现出P′′γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ。另一方面，与PAO1一样，phoB和phoU单突变体是ctpH::lacZ表达的组成部分。因此，ctpL pho U突变体，而不是ctpL phoB突变体和PAO1，构成了p220的趋近性，这一证据提高了PhoU在转录后水平上对CtpH检测p220的可能性。用n -甲基-n′-硝基-n -亚硝基胍（NTG）处理阴沟肠杆菌IFO3320后，分离到一株趋化缺陷突变体EC1。计算机辅助毛细管化验显示,他们未能显示趋化反应蛋白胨和PィイD2iィエD2克隆和序列分析表明,这两个EC1雪儿突变。这些结果表明，阴沟肠杆菌对P的趋化作用依赖于甲基接受趋化蛋白（MCPs）。进一步用NTG诱变EC1，分离出cheR pstS和cheR pstA双突变体，分别命名为ECAP1和ECAP2。pECT01.1含有大肠泄殖腔雪儿基因恢复两双突变体的能力表现出趋药性的反应对蛋白胨但不是对PィイD2iィエD2的这些结果同时证实,雪儿和Pst所需PィイD2iィエD2大肠泄殖腔的出租车。少

项目成果

Hong Wo, et al.: "Identification and characterization of two chemotactic transducers for inorganic phosphate in Pseudomonas aeruginosa."Journal of Bioteriology. (in press).

Hong Wo 等人：“铜绿假单胞菌中无机磷酸盐的两种趋化传感器的鉴定和表征。”生物技术杂志。

Junichi Kato, et al.: "Cloning and characterization of chemotaxis genes in Pseudomonas aeruginosa."Bioscience, Biotechnology, and Biochemistry. Vol. 63. 155-161 (1999)

Junichi Kato 等人：“铜绿假单胞菌趋化基因的克隆和表征。”生物科学、生物技术和生物化学。

Junichi Kato: "Cloning and characterization of chemitaxis genes in Pseudomondo earuginosa"Biochemica Biotechnology,and Biochemistry. 63. 155-161 (1999)

Junichi Kato：“Pseudomondoearuginosa 趋化基因的克隆和表征”Biochemica 生物技术和生物化学。

H.Wu: "Cloning and characterization of Pseudomonas putida genes encoding the posphate-specific transport system" J.Biosci.Biotechnol.in press.

H.Wu：“编码磷酸盐特异性转运系统的恶臭假单胞菌基因的克隆和表征”J.Biosci.Biotechnol.in press。

H. Wo.: "Indentification and characterization of two chemotactic transducers for inorganic phosphate in Pseudomonas aeruginosa"Journal of Bacteriology. (印刷中). (2000)

H. Wo.：“铜绿假单胞菌中两种无机磷酸盐趋化传感器的鉴定和表征”细菌学杂志（2000 年出版）。