Studies on the transcriptional regulatory system for sensing of environmental pollutants

环境污染物传感转录调控系统的研究

• 批准号: 12460043
• 负责人: FUKUDA Masao
• 金额: $ 7.3万
• 依托单位: Nagaoka University of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460043/
• 关键词: environmental pollution; sensor; polychlorinated biphenyl; transcriptional regulation; two component system; biphenyl; Rhodococcus; Rhodococcus

We characterized the gene regulation system by BphS1/BphT1 and BphS2/T2, which respond to the aromatic compounds and activate the transcription of biphenyl degradation genes in Rhodococcus sp. strain RHA1.1. The substrate specificities of BphS1 and BphS2, which have high similarities each other, were investigated. Both BphSs responded to a wide range of aromatic compounds. The substrate specificities of them were alike, except for the case of biphenyl. Towards biphenyl, only BphS1 responded. The construction and analysis of BphS1-BphS2 hybrid sensor kinase revealed that the N terminus region of BphS (to amino acid number 405) is responsible for the response towards biphenyl.2. The signal transduction system between BphS and BphT were investigated. Mutants of both BphSs were constructed by the substitution of His 1411 to Arg. His 1411 is a amino acid residue which corresponds to the conserved His which act as phosporylation site among many sensor kinases. Both mutants were expressed as soluble proteins, but lost their activities, which showed their importance in the phospho-relay to the BphTs. We also observed the cross-talk between BphS1 and BphT2, and BphS2 and BphT1, respectively.3. The upregulation activity of BphSTs towards various promoters of biphenyl degradation genes (etbA1, ebdA1, etbD1, and bphA4-2) were investigated. The results showed that all the promoters mentioned above were regulated by BphST, and this suggested that these promoters have consensus sequences for BphT binding. The transcriptional start points of these promoters were determined by the primer extension analysis, and the results revealed a consensus motif that exists around -35 of each transcriptional start point. The overexpression and the purification of BphT protein were also performed by using E. coli as a host.

我们通过BphS1/BphT1和BphS2/T2对Rhodococcus sp. RHA1.1中的芳香族化合物作出反应并激活联苯降解基因的转录，对基因调控系统进行了表征。研究了具有高度相似性的BphS1和BphS2的底物特异性。这两种BphSs都对多种芳香族化合物有反应。除联苯外，它们的底物特异性相似。对于联苯，只有BphS1有反应。BphS1-BphS2杂交传感器激酶的构建和分析表明，bphs1的N端区域（氨基酸号为405）负责对联苯2的响应。研究了BphS与BphT之间的信号转导系统。通过将His 1411替换为Arg，构建了两种BphSs的突变体。His 1411是一个氨基酸残基，与保守的His相对应，在许多传感器激酶中起磷酸化作用。这两个突变体都以可溶性蛋白的形式表达，但失去了活性，这表明它们在磷酸化传递到bpht的过程中很重要。我们还观察到了BphS1与BphT2、BphS2与BphT1之间的串扰。研究了BphSTs对联苯降解基因（etbA1、ebdA1、etbD1和bphA4-2）启动子的上调活性。结果表明，上述启动子均受BphST调控，说明这些启动子具有BphT结合的一致序列。通过引物延伸分析确定了这些启动子的转录起始点，结果显示每个转录起始点的-35处存在一个一致的基序。以大肠杆菌为宿主进行了BphT蛋白的过表达和纯化。