Characterization of MobR, the 3-hydroxybenzoate-responsive transcriptional regulator for the 3-hydroxybenzoate hydroxylase gene of Comamonas testosteroni KH122-3s
Characterization of MobR, the 3-hydroxybenzoate-responsive transcriptional regulator for the 3-hydroxybenzoate hydroxylase gene of Comamonas testosteroni KH122-3s
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DOI：
10.1016/j.jmb.2006.08.098
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发表时间：
2006-12-15
影响因子：
5.6
通讯作者：
Fujiwara, Shinsuke
中科院分区：
文献类型：
--
作者：
Hiromoto, Takeshi;Matsue, Hanako;Fujiwara, Shinsuke
睾丸酮丛毛单胞菌KH 122 -3s是一种以3-羟基苯甲酸酯为唯一碳源和能源的好氧土壤细菌。在该菌株中，3-羟基苯甲酸羟化酶（MobA）作用于降解的初始步骤以产生3，4-二羟基苯甲酸酯，其随后经历间位裂解途径，导致三羧酸循环中间体。对mobA上游区域的基因步移分析揭示了一个开放阅读框（mobR），其编码马尔R家族的转录调节因子。在这里，我们报告说，MobR负调控的表达mobA，并通过结合3-羟基苯甲酸酯，底物MobA的抑制被解除。进行引物延伸实验以确定mobA的转录起始位点，并在mobA起始密码子上游83 bp处鉴定出它，伴随有典型的sigma（70）型启动子。在大肠杆菌中表达了mobR基因，并纯化了重组产物。凝胶迁移率变化分析和DNA酶I足迹分析表明，MobR作为同源二聚体结合到mobA-mobR基因间区域内的不完全反向重复序列，表观解离常数为11.5（+/- 0.5）nM。操作位点位于起始密码子和启动子区域之间的mobA，这表明MobR的功能作为一个转录阻遏物的mobA表达。效应器结合试验的结果表明，MobR，而不是其异构体4-羟基苯甲酸酯和水杨酸酯，是从操作网站通过添加3-羟基苯甲酸酯释放。这个解离过程是高度合作的，希尔系数近似于2。此外，CD光谱研究表明，MobR采用两种构象状态对应的effectorbound和unbound形式。这些结果表明，MobR二聚体具有至少两个效应器结合位点，并且与MobR结合的效应器诱导蛋白质-DNA复合物解离所需的变构构象变化。(c)2006爱思唯尔有限公司版权所有。
Comamonas testosteroni KH122-3s is an aerobic soil bacterium that utilizes 3-hydroxybenzoate as a sole carbon and energy source. In this strain, 3-hydroxybenzoate hydroxylase (MobA) acts on the initial step of the degradation to produce 3,4-dihydroxybenzoate, which is subsequently subjected to the meta-cleavage pathway leading to tricarboxylic acid cycle intermediates. Gene walking analysis of the upstream region of mobA revealed an open reading frame (mobR) that encodes a transcriptional regulator of the MarR family. Here, we report that MobR negatively regulates the expression of mobA, and that the repression is relieved by binding of 3-hydroxybenzoate, the substrate for MobA. A primer extension experiment was performed to determine the transcription start site for mobA and identified it at 83 bp upstream of the mobA start codon, accompanied by 70 a typical sigma(70)-type promoter. The mobR gene was expressed in Escherichia coli cells and the recombinant product was purified to homogeneity. Gel mobility-shift assays and DNase I footprinting analyses indicated that MobR binds as a homodimer to an imperfect inverted repeat within the mobA-mobR intergenic region, with an apparent dissociation constant of 11.5(+/- 0.5) nM. The operator site is located between the start codon and the promoter region for mobA, suggesting that MobR functions as a transcriptional repressor for mobA expression. The results of effector-binding assays indicated that MobR, but not its isomers 4-hydroxybenzoate and salicylate, is released from the operator site by the addition of 3-hydroxybenzoate. This dissociation process is highly cooperative, with a Hill coefficient of similar to 2. In addition, CD spectroscopic studies demonstrated that MobR adopts two conformational states corresponding to the effectorbound and unbound forms. These results suggest that the MobR dimer possesses at least two effector-binding sites, and that the effector binding to MobR induces an allosteric conformational change required for dissociation of the protein-DNA complex. (c) 2006 Elsevier Ltd. All rights reserved.