Characterization of a two-component signal transduction system that controls arsenite oxidation in the chemolithoautotroph NT-26
Characterization of a two-component signal transduction system that controls arsenite oxidation in the chemolithoautotroph NT-26
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DOI：
10.1111/j.1574-6968.2010.02121.x
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发表时间：
2010-12-01
影响因子：
2.1
通讯作者：
Djordjevic, Snezana
中科院分区：
文献类型：
--
作者：
Sardiwal, Sunita;Santini, Joanne M.;Djordjevic, Snezana
NT-26是一种化学自养型亚砷酸盐氧化剂。了解NT-26对亚砷酸盐信号、耐受和氧化的机制，将对其在生物修复和亚砷酸盐传感方面的应用具有重要意义。我们已经确定了组氨酸激酶(AROS)和同源反应调节因子(AroR)参与了砷依赖的亚砷酸氧化酶aroBA操纵子的转录调控。AROS含有一个单一的周质感受域，通过跨膜螺旋连接到HAMP结构域，HAMP结构域将信号传递到蛋白质的激酶核心。AroR属于AAA+转录调控因子家族，通过螺旋-转角-螺旋结构域与DNA相互作用。AAA+结构域和RNA聚合酶Sigma 54-相互作用序列基序的存在表明，该蛋白通过与RNA聚合酶以Sigma 54依赖的方式相互作用来调节转录。异源表达和纯化了AROS的激酶核心和AroR的受体结构域，并证实了它们的自磷酸化和转磷酸化活性。利用定点突变，我们已经确定了这两种蛋白质上的磷酸化位点。NT-26中的突变分析证实，这两种蛋白都是亚砷酸盐氧化所必需的，并且aros突变体影响了亚砷酸盐的生长，也表明它参与了亚砷酸盐耐受性的调节。最后，亚砷酸盐传感似乎不涉及硫醇化学。
NT-26 is a chemolithoautotrophic arsenite oxidizer. Understanding the mechanisms of arsenite signalling, tolerance and oxidation by NT-26 will have significant implications for its use in bioremediation and arsenite sensing. We have identified the histidine kinase (AroS) and the cognate response regulator (AroR) involved in the arsenite-dependent transcriptional regulation of the arsenite oxidase aroBA operon. AroS contains a single periplasmic sensory domain that is linked through transmembrane helices to the HAMP domain that transmits the signal to the kinase core of the protein. AroR belongs to a family of AAA+ transcription regulators that interact with DNA through a helix-turn-helix domain. The presence of the AAA+ domain as well as the RNA polymerase Sigma 54-interaction sequence motif suggests that this protein regulates transcription through interaction with RNA polymerase in a Sigma 54-dependent fashion. The kinase core of AroS and the receiver domain of AroR were heterologously expressed and purified and their autophosphorylation and transphosphorylation activities were confirmed. Using site-directed mutagenesis, we have identified the phosphorylation sites on both proteins. Mutational analysis in NT-26 confirmed that both proteins are essential for arsenite oxidation and the AroS mutant affected growth with arsenite, also implicating it in the regulation of arsenite tolerance. Lastly, arsenite sensing does not appear to involve thiol chemistry.