Genetic Analysis of Complementary Chromatic Adaptation

互补色适应的遗传分析

• 批准号: 0084297
• 负责人: David Kehoe
• 金额: $ 33万
• 依托单位: Indiana University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0084297&HistoricalAwards=false
• 关键词: Genetic; Analysis; Complementary; Chromatic; Adaptation

The major photosynthetic light harvesting structures of cyanobacteria are phycobilisomes, complexes that can constitute 40% of the total soluble protein. The composition of these structures is altered by numerous environmental conditions, including changes in the wavelength (color) of the ambient light. A process called complementary chromatic adaptation allows many filamentous cyanobacteria to respond to such changes by restructuring both the chromophore and protein composition of their phycobilisomes, permitting them to continually maximize the absorption of the predominant wavelengths of light available for photosynthesis. Numerous mutants that are aberrant in chromatic adaptation have been isolated, based on their color phenotypes, from the filamentous cyanobacterium Fremyella diplosiphon. The complementation of black mutants resulted in the discovery of RcaE, the first of a new class of prokaryotic photoreceptors. RcaE contains a transmitter module found in sensor kinase proteins of two component regulatory systems, and a chromophore binding domain found in plant photoreceptors called phytochromes. Complementation of two different classes of red mutants led to the identification of two separate response regulator proteins called RcaF and RcaC. RcaF is a small, single domain, CheY-like protein; RcaF is much larger and contains two response regulator input domains, a DNA binding motif, and a HPt motif, the hallmark of complex types of two component systems. A preliminary model has been proposed for the early steps in the chromatic adaptation regulatory pathway: RcaE is proposed to be a photoreceptor and act upon RcaF, which in turn regulates RcaC. Based on in vivo site directed mutagenesis studies, this pathway is proposed to be phosphorylated in red light and dephosphorylated in green light. Finally, recent biochemical evidence has demonstrated that a tetrapyrrole chromophore is covalently attached to RcaE and functional studies suggest that another, red-light absorbing chromophore may be involved in regulating chromatic adaptation. The goal of this project is to add to our understanding of the regulation of chromatic adaptation. This will be done by detailed analyses of RcaE and its interactions with RcaF and RcaC, as well as through the identification of new regulatory elements controlling this process. There are three specific objectives in this project. First, the hypothesis that a second chromophore, and photoreceptor, controls chromatic adaptation will be tested by gel filtration and immunoprecipitation studies. If a second photoreceptor is found to regulate this process, a putative chromoprotein encoded by a gene located in the genome near rcaE will be tested, using site directed mutagenesis and gene replacement studies, for a possible role in chromatic adaptation. Second, in vivo biochemical studies will be initiated to test the hypothesis that RcaE is a light-regulated histidine kinase and that phosphoryl group transfer occurs between specific histidine and aspartate residues within RcaE, RcaF, and RcaC. Third, genes containing lesions will be identified, isolated, and characterized from currently existing chromatic adaptation regulatory mutants using transformation approaches. Proteins related to both RcaE and the response regulators isolated thus far have also been found in a wide range of non-photosynthetic prokaryotes and photosynthetic eukaryotes. Thus, the findings from this work are expected to have broad implications for advancing our understanding of signal transduction processes in both bacteria and land plants.

蓝藻的主要光合捕光结构是藻胆体，复合物可以构成总可溶性蛋白质的40%。这些结构的组成会因许多环境条件而改变，包括环境光的波长（颜色）的变化。一个被称为互补色适应的过程允许许多丝状蓝藻通过重组藻胆体的发色团和蛋白质组成来应对这种变化，使它们能够不断最大限度地吸收光合作用可用的主要波长的光。许多突变体，是异常的色适应已被分离，根据其颜色表型，从丝状蓝藻Fremyella diplosiphon。黑色突变体的互补导致了RcaE的发现，RcaE是一类新的原核光感受器。RcaE包含一个在双组分调节系统的传感器激酶蛋白中发现的发射器模块，以及一个在植物光感受器中发现的称为光敏色素的发色团结合结构域。两种不同类型的红色突变体的互补导致了两种独立的反应调节蛋白RcaF和RcaC的鉴定。RcaF是一个小的，单结构域，CheY样蛋白; RcaF大得多，包含两个反应调节器输入结构域，一个DNA结合基序，和一个HPt基序，复杂类型的双组分系统的标志。色适应调节途径的早期步骤已经提出了一个初步的模型：RcaE被认为是一个光感受器，作用于RcaF，RcaF反过来调节RcaC。基于体内定点突变研究，提出该途径在红光下磷酸化，在绿色光下去磷酸化。最后，最近的生化证据表明，四吡咯发色团共价连接到RcaE和功能研究表明，另一个，红光吸收发色团可能参与调节色适应。这个项目的目标是增加我们对色彩适应调节的理解。这将通过详细分析RcaE及其与RcaF和RcaC的相互作用，以及通过确定控制这一过程的新的监管要素来完成。该项目有三个具体目标。首先，假设第二个发色团，感光体，控制色适应将测试凝胶过滤和免疫沉淀研究。如果发现第二个光感受器调节这一过程，将使用定点诱变和基因置换研究，测试由位于基因组中rcaE附近的基因编码的推定的色蛋白在色适应中的可能作用。第二，将启动体内生物化学研究，以检验RcaE是一种光调节组氨酸激酶，并且在RcaE、RcaF和RcaC内的特定组氨酸和天冬氨酸残基之间发生磷酰基转移的假设。第三，基因含有病变将被确定，分离，并从目前存在的色适应调节突变体的转化方法的特点。与RcaE和迄今为止分离的反应调节剂相关的蛋白质也已在广泛的非光合原核生物和光合真核生物中发现。因此，这项工作的发现预计将对促进我们对细菌和陆地植物中信号转导过程的理解产生广泛的影响。