Genetic Analysis of Complementary Chromatic Adaptation

蓝藻的主要光合光收集结构是藻胆体，这种复合体可以构成总可溶性蛋白质的40%。这些结构的组成受到许多环境条件的影响，包括环境光的波长（颜色）的变化。一种被称为互补色适应的过程允许许多丝状蓝藻通过重组其藻胆体的发色团和蛋白质组成来应对这种变化，使它们能够不断地最大化地吸收可用于光合作用的主要波长的光。根据其颜色表型，已经从丝状蓝藻freyella diplosiphon中分离出许多在颜色适应方面异常的突变体。黑色突变体的互补导致了RcaE的发现，这是一类新的原核光感受器中的第一个。RcaE包含一个在双组分调节系统的传感激酶蛋白中发现的传递模块，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域。两种不同类型的红色突变体的互补导致鉴定出两种不同的反应调节蛋白，称为RcaF和RcaC。RcaF是一种小的、单域的、chey样蛋白；RcaF更大，包含两个响应调节输入域，一个DNA结合基序和一个HPt基序，这是复杂类型双组分系统的标志。对于颜色适应调控途径的早期步骤，已经提出了一个初步的模型：RcaE被认为是一种光感受器，作用于RcaF， RcaF反过来调节RcaC。基于体内定向诱变研究，该途径在红光下被磷酸化，在绿光下被去磷酸化。最后，最近的生化证据表明，四吡咯色团与RcaE共价连接，功能研究表明，另一种红光吸收色团可能参与调节颜色适应。这个项目的目标是增加我们对颜色适应调节的理解。这将通过详细分析RcaE及其与RcaF和RcaC的相互作用，以及通过识别控制这一过程的新调控元件来完成。这个项目有三个具体目标。首先，第二个发色团和光感受器控制颜色适应的假设将通过凝胶过滤和免疫沉淀研究进行验证。如果发现第二个光感受器调节这一过程，则将使用位点定向诱变和基因替代研究，对位于rcaE附近的基因组中一个基因编码的假设的色蛋白进行测试，以确定其在颜色适应中的可能作用。其次，将启动体内生化研究，以验证RcaE是一种光调节的组氨酸激酶，并且在RcaE、RcaF和RcaC中特定的组氨酸和天冬氨酸残基之间发生磷酸化基团转移的假设。第三，将使用转化方法从现有的颜色适应调节突变体中鉴定、分离和表征含有病变的基因。与RcaE和反应调节因子相关的蛋白质目前已在广泛的非光合原核生物和光合真核生物中被发现。因此，这项工作的发现有望对促进我们对细菌和陆地植物信号转导过程的理解具有广泛的意义。

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.