Biosynthesis of phycoerythrins, photoysnthetic light harvesting proteins in cyanobacteria

智力优势：小型单细胞蓝藻，如聚球菌RS9916，估计贡献了海洋总生产力的25-30%。聚藻球菌可能是世界海洋中最普遍存在的浮游植物生物之一，在这个生态系统中，在数量上是第二丰富的海洋光养生物。这种普遍性和丰度在很大程度上是由于其在使用多种光合色素方面的显着表型可塑性，这是由一组复杂的基因和调控系统维持的，这些基因和调控系统可以在世系之间横向转移。这种多样性大部分发生在蛋白质藻红蛋白（PE）中，它在六个半胱氨酸残基上附着五个藻红蛋白（PEB）或藻红蛋白（PUB）发色团。这些翻译后修饰是由十亿蛋白裂解酶/异构酶催化的。由于蓝光是贫营养开放海洋中存在的主要波长，能够通过增加PE上的PUB含量来适应称为IV型色驯化（CA4）过程的菌株通过一种新机制：诱导十亿蛋白裂解酶/异构酶来利用光通过水的过滤。该项目的研究将有助于确定这些PE十亿蛋白裂解酶/异构酶的功能，这些酶对这种多样化的光合作用原核生物的成功和适应至关重要。更好地理解这种新型的色驯化（CA4）将实现。第一个目标是表征一个“简单”聚乙烯的生物合成在freremyella diplosiphon。与已知的胆磷脂裂解酶相似的6个基因被植红蛋白结构基因上调。第二个目标是表征聚球菌RS9916中PEI和PEII特异的十亿蛋白裂解酶的功能，重点是表征导致CA4变化的裂解酶。将对十亿蛋白裂解酶敲除突变体中产生的PE进行分析，并在大肠杆菌中表达重组蛋白，以测试十亿蛋白连接和异构化活性。这些研究不仅有助于了解这些生态上重要的生物如何在海洋中适应并茁壮成长，而且还将对酶进行生物化学表征，这些酶将具有非常重要的潜在生物技术和细胞生物学应用，将植物红蛋白用作荧光标记。更广泛的影响：该项目在培养本科生和研究生方面有着良好的记录，特别是在科学领域中代表性不足的群体的成员，这些努力将在本次资助期间继续进行。PI将把她的研究纳入一门名为“生物研究工具”的课程中，学生将学习大肠杆菌中藻胆蛋白的基本分子克隆和蛋白质表达/纯化。至少两名本科生（在资助期间）将在夏季前往印第安纳大学David Kehoe博士的实验室（在Schluchter实验室工作一年后），为期6-8周，以学习产生清洁缺失的技术，并研究与CA4合作项目相关的基因调控。这些学生很可能是一个代表性不足的群体的成员（PI过去的本科生中有50%是少数民族），这些交换的目的是让这些学生接触到更大的研究型大学。

Intellectual Merit: Small unicellular cyanobacteria such as Synechococcus RS9916 are estimated to contribute 25-30% of the total marine productivity. Synechococcus is likely to be one of the most ubiquitous phytoplanktonic organisms in the world's oceans and numerically the second most abundant marine phototroph in this ecosystem. This ubiquity and abundance is in large part due to its remarkable phenotypic plasticity in its use of a diverse set of photosynthetic pigments, which is sustained by a complex set of genes and regulatory systems that can be laterally transferred between lineages. Much of this diversity occurs within the protein Phycoerythrin (PE), which has five phycoerythrobilin (PEB) or phycourobilin (PUB) chromophores attached at six cysteine residues. These posttranslational modifications are catalyzed by bilin lyase/isomerase enzymes. As blue light is the dominating wavelength present in the oligotrophic open ocean, strains capable of a process called Type IV chromatic acclimation (CA4) have adapted by increasing the PUB content on their PE to take advantage of the light filtering through the water by a novel mechanism: induction of a bilin lyase/isomerase. The research in this project will contribute to defining the function of these PE bilin lyase/isomerase enzymes that are critical to the success and adaptation of this diverse group of photosynthetic prokaryotes. A better understanding of this novel type of chromatic acclimation (CA4) will be achieved. The first objective is to characterize the biosynthesis of a "simple" PE in Fremyella diplosiphon. Six genes with similarity to known bilin lyase enzymes are upregulated with phycoerythrin structural genes. The second objective involves characterizing the function of bilin lyases specific for PEI and PEII in Synechococcus RS9916, focusing on the characterization of lyases responsible for CA4 changes. Analyses of PE produced in the bilin lyase knockout mutants will be performed, and recombinant protein expression in E. coli to test for bilin ligation and isomerization activity will also be used. Not only will these studies contribute to understanding how these ecologically important organisms acclimate to thrive in the ocean, they will also biochemically characterize enzymes which will have very important potential biotechnological and cell biology applications for the use of phycoerythrins as fluorescent tags.Broader impacts: The PI has a strong track record of training undergraduates and graduate students, especially members of under-represented groups in science, and these efforts will continue during this granting period. The PI will incorporate her research into a course called Biological Research Tools where students learn basic molecular cloning and protein expression/purification of phycobiliproteins in E. coli. At least two undergraduates (over the course of the grant) will be selected to go to the laboratory of Dr. David Kehoe at Indiana University (after working in the Schluchter lab for a year first) for 6-8 weeks in the summer in order to learn techniques to generate clean deletions and to study gene regulation related to the CA4 collaboration project. The student is likely to be a member of an under-represented group (50% of the PI's past undergraduates have been minorities), and the purpose of these exchanges is to expose these students to a larger, research university.