Analysis of a Stress-Inducible DNA-Binding Protein in a Cyanobacterium

9634049 Bullerjahn本项目是对蓝藻聚球藻的一种机制的研究。PCC7942菌株能够适应氧化应激。编码一种应激诱导的DNA结合蛋白的基因dpsA已经被克隆和测序。以前的工作已经表明，DpsA蛋白在营养胁迫条件下以及在生长曲线的稳定期积累。这种蛋白质形成非常稳定的六聚体复合体，与DNA结合；对进化上不同的大肠杆菌基因及其产物的研究表明，DpsA蛋白在长期营养枯竭和接触过氧化氢期间保护染色体免受氧化应激的影响。然而，大肠杆菌和聚球菌多肽之间的一个主要区别是在聚球藻DpsA蛋白的C-末端半部分存在一个血红素结合域。推测血红素的作用是通过过氧化氢酶机制消耗过量的过氧化氢。蓝藻等产氧光养生物对氧的解毒活性物种有潜在的更高需求。在生产DpsA方面有缺陷的突变株将被检查其在光氧化损伤和过氧化氢处理条件下的生存能力。截短形式和嵌合形式的dpsA也将被引入聚球藻的dpsA缺失突变体中，以测试是否存在血红素结合结构域在生存中发挥重要作用。RNA印迹和启动子融合将用于确定有助于dpsA转录的环境条件。免疫细胞化学方法将被用来试图确定DpsA/DNA复合体在适应到静止阶段期间在细胞中的位置。这项研究可能有助于确定类核是否存在一个明显的结构区域，该区域优先受到DpsA蛋白复合体的保护。最后，酵母双杂交系统将被用于筛选可能参与调节稳定的DpsA复合体的组装和周转的蛋白质-蛋白质相互作用。总体而言，这项工作将有助于揭示蓝藻和其他光养生物的全球应激反应机制。%本研究是关于蓝藻聚球藻。PCC7942菌株能够适应对细胞成分造成氧化损伤的条件。将研究dpsA基因及其编码的蛋白质。DpsA通过一种不寻常的机制帮助保护细胞免受氧化损伤。这项提案将集中在光照和其他环境条件如何控制DpsA的生产。这些研究将提供有关光合作用有机体如何应对氧化压力和变化的新信息。***

9634049 Bullerjahn This project is an investigation of a mechanism by which the cyanobacterium Synechococcus sp. strain PCC 7942 can adapt to oxidative stress. The gene dpsA, encoding a stress-induced DNA-binding protein has been cloned and sequenced. Previous work has shown that the DpsA protein accumulates under conditions of nutrient stress as well during the stationary phase of the growth curve. The protein forms extremely stable hexameric complexes that bind DNA; studies of the evolutionarily divergent Escherichia coli gene and its product indicate the DpsA protein serves to protect the chromosome from oxidative stress during long-term nutrient depletion and exposure to peroxide. However, a major difference between the E. coli and Synechococcus polypeptides is the presence of a heme-binding domain in the C-terminal half of the Synechococcus DpsA protein. It is suggested that the function of heme is to consume excess peroxide by a catalase mechanism. There is potentially a higher demand for detoxifying reactive species of oxygen in oxygen-producing phototrophs such as cyanobacteria. Mutants defective in the production of DpsA will be examined with respect to their ability to survive conditions of photooxidative damage and peroxide treatment. Truncated and chimeric forms of dpsA will also be introduced into dpsA null mutants of Synechococcus in order to test whether the presence of the heme-binding domain plays an important role in survival. RNA blots and promoter fusions will be used to determine the environmental conditions contributing to dpsA transcription. Immunocytochemical methods will be used to attempt to determine the location of the DpsA/DNA complex in the cells during adaptation to stationary phase. This study may help determine whether there is a distinct structural region of the nucleoid that is preferentially protected by the DpsA protein complex. Lastly, the yeast two-hybrid system will be employed to screen for possible protein-protein interactions that might be involved in regulating assembly and turnover of the stable DpsA complex. Overall, this work will help reveal mechanisms involved in global stress responses in cyanobacteria, and other phototrophs. %%% This study is concerned with how the cyanobacterium Synechococcus sp. strain PCC 7942 can adapt to conditions which cause oxidative damage to cell components. The gene dpsA and the protein it encodes DpsA will be studied. DpsA helps protect the cells against oxidative damage by an unusual mechanism. This proposal will focus on how light and other environmental conditions control production of DpsA. These studies will provide new information about how photosynthetic organisms respond to oxidative stress and change. ***