Linking second messenger nucleotide signalling with CO2 homeostasis in cyanobacteria: unravelling the SbtB-based network

蓝藻是一种富氧的光养生物，能够将二氧化碳同化为有机化合物。然而，水生生境中的二氧化碳水平往往是有限的，可能会迅速变化。因此，蓝藻需要复杂的系统来适应二氧化碳有效性的波动，以维持二氧化碳的固定。为了能够在低环境浓度下固定二氧化碳，蓝藻进化出一种高效的碳浓缩机制(CCM)，使高亲和力的无机碳吸收成为可能。在基因表达水平上，CCM主要受两种LysR型转录因子的调控，这两种转录因子利用代谢信号作为辅助抑制因子或辅助激活因子。然而，这些转录因子的突变只部分地取消了对不断变化的二氧化碳条件的适应，这意味着额外的调节和感觉机制是实现蓝藻细胞中二氧化碳稳态所必需的。最近，我们发现SbtB蛋白是一个潜在的二氧化碳敏感模块。SbtB在结构上与PII-蛋白有相当大的相似性，PII-蛋白在原核生物和植物的代谢碳/氮平衡中起着重要的作用。与PII蛋白类似，SbtB能够结合腺嘌呤核苷酸。然而，与PII蛋白不同的是，SbtB以更高的亲和力结合第二信使cAMP。SbtB缺陷突变体在适应不同的二氧化碳水平时会受到严重损害。众所周知，包括蓝藻在内的不同细胞中主要的cAMP合成酶--可溶性腺苷环化酶的活性受碳酸氢盐水平的调节，然而，其中的生理关联却是个谜。因此，我们得出结论，SbtB蛋白是一种新的PII类感觉分子，它依赖于蓝藻中的第二信使cAMP来感知无机碳有效性的变化。在这方面，cAMP的作用让人想起众所周知的cAMP在许多生物体中调节糖代谢的作用。最近，我们还发现SbtB突变对CO2调控的特定基因亚群的表达有显著影响，SbtB不仅能与cAMP结合，还能与环二AMP结合。本研究的目的是解开蓝藻聚球藻SbtB周围的第二信使信号网络。PCC 6803。为此，我们将确定cAMP相关的SbtB信号如何整合到二氧化碳适应的细胞网络中。此外，还将对新发现的SbtB配体c-di-AMP的功能进行分析。为了了解SbtB组成的信号转导网络，我们的目标是识别SbtB的相互作用靶点，并找出核苷酸第二信使分子是如何调控这些相互作用的。此外，SbtB还表现出一个氧化还原反应的C末端，我们的目标是通过SbtB的这个C末端片段来确定第二信使信号和可能的氧化还原控制之间的联系。

Cyanobacteria are oxygenic phototrophs able to assimilate CO2 into organic compounds. However, the CO2 levels in aquatic habitats are often limiting and may rapidly change. Therefore, cyanobacteria need sophisticated systems to acclimate to fluctuating CO2 availability to sustain CO2 fixation. To be able to fix CO2 at low ambient concentrations, cyanobacteria evolved an efficient carbon concentrating mechanism (CCM) that enables high affinity inorganic carbon uptake. At the level of gene expression, the CCM is mainly regulated by two LysR-type transcriptional factors that use metabolic signals as corepressors or coactivators. However, mutation of these transcription factors only partly abolishes the acclimation toward changing CO2 conditions, implying that additional regulatory and sensory mechanisms are necessary to achieve CO2 homeostasis in cyanobacterial cells. Recently, we identified the protein SbtB as potential CO2-sensing module. SbtB shows considerable structural similarity to PII-proteins, which play an important role in the sensing of the metabolic carbon/nitrogen balance in prokaryotes and plants. Similar to PII proteins, SbtB is able to bind adenine nucleotides. However, unlike PII proteins, SbtB binds the second messenger cAMP with even higher affinity. An SbtB-deficient mutant is severely impaired in the acclimation to varying CO2 levels. The activity of the major cAMP-synthesizing enzyme, the soluble adenylate cyclase, is long-known to be modulated by bicarbonate levels in different cells including cyanobacteria, however, the physiological relevance was enigmatic. Therefore, we concluded that the SbtB protein represents a new PII-like sensory molecule that senses changes in inorganic carbon availability depending on the second messenger cAMP among cyanobacteria. In this respect, the role of cAMP is reminiscent to the well-known roles of cAMP in many organisms for regulating sugar metabolism. Recently, we also found that sbtB mutation has marked impact on the expression of specific subset of CO2-regulated genes and that SbtB can not only bind cAMP but also cyclic-di-AMP. The aim of this proposal is to unravel the second messenger signaling network around SbtB in the model cyanobacterium Synechocystis sp. PCC 6803. For this purpose, we will determine how the cAMP-related SbtB signaling is integrated into the cellular network of CO2 acclimation. Furthermore, the function of the newly identified SbtB ligand c-di-AMP will be analyzed. To understand the signal transduction network organized by SbtB, we aim to identify interaction targets of SbtB and to find out, how these interactions are modulated by nucleotide second messenger molecules. In addition, SbtB exhibits a redox-responsive C-terminus and we aim to identify the link between second messenger signaling and the presumed redox control via this C-terminal segment of SbtB.