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

蓝细菌是能够将CO2同化为有机化合物的光合氧生物。然而，水生生境中的二氧化碳水平往往是有限的，并可能迅速变化。因此，蓝藻需要复杂的系统来适应波动的CO2可用性，以维持CO2固定。为了能够在低环境浓度下固定CO2，蓝藻进化出一种有效的碳浓缩机制（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主要受两个LysR型转录因子的调控，这两个转录因子利用代谢信号作为辅抑制子或辅激活子。然而，这些转录因子的突变，只有部分废除了适应不断变化的CO2条件，这意味着额外的监管和感官机制是必要的，以实现CO2在蓝藻细胞内的稳态。最近，我们确定了蛋白SbtB作为潜在的CO2传感模块。SbtB显示出与PII蛋白相当大的结构相似性，PII蛋白在原核生物和植物的代谢碳/氮平衡的传感中起重要作用。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，SbtB以甚至更高的亲和力结合第二信使cAMP。SbtB缺陷型突变体在适应不同CO2水平时严重受损。主要cAMP合成酶，可溶性腺苷酸环化酶的活性，是长期已知的由碳酸氢盐水平在不同的细胞，包括蓝藻，然而，生理相关性是谜。因此，我们得出结论，SbtB蛋白代表了一种新的PII样感觉分子，其感觉无机碳可用性的变化取决于蓝藻中的第二信使cAMP。在这方面，cAMP的作用使人想起cAMP在许多生物体中调节糖代谢的众所周知的作用。最近，我们还发现sbtB突变对CO2调节基因的特定子集的表达有显著影响，并且SbtB不仅可以结合cAMP，还可以结合cyclic-di-AMP。 该建议的目的是解开SbtB周围的第二信使信号网络的模式蓝藻集胞藻属PCC 6803。为此，我们将确定cAMP相关的SbtB信号是如何整合到CO2驯化的细胞网络中的。此外，将分析新鉴定的SbtB配体c-di-AMP的功能。为了了解由SbtB组织的信号转导网络，我们的目标是确定SbtB的相互作用靶点，并找出这些相互作用是如何被核苷酸第二信使分子调节的。此外，SbtB表现出氧化还原反应的C-末端，我们的目标是确定第二信使信号和假定的氧化还原控制之间的联系，通过这个C-末端SbtB片段。

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.