Linking PII regulation with central carbon control in Synechocystis PCC 6803

细胞精确感知碳/氮（C/N）平衡并在扰动时执行适当反应的能力是维持细胞稳态的关键要求。在原核生物中，多功能PII信号蛋白在这方面是重要的参与者。它们充当多任务信号处理器，通过相互依赖的ATP/ADP结合将来自C/N状态报告代谢物2-OG的信号与来自细胞能量状态的信号整合。根据整合的信号，PII通过与各种靶点结合来协调代谢活动以响应环境变化。过去十年的研究表明，PII信号传导的原理在进化过程中保持保守，而不同的分子机器（酶，转运蛋白，转录因子）已经获得了读取代谢信息的能力，这些信息是通过与PII的各种相互作用由PII蛋白整合和计算的。最近，我们在蓝细菌集胞藻PCC 6803模型中发现了几个新的PII靶点，这表明PII协调细胞活动的范围比迄今为止所认为的更广。在这些靶标中，有三种主要氮源的转运系统，铵转运蛋白AMT 1、ABC型硝酸盐/亚硝酸盐摄取系统NRT和ABC型尿素摄取系统URT，此外还有磷酸烯醇式丙酮酸羧化酶，以及两种功能未知的小肽Sll 0944和Ssr 0692。初步数据表明，Sll 0944在蓝藻驯化氮剥夺中具有关键作用。Sll 0944的突变导致在N-饥饿下逐渐丧失生存能力，并导致伴随的聚羟基丁酸酯的大量过度积累，以前从未报道过这种程度。通过下拉分析，我们可以确定中心碳分布酶PGAM（2-3磷酸甘油酸独立磷酸甘油酸酯酶）作为Sll 0944的主要靶标，可能涉及羧基体相关蛋白CcmP。在目前的建议中，我们的目标是确定这些蛋白质在集胞藻氮驯化和PII相互作用的功能意义的作用。因此，我们提出了一个项目，系统地剖析和表征，从活动到结构水平，PII/Sll 0944/PGAM/CcmP相互作用网络的相互作用。由于这些蛋白质在蓝藻中高度保守，我们将获得细菌代谢控制的全局相关性的基本见解。

The ability of cells to precisely sense the carbon/nitrogen (C/N) balance and execute appropriate responses upon perturbation is a key requirement for maintenance of cellular homeostasis. In prokaryotes, the versatile PII signaling proteins are important players in this respect. They act as multitasking signal processors, integrating the signal from the C/N status reporter metabolite 2-OG with that from the energy state of the cell through interdependent ATP/ADP binding. Depending on the integrated signals, PII orchestrates metabolic activities in response to environmental changes through binding to various targets. Research in the last decade has revealed that the principle of PII signalling has remained conserved during evolution, whereas different molecular machines (enzymes, transporters, transcription factors) have acquired the ability to read out the metabolic information that has been integrated and calculated by PII proteins through diverse interactions with PII. Recently, we discovered several novel targets of PII in the model cyanobacterium Synechocystis PCC 6803, which indicates that PII orchestrates cellular activities even wider than thought so-far. Among these targets are three transport systems for the major nitrogen sources, the ammonium transporter AMT1, the ABC-type nitrate/nitrite uptake system NRT and the ABC-type urea uptake system URT, furthermore phosphoenolpyruvate carboxylase, as well as two small peptides of unknown function, Sll0944 and Ssr0692. Preliminary data indicate that Sll0944 has a pivotal role in the acclimation of cyanobacteria to nitrogen deprivation. Mutation of Sll0944 leads to gradual loss of viability under N-starvation and to a concomitant massive over-accumulation of polyhydroxybutyrate, never reported to this extent before. By pull-down analysis, we could identify the central carbon-distributing enzyme PGAM (2-3 phosphoglycerate independent phosphoglycerate mutase) as dominant target of Sll0944, with potential involvement of the carboxysome associated protein CcmP. In the current proposal, we aim to identify the role of these proteins in Synechocystis nitrogen acclimation and the functional implication of PII interaction. Therefore, we propose a project that systematically dissects and characterizes, from activity to structural level, the interactions of the PII/Sll0944/PGAM/CcmP interaction network. Since these proteins are highly conserved in cyanobacteria, we will obtain fundamental insights in bacterial metabolic control of global relevance.