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内稳态的关键要求。在原核生物中，多功能PII信号蛋白是这方面的重要参与者。它们作为多任务信号处理器，通过相互依赖的ATP/ADP结合，整合来自C/N状态报告代谢物2-OG和来自细胞能量状态的信号。PII根据整合的信号，通过与各种靶标结合，协调代谢活动以响应环境变化。近十年的研究表明，PII信号传导原理在进化过程中一直保持保守，而不同的分子机器（酶、转运体、转录因子）通过与PII的不同相互作用，获得了读出PII蛋白整合和计算的代谢信息的能力。最近，我们在蓝藻聚胞菌PCC 6803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认为的要广。这些靶点包括三个主要氮源转运系统，铵转运体AMT1， abc型硝酸盐/亚硝酸盐吸收系统NRT和abc型尿素吸收系统URT，此外还有磷酸烯醇丙酮酸羧化酶，以及两个功能未知的小肽Sll0944和Ssr0692。初步数据表明，Sll0944在蓝藻对氮剥夺的适应中起着关键作用。Sll0944突变导致在n -饥饿条件下逐渐丧失生存能力，并伴有聚羟基丁酸盐的大量过度积累，这在以前从未报道过。通过下拉分析，我们可以确定中心碳分布酶PGAM（2-3磷酸甘油酸非依赖性磷酸甘油酸突变酶）是Sll0944的主要靶点，羧基体相关蛋白CcmP可能参与其中。在目前的提案中，我们的目标是确定这些蛋白在聚囊藻氮驯化中的作用以及PII相互作用的功能含义。因此，我们提出了一个项目，从活动到结构水平，系统地剖析和表征PII/Sll0944/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，我们将获得全球相关细菌代谢控制的基本见解。

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.