Linking PII regulation with central carbon control in Synechocystis PCC 6803

细胞精确感知碳/氮(C/N)平衡并在扰动时做出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，功能多样的PII信号蛋白在这方面发挥着重要作用。它们作为多任务信号处理器，通过相互依赖的ATP/ADP结合，将来自C/N状态报告代谢产物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通过与不同的靶点结合来协调代谢活动以响应环境的变化。过去十年的研究表明，PII信号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)已经获得了读出PII蛋白通过与PII的不同相互作用而整合和计算的代谢信息的能力。最近，我们在模型蓝藻聚球藻PCC6803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目前认为的更广泛。在这些靶标中，有三个主要氮源的运输系统，即ABC型硝酸盐/亚硝酸盐吸收系统NRT和ABC型尿素吸收系统URT，此外还有磷酸烯醇式丙酮酸羧化酶，以及两个功能未知的小肽S110944和Ssr0692。初步数据表明，S110944在蓝藻适应缺氮的过程中起着关键作用。S110944的突变导致在N饥饿下逐渐丧失活力，并伴随着聚羟基丁酸的大量过度积累，这是以前从未报道过的。通过下拉分析，我们可以确定中心碳分配酶PGAM(2-3磷酸甘油非依赖性磷酸甘油酸变位酶)是S110944的主要靶点，并可能参与羧体相关蛋白CCMP的作用。在目前的提案中，我们的目标是确定这些蛋白在聚球藻氮驯化中的作用以及PII相互作用的功能意义。因此，我们提出了一个从活动到结构水平系统地剖析和表征PII/S110944/PGAM/CCMP相互作用网络的项目。由于这些蛋白质在蓝藻中高度保守，我们将获得具有全球相关性的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.