The role of small proteins in metabolic regulation of cyanobacteria

在这一对环境至关重要的初级生产者群体中，藻蓝共生菌（synnechocystis sp.）菌株PCC 6803充当了含氧光合作用和代谢调节的模型。最近，在聚囊藻基因组中发现了以前未知的转录本，这些转录本可以编码小蛋白质。在SPP2002中，我们的目标是分析新发现的小蛋白是否参与蓝藻的代谢调节。选取三个小蛋白Norf4 （31 AA）、pSYS_ORF3/AcnSP （44 AA）和HliR1 （37 AA）进行功能注释。使用突变体（敲除和选择的位点特异性突变体消除小蛋白表达）和异位表达小蛋白的菌株进行生化和生理分析将揭示它们是否真的具有重要的功能。我们推测这些小蛋白可以直接或间接调节特定的酶如甘油醛3-磷酸脱氢酶（Gap）和乌头酸脱氢酶（AcnB）或参与蓝藻的应激保护调节。在第一个资助期内，我们证明了pSYS_ORF3调节三羧酸酶AcnB的生化活性，因此它被重命名为AcnSP。我们的研究结果表明，小蛋白AcnSP影响碳流进入开放蓝藻TCA循环的氧化分支，从而调节聚囊藻的整体C/N代谢。此外，我们的研究结果表明，sorf甚至可以起源于看似不规则的基因片段复制，并在AcnB周围的复杂调控系统中添加了另一个元素。此外，我们获得了明确的提示，AcnSP也参与铁胁迫反应的调节，这与AcnB在许多其他生物中作为TCA循环酶和铁感知模块的双重作用是一致的。揭示潜在的机制，即AcnSP如何参与对铁胁迫的适应，可能参与sRNAs和RNase E作用于acnB mRNA稳定性的网络，将是即将到来的项目期的重点。我们对Norf4和HliR1的研究证实了这些小蛋白确实参与了聚囊藻的代谢调节或应激适应。norf4突变体不能在黑暗中与葡萄糖一起生长，并表现出对储存的糖原利用的改变。这些发现与我们最初的假设一致，即Norf4调节糖酵解关键酶Gap1的活性。最后，由于超氧化物歧化酶B （SodB）活性降低，hliR1突变体表现出明显的高光敏感表型。这一发现支持了我们最初的假设，即HliR1调节SodB， SodB在蓝藻适应由强光或铁稳态紊乱引起的氧化应激中起着至关重要的作用。Norf4-和hlir1介导的调控的分子机制将在未来进行分析。

The cyanobacterium Synechocystis sp. strain PCC 6803 serves as the model for oxygenic photosynthesis as well as metabolic regulation within this environmentally important group of primary producers. Recently, previously unknown transcripts were identified in the Synechocystis genome that could encode small proteins. Within SPP2002, we aim to analyze whether or not the newly discovered small proteins are involved in metabolic regulation of cyanobacteria. Three small proteins namely Norf4 (31 AA), pSYS_ORF3/AcnSP (44 AA) and HliR1 (37 AA) were chosen for functional annotation. Biochemical and physiological analysis using mutants (knock out and selected site-specific mutants abolishing the small protein expression) and strains ectopically expressing the small proteins will reveal if they are really functionally important. We hypothesize that these small proteins could directly or indirectly regulate specific enzymes such as glyceraldehyde 3-phosphate dehydrogenase (Gap) and aconitate dehydratase (AcnB) or participate in the regulation of stress protection of cyanobacteria. During the first funding period we demonstrated that pSYS_ORF3 regulates the biochemical activity of the tricarboxylic acid enzyme AcnB, therefore it was renamed as AcnSP. Our results indicated that the small protein AcnSP impacts the carbon flow into the oxidative branch of the open cyanobacterial TCA cycle thereby regulating the overall C/N metabolism in Synechocystis. Furthermore, our results demonstrated that sORFs can originate even from seemingly irregular gene fragment duplications and add another element to the complex regulatory system around AcnB. Moreover, we obtained clear hints that AcnSP is also involved in the regulation of iron-stress response, which is consistent with the dual role of AcnB as TCA cycle enzyme and iron-sensory module in many other organisms. Unravelling the underlying mechanisms, i.e. how AcnSP is involved in the acclimation to iron stress probably participating in a network with sRNAs and RNase E action on acnB mRNA stability, will be in the focus of the upcoming project period.Our work on Norf4 and HliR1 verified that these small proteins are indeed involved in metabolic regulation or stress acclimation of Synechocystis. The norf4 mutant is unable to grow with glucose in darkness and shows alterations in the utilization of stored glycogen. These findings are consistent with our initial hypothesis that Norf4 is regulating the activity of the glycolytic key enzyme Gap1. Finally, the hliR1 mutant showed a clear high-light-sensitive phenotype due to reduced superoxide dismutase B (SodB) activity. This finding is supporting our initial hypothesis that HliR1 is regulating SodB, which plays a crucial role in the acclimation of cyanobacteria towards oxidative stress induced by high-light or disturbed iron homeostasis. The molecular mechanisms of the Norf4- and HliR1-mediated regulations will be analyzed in the future.