Novel regulators and interactions within the regulatory networks controlling the autotrophy -heterotrophy switch in cyanobacterial carbon metabolism

一些证据表明，到目前为止，控制蓝藻初级代谢的重要调节因子、活性和信号转导链仍未确定。作为SCyCode研究小组的一部分，我们的目标是专注于调节器和调节元件的表征，到目前为止，这些调节器和调节元件在蓝藻的自养-异养转换过程中可能起到控制作用。在第一个资助期，我们能够确定sRNA调控基因ApcZ和转录因子Fura的调控基因。此外，在Grad-Seq和PROMIS4Cyanos中，我们建立了两种方法，通过确定蛋白质-RNA复合体和蛋白质-代谢物复合体的组成和相关性分析，通过对蛋白质-RNA复合体和蛋白质-代谢物复合体的分级来确定调节初级代谢复合体的新候选者。在这些结果的基础上，我们将在第二个资助期识别这些假定的调节器和调节元件，或使用四种不同的方法更精确地描述它们的功能。在第一种方法中，我们使用已经建立的PROMIS4Cyanos工作流程来识别蛋白质和蛋白质复合体以及共分离的代谢物，重点是对无机碳缺乏的适应，以及在光/暗周期中RpaA缺失突变体以及已知的2-磷酸乙醇酸的过度积累。选定的可能参与初级代谢调节的新候选者，例如通过D-3-磷酸甘油酸脱氢酶，将得到生化验证。在第二种方法中，我们想要研究新发现的潜在调节因子，即蛋白质以及小的调节RNA，并识别它们控制的基因或蛋白质或扩展已知的调节因子。在第三种蛋白质基因组学方法中，我们希望通过检测模型系统聚球藻sp.中以前未知的蛋白质来扩大此类研究的基础。PCC 6803。在第4个工作包中，我们希望利用广泛的现有数据集，对替代启动子和转录起始点进行全基因组表征，以定义由反应调节因子RpaA控制的调节子。我们将与研究小组的其他成员密切合作，研究所有四种方法，并使用蓝藻聚胞藻的培养物。PCC 6803在受控条件下，使我们能够专注于从自养模式到异养模式转换过程中的调控事件，在昼夜光暗转变、从高无机到低无机转变和/或氮素供应下降之后。

Several lines of evidence indicate that important regulators, activities and signal transduction chains in the control of the cyanobacterial primary metabolism have so far remained unidentified. As part of the SCyCode research group, we aim to focus on the characterization of regulators and regulatory elements that have remained largely uncharacterized thus far but are likely to play a role in the control of the autotrophy-heterotrophy switching process in cyanobacteria. During the first funding period we were able to characterize the sRNA regulator ApcZ and the regulon of the transcription factor FurA. Furthermore, with Grad-seq and PROMIS4Cyanos, we have established two methods to identify new candidates for the regulation of complexes of the primary metabolism via the fractionation of protein-RNA complexes and protein-metabolite complexes after determination of the respective composition and correlation analysis.Building on these results, we will in the 2nd funding period identify such putative regulators and regulatory elements or characterize them functionally more precisely using four different approaches. In the first approach, we identify proteins and protein complexes and co-fractionating metabolites using the established PROMIS4Cyanos workflow with a focus on the acclimation to inorganic carbon deficiency and the RpaA deletion mutant in the light/dark cycle and the already known over-accumulation of 2-phosphoglycolate in it. Selected novel candidates likely involved in the regulation of primary metabolism, for example through the enzyme D-3-phosphoglycerate dehydrogenase, will be biochemically validated. In the second approach, we want to investigate newly identified potential regulators, i.e. proteins as well as small regulatory RNAs, and identify the genes or proteins controlled by them or expand the known regulons. In a third, proteogenomic approach, we would like to expand the basis for such investigations by examining previously unknown proteins in the model system Synechocystis sp. PCC 6803. In the 4th work package we would like to utilize the extensive existing data sets for the genome-wide characterization of alternative promoters and transcription start sites in order to define the regulon controlled by the response regulator RpaA. We will pursue all four approaches in close cooperation with the other members of the research group and use cultures of the cyanobacterium Synechocystis sp. PCC 6803 under controlled conditions that allow us to focus on the regulatory events during the switch from autotrophic to heterotrophic modes after diurnal light to dark shifts, shifts from high to low inorganic, and/or down-shifts in nitrogen supply.