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

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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.