Identification and Characterization of Novel Interaction Partners of the Signaling Protein PII in the Cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria are a diverse group of gram-negative procaryotes that shaped the atmosphere of today’s earth. While some cyanobacteria are multicellular and even consist of different specialized cell types, others such as the model organism Synechocystis sp. PCC 6803 are unicellular. Synechocystis can grow autotrophically, heterotrophically, or mixotrophically. Like other unicellular cyanobacteria, Synechocystis is non-diazotrophic and relies on combined nitrogen sources. These are processed in the GS/GOGAT cycle where glutamate is formed using 2-oxoglutarate (2-OG) as carbon skeleton for amination. 2-OG, being an intermediate of the TCA cycle, can be considered the interconnection of nitrogen and carbon metabolism, making it the ideal status reporter of the carbon/nitrogen balance. When depleted of nitrogen, Synechocystis switches to a highly reduced metabolic state, called chlorosis. During chlorosis, glycogen is slowly degraded resulting in the formation of polyhydroxybutyrate (PHB), a biopolymer of industrial relevance. In Synechocystis and other organisms, the signaling protein PII controls a variety of cellular processes. PII proteins can sense the energy and nitrogen state of the cell by binding either ATP or ADP and additionally 2-OG in the ATP bound state. Depending on these effectors, PII can interact with its targets, i.e., enzymes, co-transcription factors, transporters, or small proteins controlling other targets. Understanding the PII interactome is therefore crucial for understanding the cyanobacterial metabolism. Hence the main task of this work was to identify and characterize new PII interaction partners based on preliminary pulldown results. Indeed, two previously unknown PII targets – phosphoenolpyruvate carboxylase (PEPC) and the PII interacting regulator of carbon metabolism (PirC) – were successfully identified. The mode of interaction with PII, influence of effectors, binding constants and physiological and metabolic effects of PII regulation were described. PirC was identified as inhibitor of phosphoglycerate mutase (PGAM), thereby controlling the carbon flux towards either glycogen synthesis or the TCA cycle as shown by a PirC deficient mutant. During chlorosis, the mutant strain utilized glycogen at a higher rate than the wild type while simultaneously producing unusually high amounts of PHB. Moreover, these results were brought to practical use with the creation of the PHB overproducing strain PPT1.