The Novel P _II -Interacting Protein PirA Controls Flux into the Cyanobacterial Ornithine-Ammonia Cycle

Abstract

Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-l-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it PII-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-PII interaction requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell. IMPORTANCE Cyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g., as major primary producers. Due to their photosynthetic lifestyle, cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis. Besides its role as a proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. Therefore, the obtained results will not only enhance our understanding of flux control in these organisms but also help to provide a scientific basis for targeted metabolic engineering and, hence, the design of photosynthesis-driven biotechnological applications.

New views on PII signaling: from nitrogen sensing to global metabolic control

“…In vitro, PirA binds to PII-ADP to tune down the PII-promoted activation of NAGK, the PII-controlled enzyme of arginine biosynthesis [6,8,[21][22][23]. In agreement with these findings, overproduction of PirA reduced arginine levels, whereas in the absence of PirA overproduction of arginine and related metabolites was observed [66]. Together, these data suggest that, in response to ammonium-upshift conditions, PirA antagonizes the PII-promoted activation of NAGK to re-equilibrate arginine levels (Figure 3).…”

“…The second small protein that interacts with PII, product of ssl0692 in Synechocystis, is 51 amino acids long and is named PirA [66]. PirA shows the inverse expression compared with PirC, being repressed by NtcA and accumulating following ammonium upshift.…”

The sRNA NsiR4 fine-tunes arginine synthesis in the cyanobacterium Synechocystis sp. PCC 6803 by post-transcriptional regulation of PirA

“…In particular, the role of small proteins as effectors for the activity of key enzymes receives more and more attention (for a review see [ 31 ]). One such small protein is PirA ( P II - i nteracting r egulator of a rginine synthesis) which has recently been shown to modulate arginine synthesis via competition with NAGK for P II binding [ 32 ]. As binding of NAGK to the P II protein reduces the effect of feedback inhibition, PirA interferes with arginine accumulation and acts as indirect inhibitor of NAGK.…”

“…As binding of NAGK to the P II protein reduces the effect of feedback inhibition, PirA interferes with arginine accumulation and acts as indirect inhibitor of NAGK. Similar to gifA and gifB , which encode the two known proteinaceous GS inhibitors IF7 and IF17, expression of the pirA gene (Gene ID: ssr0692 ) increases with rising N availability due to relief from NtcA-mediated transcriptional repression [ 24 , 32 ]. Moreover, novel RNA-based mechanisms regulating N assimilation have been described in Synechocystis and other cyanobacteria [ 33–35 ].…”

Alterations in the CO2 availability induce alterations in the phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803

“…It has been shown that the downregulation of genes related to nitrogen assimilation is mediated via the nitrogen‐sensing protein P II (Schwarz et al ., 2014), which was found to be static at protein levels but with an altered phosphorylation state (discussed below). Interestingly, the P II interaction regulator of carbon storage metabolism, PirC, is highly abundant at HC conditions and is downregulated at LC conditions, whereas the P II interaction regulator of the arginine synthesis, PirA, shows opposite trends (Bolay et al ., 2021; Orthwein et al ., 2021). Furthermore, proteins involved in the biosynthesis of the molybdopterin cofactor that is part of the nitrate reductase (Lin & Stewart, 1998) are also found among the downregulated proteins in LC‐shifted cells.…”