Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essential PLP-holoenzymes or synthetic lethality with cysK. In addition, the related phenotypes of pipY and pipX mutants are consistent with genetic interactions in the contexts of survival to PLP-targeting antibiotics and transcriptional regulation. We also showed that PipY overexpression increased the length of S. elongatus cells. Taken together, our results support a universal regulatory role for COG0325 proteins, paving the way to a better understanding of these proteins and of their connections with other biological processes.

Cyanobacteria, phototrophic organisms that perform oxygenic photosynthesis, perceive nitrogen status by sensing 2-oxoglutarate levels. PII, a widespread signaling protein, senses and transduces nitrogen and energy status to target proteins, regulating metabolism and gene expression. In cyanobacteria, under conditions of low 2-oxoglutarate, PII forms complexes with the enzyme N-acetyl glutamate kinase, increasing arginine biosynthesis, and with PII-interacting protein X (PipX), making PipX unavailable for binding and co-activation of the nitrogen regulator NtcA. Both the PII-PipX complex structure and in vivo functional data suggested that this complex, as such, could have regulatory functions in addition to PipX sequestration. To investigate this possibility we performed yeast three-hybrid screening of genomic libraries from Synechococcus elongatus PCC7942, searching for proteins interacting simultaneously with PII and PipX. The only prey clone found in the search expressed PlmA, a member of the GntR family of transcriptional regulators proven here by gel filtration to be homodimeric. Interactions analyses further confirmed the simultaneous requirement of PII and PipX, and showed that the PlmA contacts involve PipX elements exposed in the PII-PipX complex, specifically the C-terminal helices and one residue of the tudor-like body. In contrast, PII appears not to interact directly with PlmA, possibly being needed indirectly, to induce an extended conformation of the C-terminal helices of PipX and for modulating the surface polarity at the PII-PipX boundary, two elements that appear crucial for PlmA binding. Attempts to inactive plmA confirmed that this gene is essential in S. elongatus. Western blot assays revealed that S. elongatus PlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon. In silico studies showed that PlmA is universally and exclusively found in cyanobacteria. Based on interaction data, on the relative amounts of the proteins involved in PII-PipX-PlmA complexes, determined in western assays, and on the restrictions imposed by the symmetries of trimeric PII and dimeric PlmA molecules, a structural and regulatory model for PlmA function is discussed in the context of the cyanobacterial nitrogen interaction network.

PII, a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. PII sensing is mediated by the PII allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of PII function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a PII regulatory network. Studies on these organisms yielded a first structure of a PII complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how PII can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which PII can control gene expression. A small adaptor protein, PipX, is sequestered by PII when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of PII-NAGK, PII-PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally PII regulatory functions and reveal the changing shapes and interactions of the T-loops of PII depending on the partner and on the allosteric effectors bound to PII. Cyanobacterial studies have also revealed that in the PII-PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components of this PII-PipX regulatory network and of KD values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs.