In Synechococcus elongatus sp. PCC 7942, PipX forms complexes with P II , a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, and with the cyanobacterial nitrogen regulator NtcA. We recently showed that previous inactivation of pipX facilitates subsequent inactivation of the glnB gene. Here, we show that the three spontaneous pipX point mutations pipX-92delT, pipX160C.T and pipX194T.A, initially found in different glnB strains, are indeed suppressor mutations. When these mutations were reconstructed in the wild-type background, the glnB gene could be efficiently inactivated. Furthermore, the point mutations have different effects on PipX levels, coactivation of NtcA-dependent genes and protein-protein interactions. Further support for an in vivo role of PipX-P II complexes is provided by interaction analysis with the in vivo-generated P II T-loop+7 protein, a P II derivative unable to interact with its regulatory target N-acetyl-L-glutamate kinase, but which retains the ability to bind to PipX. The implications of these results are discussed.

PipX provides a functional link between the cyanobacterial global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as integrators of signals of the nitrogen and carbon balance. PipX, which is toxic in the absence of PII, can form alternative complexes with NtcA and PII and these interactions are respectively stimulated and inhibited by 2-oxoglutarate, providing a mechanism by which PII can modulate expression at the NtcA regulon. Structural information on PipX-NtcA complexes suggests that PipX coactivates NtcA controlled genes by stabilizing the active conformation of NtcA bound to 2-oxoglutarate and by possibly helping recruit RNA polymerase. To get insights into PipX functions, we perform here a mutational analysis of pipX informed by the structures of PipX-PII and PipX-NtcA complexes and evaluate the impact of point mutations on toxicity and gene expression. Two amino acid substitutions (Y32A and E4A) were of particular interest, since they increased PipX toxicity and activated NtcA dependent genes in vivo at lower 2-oxoglutarate levels than wild type PipX. While both mutations impaired complex formation with PII, only Y32A had a negative impact on PipX-NtcA interactions.

The P II proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of P II proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which P II proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where P II forms complexes with the NtcA coactivator PipX, attempts to engineer P II -deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that P II is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated.Cyanobacteria are phototrophic organisms that perform oxygenic photosynthesis. Autotrophic growth requires the constant assimilation of ammonium via the coordinated action of glutamine synthetase (GS) and glutamate synthase, also known as the GS-GOGAT cycle, resulting in consumption of 2-oxoglutarate (34). Due to the lack of 2-oxoglutarate dehydrogenase in cyanobacteria, synthesis of 2-oxoglutarate represents the final step in the oxidative branch of the trichloroacetic acid cycle and directly links 2-oxoglutarate levels to nitrogen assimilation (35). Thus, 2-oxoglutarate accumulates during nitrogen starvation, making this metabolite an excellent indicator of the intracellular carbon-nitrogen balance (12,25).In cyanobacteria, multiple metabolic and developmental processes are induced by nitrogen starvation. NtcA, the global regulator for nitrogen control, activates genes involved in nitrogen assimilation, heterocyst differentiation, and acclimation to nitrogen starvation (20,30,41). 2-Oxoglutarate, the signal of nitrogen deficiency, stimulates binding of NtcA to target sites (45), transcription activation in vitro (44), and complex formation between the global nitrogen regulator NtcA and its coactivator factor PipX, a regulatory protein conserved in cyanobacteria (5, 9). The interaction between PipX and NtcA is known to be relevant for maximal activation of NtcA-dependent genes under nitrogen limitation (9, 10). PipX-deficient cultures of Synechococcus elongatus PCC 7942 showed reduced activity of nitrogen assimilation enzymes, retarded nitrogen induction, a slower rate of nitrate consumption, and when subjected to nitrogen starvation, retarded phycobilisome degradation and faster reduction of the chlorophyll content (10).The homotrimeric P II protein is one of the most conserved and widespread signal transduction proteins in nature and plays key roles in nitrogen assimilatory processes (28). P II proteins contain three binding sites (one per subunit) for 2-oxoglutarate and ATP, and their primary function is to regulate, by direct protein-protein interactions, the activity of proteins implicated in nitrogen metabolism (reviewed in reference 13). In cyanobacteria, several proteins are k...

Nitrogen regulation involves the formation of different types of protein complexes between signal transducers and their transcriptional or metabolic targets. In oxygenic phototrophs, the signal integrator P II activates the enzyme N-acetyl-L-glutamate kinase (NAGK) by complex formation. P II also interacts with PipX, a protein with a tudor-like domain that mediates contacts with P II and with the transcriptional regulator NtcA, to which it binds to increase its activity. Here, we use a combination of in silico, yeast two-hybrid and in vitro approaches to investigate the nitrogen regulation network of Synechococcus WH5701, a marine cyanobacterium with two P II (GlnB_A and GlnB_B) and two PipX (PipX_I and PipX_II) proteins. Our results indicate that GlnB_A is functionally equivalent to the canonical P II protein from Synechococcus elongatus. GlnB_A interacted with PipX and NAGK proteins and stimulated NAGK activity, counteracting arginine inhibition. GlnB_B had only a slight stimulatory effect on NAGK activity, but its potential to bind effectors and form heterotrimers in Synechococcus WH5701 indicates additional regulatory functions. PipX_II, and less evidently PipX_I, specifically interacted with GlnB_A and NtcA, supporting a role for both Synechococcus WH5701 PipX proteins in partner swapping with GlnB_A and NtcA.