Analysing the Cyanobacterial PipX Interaction Network Using NanoBiT Complementation in Synechococcus elongatus PCC7942.

The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding to them, in vivo studies are required to understand the physiological significance and contribution of environmental factors to the regulation of PipX complexes. Here, we have used the NanoBiT complementation system to analyse the regulation of complex formation in Synechococcus elongatus PCC 7942 between PipX and each of its two best-characterized partners, PII and NtcA. Our results confirm previous in vitro analyses on the regulation of PipX-PII and PipX-NtcA complexes by 2-oxoglutarate and on the regulation of PipX-PII by the ATP/ADP ratio, showing the disruption of PipX-NtcA complexes due to increased levels of ADP-bound PII in Synechococcus elongatus. The demonstration of a positive role of PII on PipX-NtcA complexes during their initial response to nitrogen starvation or the impact of a PipX point mutation on the activity of PipX-PII and PipX-NtcA reporters are further indications of the sensitivity of the system. This study reveals additional regulatory complexities in the PipX interaction network, opening a path for future research on cyanobacteria.

The Signal Transduction Protein PII Controls the Levels of the Cyanobacterial Protein PipX.

Cyanobacteria, microorganisms performing oxygenic photosynthesis, must adapt their metabolic processes to environmental challenges such as day and night changes. PipX, a unique regulatory protein from cyanobacteria, provides a mechanistic link between the signalling protein PII, a widely conserved (in bacteria and plants) transducer of carbon/nitrogen/energy richness, and the transcriptional regulator NtcA, which controls a large regulon involved in nitrogen assimilation. PipX is also involved in translational regulation through interaction with the ribosome-assembly GTPase EngA. However, increases in the PipX/PII ratio are toxic, presumably due to the abnormally increased binding of PipX to other partner(s). Here, we present mutational and structural analyses of reported PipX-PII and PipX-NtcA complexes, leading to the identification of single amino acid changes that decrease or abolish PipX toxicity. Notably, 4 out of 11 mutations decreasing toxicity did not decrease PipX levels, suggesting that the targeted residues (F12, D23, L36, and R54) provide toxicity determinants. In addition, one of those four mutations (D23A) argued against the over-activation of NtcA as the cause of PipX toxicity. Most mutations at residues contacting PII decreased PipX levels, indicating that PipX stability would depend on its ability to bind to PII, a conclusion supported by the light-induced decrease of PipX levels in Synechococcus elongatus PCC7942 (hereafter S. elongatus).

The ribosome assembly GTPase EngA is involved in redox signaling in cyanobacteria.

Photosynthetic organisms must cope with environmental challenges, like those imposed by the succession of days and nights or by sudden changes in light intensities, that trigger global changes in gene expression and metabolism. The photosynthesis machinery is particularly susceptible to environmental changes and adaptation to them often involves redox-sensing proteins that are the targets of reactive oxygen species generated by photosynthesis activity. Here we show that EngA, an essential GTPase and ribosome-assembly protein involved in ribosome biogenesis in bacteria and chloroplasts, also plays a role in acclimatization to environmentally relevant stress in Synechococcus elongatus PCC7942 and that PipX, a promiscuous regulatory protein that binds to EngA, appears to fine-tune EngA activity. During growth in cold or high light conditions, the EngA levels rise, with a concomitant increase of the EngA/PipX ratio. However, a sudden increase in light intensity turns EngA into a growth inhibitor, a response involving residue Cys122 of EngA, which is part of the GD1-G4 motif NKCES of EngA proteins, with the cysteine conserved just in the cyanobacteria-chloroplast lineage. This work expands the repertoire of ribosome-related factors transmitting redox signals in photosynthetic organisms and provides additional insights into the complexity of the regulatory interactions mediated by EngA and PipX.

Pleiotropic effects of PipX, PipY, or RelQ overexpression on growth, cell size, photosynthesis, and polyphosphate accumulation in the cyanobacterium Synechococcus elongatus PCC7942.

The cyanobacterial protein PipY belongs to the Pyridoxal-phosphate (PLP)-binding proteins (PLPBP/COG0325) family of pyridoxal-phosphate-binding proteins, which are represented in all three domains of life. These proteins share a high degree of sequence conservation, appear to have purely regulatory functions, and are involved in the homeostasis of vitamin B6 vitamers and amino/keto acids. Intriguingly, the genomic context of the pipY gene in cyanobacteria connects PipY with PipX, a protein involved in signaling the intracellular energy status and carbon-to-nitrogen balance. PipX regulates its cellular targets via protein-protein interactions. These targets include the PII signaling protein, the ribosome assembly GTPase EngA, and the transcriptional regulators NtcA and PlmA. PipX is thus involved in the transmission of multiple signals that are relevant for metabolic homeostasis and stress responses in cyanobacteria, but the exact function of PipY is still elusive. Preliminary data indicated that PipY might also be involved in signaling pathways related to the stringent stress response, a pathway that can be induced in the unicellular cyanobacterium Synechococcus elongatus PCC7942 by overexpression of the (p)ppGpp synthase, RelQ. To get insights into the cellular functions of PipY, we performed a comparative study of PipX, PipY, or RelQ overexpression in S. elongatus PCC7942. Overexpression of PipY or RelQ caused similar phenotypic responses, such as growth arrest, loss of photosynthetic activity and viability, increased cell size, and accumulation of large polyphosphate granules. In contrast, PipX overexpression decreased cell length, indicating that PipX and PipY play antagonistic roles on cell elongation or cell division. Since ppGpp levels were not induced by overexpression of PipY or PipX, it is apparent that the production of polyphosphate in cyanobacteria does not require induction of the stringent response.

The Conserved Family of the Pyridoxal Phosphate-Binding Protein (PLPBP) and Its Cyanobacterial Paradigm PipY.

The PLPBP family of pyridoxal phosphate-binding proteins has a high degree of sequence conservation and is represented in all three domains of life. PLPBP members, of which a few representatives have been studied in different contexts, are single-domain proteins with no known enzymatic activity that exhibit the fold type III of PLP-holoenzymes, consisting in an α/ß barrel (TIM-barrel), where the PLP cofactor is solvent-exposed. Despite the constant presence of cofactor PLP (a key catalytic element in PLP enzymes), PLPBP family members appear to have purely regulatory functions affecting the homeostasis of vitamin B6 vitamers and amino/keto acids. Perturbation of these metabolites and pleiotropic phenotypes have been reported in bacteria and zebrafish after PLPBP gene inactivation as well as in patients with vitamin B6-dependent epilepsy that results from loss-of-function mutations at the PLPBP. Here, we review information gathered from diverse studies and biological systems, emphasizing the structural and functional conservation of the PLPBP members and discussing the informative nature of model systems and experimental approaches. In this context, the relatively high level of structural and functional characterization of PipY from Synechococcus elongatus PCC 7942 provides a unique opportunity to investigate the PLPBP roles in the context of a signaling pathway conserved in cyanobacteria.

Regulatory Connections Between the Cyanobacterial Factor PipX and the Ribosome Assembly GTPase EngA.

Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to important environmental challenges, like those imposed by the succession of days and nights. Not surprisingly, certain regulatory proteins are found exclusively in this phylum. One of these unique proteins, PipX, provides a mechanistic link between signals of carbon/nitrogen and of energy, transduced by the signaling protein PII, and the control of gene expression by the global nitrogen regulator NtcA. PII, required for cell survival unless PipX is inactivated or downregulated, functions by protein-protein interactions with transcriptional regulators, transporters, and enzymes. PipX also functions by protein-protein interactions, and previous studies suggested the existence of additional interacting partners or included it into a relatively robust six-node synteny network with proteins apparently unrelated to the nitrogen regulation system. To investigate additional functions of PipX while providing a proof of concept for the recently developed cyanobacterial linkage network, here we analyzed the physical and regulatory interactions between PipX and an intriguing component of the PipX synteny network, the essential ribosome assembly GTPase EngA. The results provide additional insights into the functions of cyanobacterial EngA and of PipX, showing that PipX interacts with the GD1 domain of EngA in a guanosine diphosphate-dependent manner and interferes with EngA functions in Synechococcus elongatus at a low temperature, an environmentally relevant context. Therefore, this work expands the PipX interaction network and establishes a possible connection between nitrogen regulation and the translation machinery. We discuss a regulatory model integrating previous information on PII-PipX with the results presented in this work.

The default cyanobacterial linked genome: an interactive platform based on cyanobacterial linkage networks to assist functional genomics.

A database of cyanobacterial linked genomes that can be accessed through an interactive platform (https://dfgm.ua.es/genetica/investigacion/cyanobacterial_genetics/Resources.html) was generated on the bases of conservation of gene neighborhood across 124 cyanobacterial species. It allows flexible generation of gene networks at different threshold values. The default cyanobacterial linked genome, whose global properties are analyzed here, connects most of the cyanobacterial core genes. The potential of the web tool is discussed in relation to other bioinformatics approaches based on guilty-by-association principles, with selected examples of networks illustrating its usefulness for genes found exclusively in cyanobacteria or in cyanobacteria and chloroplasts. We believe that this tool will provide useful predictions that are readily testable in Synechococcus elongatus PCC7942 and other model organisms performing oxygenic photosynthesis.