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.

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.

Distinctive Features of PipX, a Unique Signaling Protein of Cyanobacteria.

PipX is a unique cyanobacterial protein identified by its ability to bind to PII and NtcA, two key regulators involved in the integration of signals of the nitrogen/carbon and energy status, with a tremendous impact on nitrogen assimilation and gene expression in cyanobacteria. PipX provides a mechanistic link between PII, the most widely distributed signaling protein, and NtcA, a global transcriptional regulator of cyanobacteria. PII, required for cell survival unless PipX is inactivated or down-regulated, functions by protein-protein interactions with transcriptional regulators, transporters, and enzymes. In addition, PipX appears to be involved in a wider signaling network, supported by the following observations: (i) PII-PipX complexes interact with PlmA, an as yet poorly characterized transcriptional regulator also restricted to cyanobacteria; (ii) the pipX gene is functionally connected with pipY, a gene encoding a universally conserved pyridoxal phosphate binding protein (PLPBP) involved in vitamin B6 and amino acid homeostasis, whose loss-of-function mutations cause B6-dependent epilepsy in humans, and (iii) pipX is part of a relatively robust, six-node synteny network that includes pipY and four additional genes that might also be functionally connected with pipX. In this overview, we propose that the study of the protein-protein interaction and synteny networks involving PipX would contribute to understanding the peculiarities and idiosyncrasy of signaling pathways that are conserved in cyanobacteria.

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.

The nitrogen regulator PipX acts in cis to prevent operon polarity.

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 factors, PipX, provides a mechanistic link between signals of carbon/nitrogen and of energy, transduced by the signalling protein PII, and the control of gene expression by the global nitrogen regulator NtcA. Here we report a new regulatory function of PipX: enhancement in cis of pipY expression, a gene encoding a universally conserved protein involved in amino/keto acid and Pyridoxal phosphate homeostasis. In Synechococcus elongatus and many other cyanobacteria these genes are expressed as a bicistronic pipXY operon. Despite being cis-acting, polarity suppression by PipX is nevertheless reminiscent of the function of NusG paralogues typified by RfaH, which are non-essential operon-specific bacterial factors acting in trans to upregulate horizontally-acquired genes. Furthermore, PipX and members of the NusG superfamily share a TLD/KOW structural domain, suggesting regulatory interactions of PipX with the translation machinery. Our results also suggest that the cis-acting function of PipX is a sophisticated regulatory strategy for maintaining appropriate PipX-PipY stoichiometry.

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles.

Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to the challenges imposed by the succession of days and nights. Two conserved cyanobacterial proteins, PII and PipX, function as hubs of the nitrogen interaction network, forming complexes with a variety of diverse targets. While PII proteins are found in all three domains of life as integrators of signals of the nitrogen and carbon balance, PipX proteins are unique to cyanobacteria, where they provide a mechanistic link between PII signalling and the control of gene expression by the global nitrogen regulator NtcA. Here we demonstrate that PII and PipX display distinct localization patterns during diurnal cycles, co-localizing into the same foci at the periphery and poles of the cells during dark periods, a circadian-independent process requiring a low ATP/ADP ratio. Genetic, cellular biology and biochemical approaches used here provide new insights into the nitrogen regulatory network, calling attention to the roles of PII as energy sensors and its interactions with PipX in the context of essential signalling pathways. This study expands the contribution of the nitrogen regulators PII and PipX to integrate and transduce key environmental signals that allow cyanobacteria to thrive in our planet.

PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network.

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.

Expanding the Cyanobacterial Nitrogen Regulatory Network: The GntR-Like Regulator PlmA Interacts with the PII-PipX Complex.

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.