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.

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions.

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a "false positive", the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.

Structure and dynamics of the cyanobacterial regulator SipA.

The small, 78-residue long, regulator SipA interacts with the non-bleaching sensor histidine kinase (NblS). We have solved the solution structure of SipA on the basis of 990 nuclear Overhauser effect- (NOE-) derived distance constraints. The average pairwise root-mean-square deviation (RMSD) for the twenty best structures for the backbone residues, obtained by CYANA, was 1.35 ± 0.21 Å, and 1.90 ± 0.16 Å when all heavy atoms were considered (the target function of CYANA was 0.540 ± 0.08). The structure is that of a ß-II class protein, basically formed by a five-stranded ß-sheet composed of antiparallel strands following the arrangement: Gly6-Leu11 (ß-strand 1), which packs against Leu66-Val69 (ß-strand 5) on one side, and against Gly36-Thr42 (ß-strand 2) on the other side; Trp50-Phe54 (ß-strand 3); and Gly57-Leu60 (ß-strand 4). The protein is highly mobile, as shown by measurements of R1, R2, NOE and ηxy relaxation parameters, with an average order parameter () of 0.70; this mobility encompasses movements in different time scales. We hypothesize that this high flexibility allows the interaction with other proteins (among them NblS), and it explains the large conformational stability of SipA.

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.

Functional and structural characterization of PII-like protein CutA does not support involvement in heavy metal tolerance and hints at a small-molecule carrying/signaling role.

The PII-like protein CutA is annotated as being involved in Cu2+ tolerance, based on analysis of Escherichia coli mutants. However, the precise cellular function of CutA remains unclear. Our bioinformatic analysis reveals that CutA proteins are universally distributed across all domains of life. Based on sequence-based clustering, we chose representative cyanobacterial CutA proteins for physiological, biochemical, and structural characterization and examined their involvement in heavy metal tolerance, by generating CutA mutants in filamentous Nostoc sp. and in unicellular Synechococcus elongatus. However, we were unable to find any involvement of cyanobacterial CutA in metal tolerance under various conditions. This prompted us to re-examine experimentally the role of CutA in protecting E. coli from Cu2+ . Since we found no effect on copper tolerance, we conclude that CutA plays a different role that is not involved in metal protection. We resolved high-resolution CutA structures from Nostoc and S. elongatus. Similarly to their counterpart from E. coli and to canonical PII proteins, cyanobacterial CutA proteins are trimeric in solution and in crystal structure; however, no binding affinity for small signaling molecules or for Cu2+ could be detected. The clefts between the CutA subunits, corresponding to the binding pockets of PII proteins, are formed by conserved aromatic and charged residues, suggesting a conserved binding/signaling function for CutA. In fact, we find binding of organic Bis-Tris/MES molecules in CutA crystal structures, revealing a strong tendency of these pockets to accommodate cargo. This highlights the need to search for the potential physiological ligands and for their signaling functions upon binding to CutA. DATABASES: Structural data are available in Protein Data Bank (PDB) under the accession numbers 6GDU, 6GDV, 6GDW, 6GDX, 6T76, and 6T7E.

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.

The PII-NAGK-PipX-NtcA Regulatory Axis of Cyanobacteria: A Tale of Changing Partners, Allosteric Effectors and Non-covalent Interactions.

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.

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.

Studies on cyanobacterial protein PipY shed light on structure, potential functions, and vitamin B6 -dependent epilepsy.

The Synechococcus elongatus COG0325 gene pipY functionally interacts with the nitrogen regulatory gene pipX. As a first step toward a molecular understanding of such interactions, we characterized PipY. This 221-residue protein is monomeric and hosts pyridoxal phosphate (PLP), binding it with limited affinity and losing it upon incubation with D-cycloserine. PipY crystal structures with and without PLP reveal a single-domain monomer folded as the TIM barrel of type-III fold PLP enzymes, with PLP highly exposed, fitting a role for PipY in PLP homeostasis. The mobile PLP phosphate-anchoring C-terminal helix might act as a trigger for PLP exchange. Exploiting the universality of COG0325 functions, we used PipY in site-directed mutagenesis studies to shed light on disease causation by epilepsy-associated mutations in the human COG0325 gene PROSC.

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.

Cross-talk and regulatory interactions between the essential response regulator RpaB and cyanobacterial circadian clock output.

The response regulator RpaB (regulator of phycobilisome associated B), part of an essential two-component system conserved in cyanobacteria that responds to multiple environmental signals, has recently been implicated in the control of cell dimensions and of circadian rhythms of gene expression in the model cyanobacterium Synechococcus elongatus PCC 7942. However, little is known of the molecular mechanisms that underlie RpaB functions. In this study we show that the regulation of phenotypes by RpaB is intimately connected with the activity of RpaA (regulator of phycobilisome associated A), the master regulator of circadian transcription patterns. RpaB affects RpaA activity both through control of gene expression, a function requiring an intact effector domain, and via altering RpaA phosphorylation, a function mediated through the N-terminal receiver domain of RpaB. Thus, both phosphorylation cross-talk and coregulation of target genes play a role in the genetic interactions between the RpaA and RpaB pathways. In addition, RpaB∼P levels appear critical for survival under light:dark cycles, conditions in which RpaB phosphorylation is environmentally driven independent of the circadian clock. We propose that the complex regulatory interactions between the essential and environmentally sensitive NblS-RpaB system and the SasA-RpaA clock output system integrate relevant extra- and intracellular signals to the circadian clock.

PipX, the coactivator of NtcA, is a global regulator in cyanobacteria.

To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA-PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria.

Negative control of cell size in the cyanobacterium Synechococcus elongatus PCC 7942 by the essential response regulator RpaB.

The essential NblS-RpaB pathway for photosynthesis regulation and acclimatization to a variety of environmental conditions is the most conserved two-component system in cyanobacteria. To get insights into the RpaB implication in cell homeostasis we investigated the phenotypic impact of altering expression of the essential rpaB gene of Synechococcus elongatus PCC 7942 and determined the in vivo levels of the RpaB and RpaB~P polypeptides. Our results implicate non-phosphorylated RpaB in controlling cell length and shape and suggest that intrinsic regulation may be important to prevent drastic variations in RpaB levels and activity.