PII, an ancient and widespread signaling protein, transduces nitrogen/carbon/energy abundance signals through interactions with target proteins. We clarify structurally how PII regulates gene expression mediated by the transcription factor NtcA, the global nitrogen regulator of cyanobacteria, shedding light on NtcA structure and function and on how NtcA is activated by 2-oxoglutarate (2OG) and coactivated by the nonenzymatic PII target, protein PipX. We determine for the cyanobacteria Synechococcus elongatus the crystal structures of the PII-PipX and PipX-NtcA complexes and of NtcA in active and inactive conformations (respective resolutions, 3.2, 2.25, 2.3, and 3.05 A). The structures and the conclusions derived from them are consistent with the results of present and prior site-directed mutagenesis and functional studies. A tudor-like domain (TLD) makes up most of the PipX structure and mediates virtually all the contacts of PipX with PII and NtcA. In the PII-PipX complex, one PII trimer sequesters the TLDs of three PipX molecules between its body and its extended T loops, preventing PipX activation of NtcA. Changes in T loop conformation triggered by 2OG explain PII-PipX dissociation when 2OG is bound. The structure of active dimeric NtcA closely resembles that of the active cAMP receptor protein (CRP). This strongly suggests that with these proteins DNA binding, transcription activation, and allosteric regulation occur by common mechanisms, although the effectors are different. The PipX-NtcA complex consists of one active NtcA dimer and two PipX monomers. PipX coactivates NtcA by stabilizing its active conformation and by possibly helping recruit RNA polymerase but not by providing extra DNA contacts.

SummaryCyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. The global nitrogen transcription factor NtcA and the signal transduction protein PII are both involved in 2-oxoglutarate sensing. PII proteins, probably the most conserved signal transduction proteins in nature, are remarkable for their ability to interact with very diverse protein targets in different systems. Despite widespread efforts to understand nitrogen signalling in cyanobacteria, the involvement of PII in the regulation of transcription activation by NtcA remains enigmatic. Here we show that PipX, a protein only present in cyanobacteria, interacts with both PII and NtcA and provides a mechanistic link between these two factors. A variety of in vivo and in vitro approaches were used to study PipX and its interactions with PII and NtcA. 2-Oxoglutarate favours complex formation between PipX and NtcA, but impairs binding to PII, suggesting that partner swapping between these nitrogen regulators is driven by the 2-oxoglutarate concentration. PipX is required for NtcA-dependent transcriptional activation in vivo, thus implying that PipX may function as a prokaryotic transcriptional coactivator.

Direct measurement of prevalence of palliative care needs on a population basis is feasible. Early identification and prevalence determination of these patients is likely to be the cornerstone of palliative care public health policies.

Palliative care (PC) has focused on patients with cancer within specialist services. However, around 75% of the population in middle-income and high-income countries die of one or more chronic advanced diseases. Early identification of such patients in need of PC becomes crucial. In this feature article we describe the initial steps of the NECPAL (Necesidades Paliativas [Palliative Needs]) Programme. The focus is on development of the NECPAL tool to identify patients in need of PC; preliminary results of the NECPAL prevalence study, which assessed prevalence of advanced chronically ill patients within the population and all socio-health settings of Osona; and initial implementation of the NECPAL Programme in the region. As first measures of the Programme, we present the NECPAL tool. The main differences from the British reference tools on which NECPAL is based are highlighted. The preliminary results of the prevalence study show that 1.45% of the total population and 7.71% of the population aged over 65 are 'surprise question' positive, while 1.33% and 7.00%, respectively, are NECPAL positive, and surprise question positive with at least one additional positive parameter. More than 50% suffer from geriatric pluri-pathology conditions or dementia. The pilot phase of the Programme consists of developing sectorised policies to improve PC in three districts of Catalonia. The first steps to design and implement a Programme to improve PC for patients with chronic conditions with a public health and population-based approach are to identify these patients and to assess their prevalence in the healthcare system.

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.

To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial P II -interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein P II , 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 P II , and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between P II 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.nitrogen regulation | transcription | translation | photosynthesis

The Synechococcus sp. PCC 7942 nitrogen regulator PipX interacts in a 2-oxoglutaratedependent manner with the global nitrogen transcription factor NtcA and the signal transduction protein P II . In vivo, PipX is involved in the NtcA-dependent induction of glnB and glnN genes. To further investigate the extent to which PipX is involved in global nitrogen control, the effect of pipX inactivation on various nitrogen-regulated processes was determined. The PipX-deficient mutant was able to use nitrate as a nitrogen source and to efficiently inhibit the nitrate transport upon ammonium addition but showed decreased nitrate and nitrite reductase activities and a delay in the induction of nitrate utilization after transfer of cultures from ammonium-to nitrate-containing media. In contrast to the wild-type, glutamine synthetase activity was not upregulated upon depletion of combined nitrogen from cultures of the mutant strain. Inactivation of pipX impaired induction of nblA and delayed phycobilisome degradation, but did not affect recovery of nitrogen-deprived cultures. Taken together, the results indicate that PipX interacts with NtcA to facilitate efficient acclimation of cyanobacteria to conditions of nitrogen limitation.

This study demonstrates that FPNS is efficacious, well accepted, and well tolerated by patients with BTCP.