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.This work was supported by the Ministerio de Ciencia e Innovación (Grant BFU2009-07371) and the Generalitat Valenciana (ACOMP2911/211)

Desde el platanal: un sinsonte y una hamaca. Estudio contrastivo de la obra teatral de Abilio Estévez

Universidad de Sevilla. Grado Filológica Hispánic

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).This work was supported by grant PID2020-118816GB-I00 from the Spanish Government (MICINN) and grants VIGROB22-126, VIGROB23-126, and GRE20-04-C from the University of Alicante

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 authors thank the Spanish Government (MINECO) for supporting grants BFU2012‐33364 and BFU2015‐66360‐P to A.C. and the University of Alicante for PhD fellowship (FPUUA59) to J.I.L

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/β 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.Work in our laboratories were supported by grants PID220‐118816GB‐I00 and BFU2015‐66360‐P from the Spanish Government (MICINN), grants GRE20‐04‐C, VIGROB20‐126, UADIF20‐34 and UADIF19‐36 from the University of Alicante (to AC) and grant CIVP‐20A6610 from the Fundación Ramón Areces (to VR)

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.Generalitat Valenciana: PROMETEO/2017/129; Universidad de Alicante: VIGROB-126/19; UAUSTI18 and FPUUA59 fellowship to J.I.L

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.This work was supported by Spanish Ministry of Economy and Competitivity Grant BFU2012-33364 (to A.C.); Generalitat Valenciana Grants ACOMP/2014/144 (to A.C.) and GV/2014/073 (to J.E.); National Institutes of Health Grant R01GM062419 (to S.S.G.); and European Molecular Biology Organization postdoctoral Fellowship ASTF 74–2013 (to J.E.)

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.This work was supported by grants from the Valencian (Gerónimo Forteza contract FPA/2015/052 to JL; PrometeoII/2014/029 to VR) and Spanish Governments (BFU2012-33364 and BFU2015-66360-P to AC; BFU2014-58229-P to VR)

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.This work was supported by grant PID2020-118816GB-I00, funded by MCIN/AEI/10.13039/501100011033, from the Spanish Government; grants VIGROB23-126 and GRE20-04-C, from the University of Alicante, both to A.C.; and grant DFG Fo195/21-1 to K.F. C.J. was the recipient of a Ph.D. fellowship (ACIF/2019/045) from Conselleria d’Innovació, Universitats, Ciència i Societat Digital of the Generalitat Valenciana. S.B. was supported by a National Grant from the Algerian Ministry of Higher Education and Scientific Research

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 β-II class protein, basically formed by a five-stranded β-sheet composed of antiparallel strands following the arrangement: Gly6-Leu11 (β-strand 1), which packs against Leu66-Val69 (β-strand 5) on one side, and against Gly36-Thr42 (β-strand 2) on the other side; Trp50-Phe54 (β-strand 3); and Gly57-Leu60 (β-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.This research was funded by Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana) [CIAICO 2021/0135 to ACA and JLN and PROMETEO/2020/012 to AM], Ministerio de Ciencia e Innovación (PID2020-118816GB-I00 to AC and PID2022-137201NB-I00 to AM) and by the European Commission NextGenerationEU fund (EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global) to AM