Mutational Analysis of the Cyanobacterial Nitrogen Regulator PipX

Laichoubi, Karim Boumediene; Espinosa, José; Castells, M.A.; Contreras, Asunción

doi:10.1371/journal.pone.0035845

Cited by 23 publications

(58 citation statements)

References 38 publications

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“…S. elongatus strains and plasmids are listed in SI Appendix, Table S1. Growth, genetic manipulations and nitrate uptake assays were as described (13,17).…”

Section: Methodsmentioning

confidence: 99%

“…and pipX E4A alleles is counteracted by P II (17,19). To provide functional evidence that the mutant proteins PipX Y32A and PipX E4A exerted their effect on class 4 genes by interacting with NtcA rather than an NtcA-independent mechanism, we tested two functions that require NtcA in S. elongatus: growth on nitrate as a nitrogen source and nitrate transport.…”

Section: Y32amentioning

confidence: 99%

“…Crystal structures of PipX-P II complexes, surface plasmon resonance, P II -stimulated NAGK activity assays, and yeast two-hybrid analysis established the importance of PipX residues Y32 and E4 for interactions with P II proteins and of Y32 for interactions with NtcA (15,17). Reporter and transcript analyses indicated that both Y32A and E4A mutations had stimulatory effects on the NtcA-activated genes glnB, glnN, and nblA but did not address differences between the in vivo action of PipX E4A and PipX…”

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confidence: 94%

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

Espinosa¹,

Rodríguez

Salinas³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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

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“…S. elongatus strains and plasmids are listed in SI Appendix, Table S1. Growth, genetic manipulations and nitrate uptake assays were as described (13,17).…”

Section: Methodsmentioning

confidence: 99%

Section: Y32amentioning

confidence: 99%

mentioning

confidence: 94%

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

Espinosa¹,

Rodríguez

Salinas³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In brief, two major targets of PII regulation have been identified at the molecular level, the transcriptional co-activator PipX and NAGK. PipX, a small protein of approximately 90 amino acids, was shown to be required for full expression of NtcA-activated genes (Espinosa et al 2006(Espinosa et al , 2007Laichoubi et al 2012). Binding of PipX to NtcA is favored by high 2-OG levels in vitro, whereas 2-OG in concert with Mg-ATP prevents binding of PipX to PII.…”

Section: Further Functions Of Pii Signaling In Oxygenic Phototrophsmentioning

confidence: 99%

From cyanobacteria to plants: conservation of PII functions during plastid evolution

Chellamuthu

Alva

Forchhammer

2012

Planta

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This article reviews the current state-of-the-art concerning the functions of the signal processing protein PII in cyanobacteria and plants, with a special focus on evolutionary aspects. We start out with a general introduction to PII proteins, their distribution, and their evolution. We also discuss PII-like proteins and domains, in particular, the similarity between ATP-phosphoribosyltransferase (ATP-PRT) and its PII-like domain and the complex between N-acetyl-L-glutamate kinase (NAGK) and its PII activator protein from oxygenic phototrophs. The structural basis of the function of PII as an ATP/ADP/ 2-oxoglutarate signal processor is described for Synechococcus elongatus PII. In both cyanobacteria and plants, a major target of PII regulation is NAGK, which catalyzes the committed step of arginine biosynthesis. The common principles of NAGK regulation by PII are outlined. Based on the observation that PII proteins from cyanobacteria and plants can functionally replace each other, the hypothesis that PII-dependent NAGK control was under selective pressure during the evolution of plastids of Chloroplastida and Rhodophyta is tested by bioinformatics approaches. It is noteworthy that two lineages of heterokont algae, diatoms and brown algae, also possess NAGK, albeit lacking PII; their NAGK however appears to have descended from an alphaproteobacterium and not from a cyanobacterium as in plants. We end this article by coming to the conclusion that during the evolution of plastids, PII lost its function in coordinating gene expression through the PipX-NtcA network but preserved its role in nitrogen (arginine) storage metabolism, and subsequently took over the fine-tuned regulation of carbon (fatty acid) storage metabolism, which is important in certain developmental stages of plants.

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“…Under the physiological range of 2-OG levels, pipX mutations specifically impairing PipX-PII complexes favor formation of PipX-NtcA complexes. PipX residues Y32 and E4 are important for interactions with PII and NtcA proteins and Y32 for interactions with NtcA and these differences affect both NtcAdependent and independent genes [3,4] . The participation of PipX in at least 3 NtcA-independent scenarios was proposed.…”

Section: Introductionmentioning

confidence: 99%

Using Multivariate Analysis and Bioinformatic Tools to Elucidate the Functions of a Cyanobacterial Global Regulator from RNA-Seq Data Obtained in Different Genetic and Environmental Backgrounds

Labella

Rodríguez

Espinosa

et al. 2015

Bioinformatics and Biomedical Engineering

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Abstract. The cyanobacterial protein PipX, known as the coactivator of the nitrogen regulator NtcA, regulates multiple operons, many of them in an NtcA-independent manner. PipX has no DNA-binding activity and must interact with additional regulators to affect gene expression. Here we follow different bioinformatic approaches to analyze previous transcriptomic data from 10 different genetic or environmental backgrounds. Using standardized residuals from 8 mutant/control comparisons a set of 331 differentially expressed genes were obtained, which were clustered according to their patterns of expression. The clusters showed significant internal coherence, supporting inferences on PipX function and providing evidence of additional complexity for in vivo interactions. The Perl based program MultiGS, designed to help searches for regulatory motifs, was used to help finding NtcA motifs within the cluster containing nitrogen assimilation genes. The results pave the way to the identification of the yet unknown transcriptional regulator(s) involved in the NtcA-independent PipX regulons.

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Mutational Analysis of the Cyanobacterial Nitrogen Regulator PipX

Cited by 23 publications

References 38 publications

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

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

From cyanobacteria to plants: conservation of PII functions during plastid evolution

Using Multivariate Analysis and Bioinformatic Tools to Elucidate the Functions of a Cyanobacterial Global Regulator from RNA-Seq Data Obtained in Different Genetic and Environmental Backgrounds

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