Effects of spontaneous mutations in PipX functions and regulatory complexes on the cyanobacterium Synechococcus elongatus strain PCC 7942

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

doi:10.1099/mic.0.037309-0

Cited by 27 publications

(66 citation statements)

References 36 publications

(36 reference statements)

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“…Importantly, Western blots showed that individual inactivation of pipX or pipY decreased the levels of PipY or PipX by 15-fold or 2-fold, respectively ( Figure 2 and Table 2 ), suggesting that the protein levels of PipY are highly dependent on pipX gene or gene product(s). In line with this, expression from the strong CK1 promoter (strain CK1X), resulting in a significant increase in the levels of PipX (Espinosa et al, 2010), produced a 3.4-fold and 4.3-fold increase in PipX and PipY, respectively ( Figure 2 ).…”

Section: Resultsmentioning

confidence: 55%

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

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 55%

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

et al. 2017

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“…In some cases, inactivation of essential genes may be facilitated by suppressor mutations, as it is the case with glnB in S. elongatus , which can be readily inactivated in the presence of loss-of-function mutations at pipX (Espinosa et al, 2009, 2010). However, attempts to obtain S. elongatus plmA null strains in different mutant backgrounds have so far been unsuccessful (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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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.

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“…Under these nitrogen regimes, particularly in ammonium, the concentration of 2-OG is expected to be relatively low and transcriptional regulation by NtcA-PipX complexes would not have widespread importance in WT cells. Because the pipX Y32A or pipX E4A allele is associated with a resistance marker (C.S3 cassette) that decreases pipX gene expression (18), a WT derivative with the same insertion in the identical position (strain CS3X) was required to perform isogenic mutant/control comparisons. Therefore, two strains were used as WT controls: WT S. elongatus for mutant strain SA591, which carries the ΔpipX allele, and CS3X for strains CS3X E4A and CS3X…”

Section: Resultsmentioning

confidence: 99%

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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Effects of spontaneous mutations in PipX functions and regulatory complexes on the cyanobacterium Synechococcus elongatus strain PCC 7942

Cited by 27 publications

References 36 publications

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

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

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

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

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