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

Abstract

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.

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles

“…To demonstrate that the localization of PipX‐GFP into foci was not an artefact, but relied on the regulated formation of PII‐PipX complexes, we next engineered and subsequently analysed in WT and pipX null backgrounds PipX‐GFP and its derivatives PipX E4A ‐GFP and PipX Y32A ‐GFP. The later proteins carry amino acid substitutions that impair interactions between PipX and PII, with Y32A having more drastic effects on PII binding than E4A (Espinosa et al ., ; Laichoubi et al ., ; Espinosa et al ., ; Labella et al ., ). In complete agreement with the specific effects of the corresponding point mutations on the binding to PII, cells expressing PipX E4A ‐GFP or, to a much greater extent, PipX Y32A ‐GFP, reduced the number of cells with foci (Fig.…”

“…PipX uses the same surface to bind to either 2‐OG‐bound NtcA, stimulating DNA binding and transcriptional activity, or to 2‐OG‐free PII to form PII‐PipX complexes (Tanigawa et al ., ; Vazquez‐Bermudez et al ., ; Llacer et al ., ; Zhao et al ., ). PII‐PipX complexes interact with the transcriptional regulator PlmA, presumably to decrease transcriptional activity (Labella et al ., ).…”

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

“…The small protein PipX is a key player, as highlighted by transcriptomic studies in Synechococcus elongatus PCC7942 (from now on S. elongatus ) . PipX regulation, mediated by the carbon/energy/nitrogen signaling protein PII and the gene expression regulator NtcA , also involves newly discovered partners as PlmA .…”

The nitrogen regulator PipX acts in cis to prevent operon polarity

“…Fluctuation of C/N and energy signals would drive partner swapping, changing the relative proportion of PII–PipX and NtcA–PipX (Espinosa et al ., , ), but still preventing PipX from making interactions with additional partners. Additional support for this idea comes from the finding that PII, which appears to be 14 times more abundant than PipX (Guerreiro et al ., ; Labella et al ., ) prevents PipX toxicity in S. elongatus (Espinosa et al ., , ; Chang et al ., ), a phenomenon further suggesting that PII binding is counteracting the interaction of PipX with yet unidentified low affinity partners.…”

“…PipX uses the same surface, which involve the tudor-like domain (TLD) (Llacer et al, 2010) to bind to either 2-OG-bound NtcA, stimulating DNA binding and transcriptional activity, or to 2-OG-free PII to form PII-PipX complexes (Tanigawa et al, 2002;Vazquez-Bermudez et al, 2002;Llacer et al, 2010;Zhao et al, 2010). PII-PipX complexes interact with the transcriptional regulator PlmA, presumably to decrease transcriptional activity (Labella et al, 2016). When considering the affinities between PipX and partners NtcA (Kd of 85 nM) (Forcada-Nadal et al, 2014) and PII (Kd of 7 μM in the absence of 2-OG and nucleotides) (Llacer et al, 2010) and the relative abundances of the individual proteins in S. elongatus (in molar terms of subunits PII is 14 times more abundant than PipX which is four times more abundant than NtcA) (Guerreiro et al, 2016;Labella et al, 2016), it appears that, under a wide range of conditions, most of the PipX protein would be forming part of proteins complexes with PII and/or NtcA.…”