Arabidopsis PII Proteins Form Characteristic Foci in Chloroplasts Indicating Novel Properties in Protein Interaction and Degradation

The PII protein is an evolutionary, highly conserved regulatory protein found in both bacteria and higher plants. In bacteria, it modulates the activity of several enzymes, transporters, and regulatory factors by interacting with them and thereby regulating important metabolic hubs, such as carbon/nitrogen homeostasis. More than two decades ago, the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behavior of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies, we found that AtPII interacts in chloroplasts with itself as well as with known interactors such as N-acetyl-L-glutamate kinase (NAGK) in dot-like aggregates, which we named PII foci. In these novel protein aggregates, AtPII also interacts with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal component of AtPII is crucial for the formation of PII foci. Altogether, the discovery and description of PII foci indicate a novel mode of interaction between PII proteins and other proteins in plants. These findings may represent a new starting point for the elucidation of physiological functions of PII proteins in plants.

Arabidopsis PII Proteins Form Characteristic Foci in Chloroplasts Indicating Novel Properties in Protein Interaction and Degradation

The PII protein is an evolutionary highly conserved regulatory protein from bacteria to higher plants. In bacteria it modulates the activity of several enzymes, transporters and regulatory factors by interacting with them and thereby regulating important metabolic hubs like carbon/nitrogen homeostasis. More than two decades ago the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behaviour of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies we found that AtPII interacts in chloroplasts with itself as well as with known interactors like NAGK in dot-like aggregates, which we named PII foci. In these novel protein aggregates AtPII interacts also with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal part of AtPII is crucial for the formation of PII foci. Altogether, the presented results indicate a novel mode of interaction for PII proteins with other proteins in plants, which may be a new starting point for the elucidation of physiological functions of PII proteins in plants.

Split NanoLuc technology allows quantitation of interactions between PII protein and its receptors with unprecedented sensitivity and reveals transient interactions

PII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed “Large BiT” and a 1.3 kDa peptide termed “Small BiT”, which only weakly associate. When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes.

Dissection of the Mechanisms of Growth Inhibition Resulting from Loss of the PII Protein in the CyanobacteriumSynechococcus elongatusPCC 7942

In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PII per se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense

ABSTRACT The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status. IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense. The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

Proteomic Insights into Starvation of Nitrogen-Replete Cells of Nostoc sp. PCC 7120 under β-N-Methylamino-L-Alanine (BMAA) Treatment

All cyanobacteria produce a neurotoxic non-protein amino acid β-N-methylamino-L-alanine (BMAA). However, the biological function of BMAA in the regulation of cyanobacteria metabolism still remains undetermined. It is known that BMAA suppresses the formation of heterocysts in diazotrophic cyanobacteria under nitrogen starvation conditions, and BMAA induces the formation of heterocyst-like cells under nitrogen excess conditions, by causing the expression of heterocyst-specific genes that are usually “silent” under nitrogen-replete conditions, as if these bacteria receive a nitrogen deficiency intracellular molecular signal. In order to find out the molecular mechanisms underlying this unexpected BMAA effect, we studied the proteome of cyanobacterium Nostoc sp. PCC 7120 grown under BMAA treatment in nitrogen-replete medium. Experiments were performed in two experimental settings: (1) in control samples consisted of cells grown without the BMAA treatment and (2) the treated samples consisted of cells grown with addition of an aqueous solution of BMAA (20 µM). In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by LC-MS/MS spectrometry. Among them, 80 proteins belonging to different functional categories were chosen for further functional analysis and interpretation of obtained proteomic data. Here, we provide the evidence that a pleiotropic regulatory effect of BMAA on the proteome of cyanobacterium was largely different under conditions of nitrogen-excess compared to its effect under nitrogen starvation conditions (that was studied in our previous work). The most significant difference in proteome expression between the BMAA-treated and untreated samples under different growth conditions was detected in key regulatory protein PII (GlnB). BMAA downregulates protein PII in nitrogen-starved cells and upregulates this protein in nitrogen-replete conditions. PII protein is a key signal transduction protein and the change in its regulation leads to the change of many other regulatory proteins, including different transcriptional factors, enzymes and transporters. Complex changes in key metabolic and regulatory proteins (RbcL, RbcS, Rca, CmpA, GltS, NodM, thioredoxin 1, RpbD, ClpP, MinD, RecA, etc.), detected in this experimental study, could be a reason for the appearance of the “starvation” state in nitrogen-replete conditions in the presence of BMAA. In addition, 15 proteins identified in this study are encoded by genes, which are under the control of NtcA—a global transcriptional regulator—one of the main protein partners and transcriptional regulators of PII protein. Thereby, this proteomic study gives a possible explanation of cyanobacterium starvation under nitrogen-replete conditions and BMAA treatment. It allows to take a closer look at the regulation of cyanobacteria metabolism affected by this cyanotoxin.

PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-L-Glutamate Kinase by L-Arginine in Corynebacterium glutamicum

ABSTRACT PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum. Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria. IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum. Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues

PII proteins comprise an ancient superfamily of signal transduction proteins, widely distributed among all domains of life. In general, PII proteins measure and integrate the current carbon/nitrogen/energy status of the cell through interdependent binding of ATP, ADP and 2-oxogluterate. In response to effector molecule binding, PII proteins interact with various PII-receptors to tune central carbon- and nitrogen metabolism. In cyanobacteria, PII regulates, among others, the key enzyme for nitrogen-storage, N-acetyl-glutamate kinase (NAGK), and the co-activator of the global nitrogen-trascription factor NtcA, the PII-interacting protein-X (PipX). One of the remarkable PII variants from Synechococcus elongatus PCC 7942 that yielded mechanistic insights in PII-NAGK interaction, is the NAGK-superactivating variant I86N. Here we studied its interaction with PipX. Another critical residue is Lys58, forming a salt-bridge with 2-oxoglutarate in a PII-ATP-2-oxoglutarate complex. Here, we show that Lys58 of PII protein is a key residue for mediating PII interactions. The K58N mutation not only causes the loss of 2-oxogluterate binding but also strongly impairs binding of ADP, NAGK and PipX. Remarkably, the exchange of the nearby Leu56 to Lys in the K58N variant partially compensates for the loss of K58. This study demonstrates the potential of creating custom tailored PII variants to modulate metabolism.