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.

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.

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.

σ54-Dependent Response to Nitrogen Limitation and Virulence in Burkholderia cenocepacia Strain H111

ABSTRACTMembers of the genusBurkholderiaare versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogenBurkholderia cenocepaciaH111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC,B. cenocepaciaH111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a σ54consensus sequence. The mapping of the σ54regulon as well as the characterization of a σ54mutant suggests an important role of σ54not only in control of nitrogen metabolism but also in the virulence of this organism.

Nitrogen Metabolism in Sinorhizobium meliloti–Alfalfa Symbiosis: Dissecting the Role of GlnD and PII Proteins

To contribute nitrogen for plant growth and establish an effective symbiosis with alfalfa, Sinorhizobium meliloti Rm1021 needs normal operation of the GlnD protein, a bifunctional uridylyltransferase/uridylyl-cleavage enzyme that measures cellular nitrogen status and initiates a nitrogen stress response (NSR). However, the only two known targets of GlnD modification in Rm1021, the PII proteins GlnB and GlnK, are not necessary for effectiveness. We introduced a Tyr→Phe variant of GlnB, which cannot be uridylylated, into a glnBglnK background to approximate the expected state in a glnD-sm2 mutant, and this strain was effective. These results suggested that unmodified PII does not inhibit effectiveness. We also generated a glnBglnK-glnD triple mutant and used this and other mutants to dissect the role of these proteins in regulating the free-living NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation was dominant to the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes. The data show that the GlnD protein has a role in free-living growth and in symbiotic nitrogen exchange that does not depend on the PII proteins, suggesting that S. meliloti GlnD can communicate with the cell by alternate mechanisms.

In vitro proof of direct regulation of glutamine synthetase by GlnK proteins in the extreme halophilic archaeon Haloferax mediterranei

Haloferax mediterranei is an extreme halophilic micro-organism belonging to the Archaea domain that was isolated from the Santa Pola solar salterns (Alicante, Spain) in 1983. The biochemistry of the proteins involved in nitrogen metabolism is being studied, but the knowledge of their regulation is very scarce at present. The PII superfamily is constituted by major regulators of nitrogen metabolism, which are widespread in prokaryotic and eukaryotic organisms. These trimeric proteins (12 kDa per subunit) have in Escherichia coli long been known to regulate GS (glutamine synthetase) activity via its adenylyltransferase/adenylyl-removing enzyme and, more recently, to be able to interact directly with this enzyme in methanogenic archaea. We have tested the possible role of PII proteins in the regulation of ammonium assimilation in our model organism and the results clearly indicate that the direct influence of GS by PII proteins can also take place in halophilic archaea, starting with the comprehension of nitrogen regulation in those organisms.