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Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the P(II)family. P(II) proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to P(II) and also by P(II) posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the P(II) protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 Å resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known P(II) complexes, the majority of contacts with the target protein do not involve the T-loop region of P(II). Hence this structure identifies a different mode of P(II) interaction with a target protein and demonstrates the potential for P(II) proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZ-DraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB.

Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the P(II) family. In this study we have characterized in vitro complex formation between the AmtB and P(II) proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB-P(II) complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg(2+) and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB-P(II) complex defines a new regulatory role for Amt proteins in Prokaryotes.

Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the PIIfamily. PII proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to PII and also by PII posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the PII protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 Å resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known PII complexes, the majority of contacts with the target protein do not involve the T-loop region of PII. Hence this structure identifies a different mode of PII interaction with a target protein and demonstrates the potential for PII proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZ-DraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB. [ABSTRACT FROM AUTHOR]

This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The PII proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of PII might play a role in the control of nitrogen metabolism in H. seropedicae. [ABSTRACT FROM AUTHOR]

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Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae. [ABSTRACT FROM AUTHOR]

Abstract: PII proteins of bacteria, archaea, and plants regulate many facets of nitrogen metabolism. They do so by interacting with their target proteins, which can be enzymes, transcription factors, or membrane proteins. A key feature of the ability of PII proteins to sense cellular nitrogen status and to interact accordingly with their targets is their binding of the key metabolic intermediate 2-oxoglutarate (2-OG). However, the binding site of this ligand within PII proteins has been controversial. We have now solved the X-ray structure, at 1.4 Å resolution, of the Azospirillum brasilense PII protein GlnZ complexed with MgATP and 2-OG. This structure is in excellent agreement with previous biochemical data on 2-OG binding to a variety of PII proteins and shows that 2-oxglutarate binds within the cleft formed between neighboring subunits of the homotrimer. The 2-oxo acid moiety of bound 2-OG ligates the bound Mg2+ together with three phosphate oxygens of ATP and the side chain of the T-loop residue Gln39. Our structure is in stark contrast to an earlier structure of the Methanococcus jannaschii GlnK1 protein in which the authors reported 2-OG binding to the T-loop of that PII protein. In the light of our new structure, three families of T-loop conformations, each associated with a distinct effector binding mode and characterized by a different interaction partner of the ammonium group of the conserved residue Lys58, emerge as a common structural basis for effector signal output by PII proteins. [Copyright &y& Elsevier]

Abstract The nitrogen metabolism of Proteobacteria is controlled by the general Ntr system in response to nitrogen quality and availability. The PII proteins play an important role in this system by modulating the cellular metabolism through physical interaction with protein partners. Herbaspirillum seropedicae , a nitrogen-fixing bacterium, has two PII proteins paralogues, GlnB and GlnK. The interaction of H. seropedicae PII proteins with its targets is regulated by allosteric ligands and by reversible post-translational uridylylation. Both uridylylation and deuridylylation reactions are catalyzed by the same bifunctional enzyme, GlnD. The mechanism of regulation of GlnD activity is still not fully understood. Here, we characterized the regulation of deuridylylation activity of H. seropedicae GlnD in vitro. To this purpose, fully modified PII proteins were submitted to kinetics analysis of its deuridylylation catalyzed by purified GlnD. The deuridylylation activity was strongly stimulated by glutamine and repressed by 2-oxoglutarate and this repression was strong enough to overcome the glutamine stimulus of enzymatic activity. We also constructed and analyzed a truncated version of GlnD, lacking the C-terminal regulatory ACT domains. The GlnDΔACT protein catalyzed the futile cycle of uridylylation and deuridylylation of PII, regardless of glutamine and 2-oxoglutarate levels. The results presented here suggest that GlnD can sense the glutamine:2-oxoglutarate ratio and confirm that the ACT domains of GlnD are the protein sensors of environment clues of nitrogen availability. Highlights • Glutamine stimulates the UR activity of Herbaspirillum seropedicae GlnD protein. • 2-OG inhibits the UR activity of H. seropedicae GlnD protein. • The glutamine and 2-OG signals are integrated by GlnD to modulate its activity. • The ACT domains of GlnD are responsible to sense the glutamine:2-OG ratio. [ABSTRACT FROM AUTHOR]

The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes. [ABSTRACT FROM AUTHOR]