glnK Nitrogen

We present a comprehensive overview of the hierarchical network of intracellular processes revolving around central nitrogen metabolism in Escherichia coli. The hierarchy intertwines transport, metabolism, signaling leading to posttranslational modification, and transcription. The protein components of the network include an ammonium transporter (AmtB), a glutamine transporter (GlnHPQ), two ammonium assimilation pathways (glutamine synthetase [GS]-glutamate synthase [glutamine 2-oxoglutarate amidotransferase {GOGAT}] and glutamate dehydrogenase [GDH]), the two bifunctional enzymes adenylyl transferase/adenylyl-removing enzyme (ATase) and uridylyl transferase/uridylyl-removing enzyme (UTase), the two trimeric signal transduction proteins (GlnB and GlnK), the two-component regulatory system composed of the histidine protein kinase nitrogen regulator II (NRII) and the response nitrogen regulator I (NRI), three global transcriptional regulators called nitrogen assimilation control (Nac) protein, leucine-responsive regulatory protein (Lrp), and cyclic AMP (cAMP) receptor protein (Crp), the glutaminases, and the nitrogen-phosphotransferase system...

UNLABELLED: Nitrogen is one of the major nutrients limiting microbial productivity in the ocean, and as a result, most marine microorganisms have evolved systems for responding to nitrogen stress. The highly abundant alphaproteobacterium "Candidatus Pelagibacter ubique," a cultured member of the order Pelagibacterales (SAR11), lacks the canonical GlnB, GlnD, GlnK, and NtrB/NtrC genes for regulating nitrogen assimilation, raising questions about how these organisms respond to nitrogen limitation...

Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters...

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. In all these organisms, P(II) proteins coordinate many facets of nitrogen metabolism by interacting with and regulating the activities of enzymes, transcription factors, and membrane transport proteins. The primary mode of signal perception by P(II) proteins derives from their ability to bind the effector molecules 2-oxoglutarate (2-OG) and ATP or ADP. The role of 2-OG as an indicator of cellular nitrogen status is well understood, but the function of ATP/ADP binding has remained unresolved...

Transcriptional changes in the nitrogen stress response (NSR) of wild type S. meliloti Rm1021, and isogenic strains missing both PII proteins, GlnB and GlnK, or carrying a ΔglnD-sm2 mutation were analyzed using whole-genome microarrays. This approach allowed us to identify a number of new genes involved in the NSR and showed that the response of these bacteria to nitrogen stress overlaps with other stress responses, including induction of the fixK2 transcriptional activator and genes that are part of the phosphate stress response...

The nitrogen is a macroelement for all alive cells, from bacteria to animals. Although NH3/NH4+ are highly toxic to animal, they are the preferred source of nitrogen for the most microorganisms and are assimilated by glutamine synthetase in the GOGAT cycle. The nitrogen limitation triggers a number of regulatory processes and activates many genes, providing the utilizing of alternative nitrogen sources. In Bacillus subtilis the genes of nitrogen metabolism are regulated by the transcription factor TnrA. In a cells it is bound to AmtB-GlnK proteins, the interaction with Glutamine synthetase (GS) represses its DNA-binding activity...

In this work we report for the first time a post-translational modification of PII homologues from the Archaea Domain. Haloferax mediterranei is the first haloarchaea whose PII proteins have been studied, it possesses two of them (GlnK1 and GlnK2 ), both encoded adjacent to a gene for the ammonia transporter Amt. An approach based on 2DE, anti-GlnK immunoblot and peptide mass fingerprint (MALDI-TOF-MS) of the reactive spots showed that GlnK proteins in H. mediterranei are post-translationally uridylylated. A third spot with lower pI suggests the existence of a non-descript post-translational modification in this protein family...

PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response...

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK(1) and glnK(2). The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK(1) and GlnK(2)...

The nitrogen-fixing Pseudomonas stutzeri strain A1501 contains two ammonium transporter genes, amtB1 and amtB2, linked to glnK. Growth of an amtB1-amtB2 double deletion mutant strain was not impaired compared to that of the wild type under any conditions tested, and it was still capable of taking up ammonium ions at nearly wild-type rates. Nitrogenase activity was repressed in wild-type strain A1501 in response to the addition of ammonium, but nitrogenase activity was only partially impaired in the amtB1 and amtB2 double mutant, suggesting that the two AmtB proteins are involved in regulating expression of nitrogenase or its activity in response to ammonium...

BACKGROUND: The assimilation of nitrogen in bacteria is achieved through only a few metabolic conversions between alpha-ketoglutarate, glutamate and glutamine. The enzymes that catalyze these conversions are glutamine synthetase, glutaminase, glutamate dehydrogenase and glutamine alpha-ketoglutarate aminotransferase. In low-GC Gram-positive bacteria the transcriptional control over the levels of the related enzymes is mediated by four regulators: GlnR, TnrA, GltC and CodY. We have analyzed the genomes of all species belonging to the taxonomic families Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae to determine the diversity in central nitrogen metabolism and reconstructed the regulation by GlnR...

ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation...

PII are signal-transducing proteins that integrate metabolic signals and transmit this information to a large number of proteins. In proteobacteria, PII are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. The uridylylation/deuridylylation cycle of PII is also regulated by carbon and energy signals such as ATP, ADP and 2-oxoglutarate (2-OG). These molecules bind to PII proteins and alter their tridimensional structure/conformation and activity. In this work, we determined the effects of ATP, ADP and 2-OG levels on the in vitro uridylylation of Herbaspirillum seropedicae PII proteins, GlnB and GlnK...

Nitrogen fixation in Herbaspirillum seropedicae is transcriptionally regulated by NifA, a σ(54) transcriptional activator with three structural domains: an N-terminal GAF domain, a catalytic AAA+ domain and a C-terminal DNA-binding domain. NifA is only active in H. seropedicae when cultures are grown in the absence of fixed nitrogen and at low oxygen tensions. There is evidence that the inactivation of NifA in response to fixed nitrogen is mediated by the regulatory GAF domain. However, the mechanism of NifA repression by the GAF domain, as well as the transduction of nitrogen status to NifA, is not understood...

The superfamily of P(II) proteins contains the most widely distributed signalling proteins in nature. Remarkable is the variety of targets whose activity is affected by protein-protein interactions. Here we identified as novel partner for interaction with GlnK an Rnf complex, known to couple the energy of ion transport to reduce ferredoxins. The endophytic diazotrophic betaproteobacterium Azoarcus sp. strain BH72 harbours two rnf-like clusters in the genome, of which only the rnf1 cluster was induced under conditions of N(2) fixation under control of the transcriptional activator NifA...

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...

GlnK proteins regulate the active uptake of ammonium by Amt transport proteins by inserting their regulatory T-loops into the transport channels of the Amt trimer and physically blocking substrate passage. They sense the cellular nitrogen status through 2-oxoglutarate, and the energy level of the cell by binding both ATP and ADP with different affinities. The hyperthermophilic euryarchaeon Archaeoglobus fulgidus possesses three Amt proteins, each encoded in an operon with a GlnK ortholog. One of these proteins, GlnK2 was recently found to be incapable of binding 2-OG, and in order to understand the implications of this finding we conducted a detailed structural and functional analysis of a second GlnK protein from A...

GlnK is an important nitrogen sensor protein in Streptomyces coelicolor. Deletion of glnK results in a medium-dependent failure of aerial mycelium and spore formation and loss of antibiotic production. Thus, GlnK is not only a regulator of nitrogen metabolism but also of morphological differentiation and secondary metabolite production. Through a comparative transcriptomic approach between the S. coelicolor wild-type and a S. coelicolor glnK mutant strain, 142 genes were identified that are differentially regulated in both strains...

The control of nitrogen metabolism in pathogenic Gram-positive bacteria has been studied in a variety of species and is involved with the expression of virulence factors. To date, no data have been reported regarding nitrogen metabolism in the odontopathogenic species Streptococcus mutans. GlnR, which controls nitrogen assimilation in the related bacterial species, Bacillus subtilis, was assessed in S. mutans for its DNA and protein binding activity. Electrophoretic mobility shift assay of the S. mutans GlnR protein indicated that GlnR binds to promoter regions of the glnRA and amtB-glnK operons...

The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C...