Parasite dodder enables transfer of bidirectional systemic nitrogen signals between host plants.

Dodder (Cuscuta spp., Convolvulaceae) is a genus of parasitic plants with worldwide distribution. Dodders are able to simultaneously parasitize two or more adjacent hosts, forming dodder-connected plant clusters. Nitrogen (N) deficiency is a common challenge to plants. To date, it has been unclear whether dodder transfers N-systemic signals between hosts grown in N-heterogeneous soil. Transcriptome and methylome analyses were carried out to investigate whether dodder (Cuscuta campestris) transfers N-systemic signals between N-replete and N-depleted cucumber (Cucumis sativus) hosts, and it was found that N-systemic signals from the N-deficient cucumber plants were rapidly translocated through C. campestris to the N-replete cucumber plants. Unexpectedly, certain systemic signals were also transferred from the N-replete to N-depleted cucumber hosts. We demonstrate that these systemic signals are able to regulate large transcriptome and DNA methylome changes in the recipient hosts. Importantly, N stress also induced many long-distance mobile mRNA transfers between C. campestris and hosts, and the bilateral N-systemic signaling between N-replete and N-depleted hosts had a strong impact on the inter-plant mobile mRNAs. Our 15N labeling experiment indicated that under N-heterogeneous conditions, N-systemic signals from the N-deficient cucumber hosts did not obviously change the N-uptake activity of the N-replete cucumber hosts; however, in plant clusters comprising C. campestris-connected cucumber and soybean (Glycine max) plants, if the soybean plants were N-starved, the cucumber plants exhibited increased N-uptake activity. This study reveals that C. campestris facilitates plant-plant communications under N-stress conditions by enabling extensive bilateral N-systemic signaling between different hosts.

Phosphoenolpyruvate carboxylase from the cyanobacterium Synechocystis sp. PCC 6803 is under global metabolic control by PII signaling.

Phosphoenolpyruvate carboxylase (PEPC) is the second major carbon-fixing enzyme in photoautotrophic organisms. PEPC is required for the synthesis of amino acids of the glutamate and aspartate family by replenishing the TCA cycle. Furthermore, in cyanobacteria, PEPC, together with malate dehydrogenase and malic enzyme, forms a metabolic shunt for the synthesis of pyruvate from PEP. During this process, CO2 is first fixed and later released again. Due to its central metabolic position, it is crucial to fully understand the regulation of PEPC. Here, we identify PEPC from the cyanobacterium Synechocystis sp. PCC 6803 (PEPC) as a novel interaction partner for the global signal transduction protein PII . In addition to an extensive characterization of PEPC, we demonstrate specific PII -PEPC complex formation and its enzymatic consequences. PEPC activity is tuned by the metabolite-sensing properties of PII : Whereas in the absence of PII, PEPC is subjected to ATP inhibition, it is activated beyond its basal activity in the presence of PII . Furthermore, PII -PEPC complex formation is inhibited by ADP and PEPC activation by PII -ATP is mitigated in the presence of 2-OG, linking PEPC regulation to the cell's global carbon/nitrogen status. Finally, physiological relevance of the in vitro measurements was proven by metabolomic analyses of Synechocystis wild-type and PII -deficient cells.

Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes.

The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation.

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. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation.

Role of NorR-like transcriptional regulators under nitrosative stress of the δ-proteobacterium, Desulfovibrio gigas.

NorR protein was shown to be responsible for the transcriptional regulation of flavorubredoxin and its associated oxidoreductase in Escherichia coli. Since Desulfovibrio gigas has a rubredoxin:oxygen oxidoreductase (ROO) that is involved in both oxidative and nitrosative stress response, a NorR-like protein was searched in D. gigas genome. We have found two putative norR coding units in its genome. To study the role of the protein designated as NorR1-like (NorR1L) in the presence of nitrosative stress, a norR1L null mutant of D. gigas was created and a phenotypic analysis was performed under the nitrosating agent GSNO. We show that under these conditions, the growth of both D. gigas mutants Δroo and ΔnorR1-like is impaired. In order to confirm that D. gigas NorR1-like may play identical function as the NorR of E. coli, we have complemented the E. coli ΔnorR mutant strain with the norR1-like gene and have evaluated growth when nitrosative stress was imposed. The growth phenotype of E. coli ΔnorR mutant strain was recovered under these conditions. We also found that induction of roo gene expression is completely abolished in the norR1L mutant strain of D. gigas subjected to nitrosative stress. It is identified in δ-proteobacteria, for the first time a transcription factor that is involved in nitrosative stress response and regulates the rd-roo gene expression.

AmtB-mediated NH3 transport in prokaryotes must be active and as a consequence regulation of transport by GlnK is mandatory to limit futile cycling of NH4(+)/NH3.

The nature of the ammonium import into prokaryotes has been controversial. A systems biological approach makes us hypothesize that AmtB-mediated import must be active for intracellular NH(4)(+) concentrations to sustain growth. Revisiting experimental evidence, we find the permeability assays reporting passive NH(3) import inconclusive. As an inevitable consequence of the proposed NH(4)(+) transport, outward permeation of NH(3) constitutes a futile cycle. We hypothesize that the regulatory protein GlnK is required to fine-tune the active transport of ammonium in order to limit futile cycling whilst enabling an intracellular ammonium level sufficient for the cell's nitrogen requirements.

Entropic mechanism of large fluctuation in allosteric transition.

A statistical mechanical model of allosteric transitions in proteins is developed by extending the structure-based model of protein folding to cases of multiple native conformations. The partition function is calculated exactly within the model and the free-energy surface reflecting allostery is derived. This approach is applied to an example protein, the receiver domain of the bacterial enhancer-binding protein NtrC. The model predicts the large entropy associated with a combinatorial number of preexisting transition routes. This large entropy lowers the free-energy barrier of the allosteric transition, which explains the large structural fluctuation observed in the NMR data of NtrC. The global allosteric transformation of NtrC is explained by the shift of preexisting distribution of conformations upon phosphorylation, but the local structural adjustment around the phosphorylation site is explained by the complementary induced-fit mechanism. Structural disordering accompanied by fluctuating interactions specific to two allosteric conformations underlies a large number of routes of allosteric transition.

NtrC-dependent regulatory network for nitrogen assimilation in Pseudomonas putida.

Pseudomonas putida KT2440 is a model strain for studying bacterial biodegradation processes. However, very little is known about nitrogen regulation in this strain. Here, we show that the nitrogen regulatory NtrC proteins from P. putida and Escherichia coli are functionally equivalent and that substitutions leading to partially active forms of enterobacterial NtrC provoke the same phenotypes in P. putida NtrC. P. putida has only a single P(II)-like protein, encoded by glnK, whose expression is nitrogen regulated. Two contiguous NtrC binding sites located upstream of the sigma(N)-dependent glnK promoter have been identified by footprinting analysis. In vitro experiments with purified proteins demonstrated that glnK transcription was directly activated by NtrC and that open complex formation at this promoter required integration host factor. Transcription of genes orthologous to enterobacterial codB, dppA, and ureD genes, whose transcription is dependent on sigma(70) and which are activated by Nac in E. coli, has also been analyzed for P. putida. Whereas dppA does not appear to be regulated by nitrogen via NtrC, the codB and ureD genes have sigma(N)-dependent promoters and their nitrogen regulation was exerted directly by NtrC, thus avoiding the need for Nac, which is missing in this bacterial species. Based upon these results, we propose a simplified nitrogen regulatory network in P. putida (compared to that in enterobacteria), which involves an indirect-feedback autoregulation of glnK using NtrC as an intermediary.

Mutations at pipX suppress lethality of PII-deficient mutants of Synechococcus elongatus PCC 7942.

The P(II) proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of P(II) proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which P(II) proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where P(II) forms complexes with the NtcA coactivator PipX, attempts to engineer P(II)-deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that P(II) is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated.

Signalling cascades integrating light-enhanced nitrate metabolism.

In higher plants, light is crucial for regulation of nitrate uptake, translocation and assimilation into organic compounds. Part of this metabolism is tightly coupled to photosynthesis because the enzymes involved, nitrite reductase and glutamate synthase, are localized to the chloroplasts and receive reducing power from photosynthetic electron transport. However, important enzymes in nitrate acquisition and reduction are localized to cellular compartments other than chloroplasts and are also up-regulated by light, i.e. transporters in cell and organellar membranes and nitrate reductase in the cytosol. This review describes the different light-dependent signalling cascades regulating nitrate metabolism at the transcriptional as well as post-transcriptional level, and how reactions in different compartments of the cell are co-ordinated. Essential players in this network are phytochrome and HY5 (long hypocotyls 5)/HYH (HY5 homologue)-dependent signalling pathways, the energy-related AMPK (AMP-activated protein kinase) protein kinase homologue SNRK1 (sucrose non-fermenting kinase 1-related kinase), chloroplastic thioredoxins and the prokaryotically originated PII protein. A complex light-dependent network of regulation emerges, which appears to be necessary for optimal nitrogen assimilation and for avoiding the accumulation of toxic intermediates and side products, such as nitrite and reactive oxygen compounds.

Chloroplast nitrite uptake is enhanced in Arabidopsis PII mutants.

In higher plants, the PII protein is a nuclear-encoded plastid protein that regulates the activity of a key enzyme of arginine biosynthesis. We have previously observed that Arabidopsis PII mutants are more sensitive to nitrite toxicity. Using intact chloroplasts isolated from Arabidopsis leaves and (15)N-labelled nitrite we show that a light-dependent nitrite uptake into chloroplasts is increased in PII knock-out mutants when compared to the wild-type. This leads to a higher incorporation of (15)N into ammonium and amino acids in the mutant chloroplasts. However, the uptake differences do not depend on GS/GOGAT activities. Our observations suggest that PII is involved in the regulation of nitrite uptake into higher plant chloroplasts.

Nitrogen control in Corynebacterium glutamicum: proteins, mechanisms, signals.

In order to utilize different nitrogen sources and to survive in a situation of nitrogen limitation, microorganisms have developed sophisticated mechanisms to adapt their metabolism to a changing nitrogen supply. In this communication, the recent knowledge of nitrogen regulation in the amino acid producer Corynebacterium glutamicum is summarized. The core adaptations of C. glutamicum to nitrogen limitation on the level of transcription are controlled by the global regulator AmtR. Further components of the signal pathway are GlnK, a P(II)-type signal transduction protein, and GlnD. Mechanisms involved in nitrogen control in C. glutamicum regulating gene expression and protein activity are repression of transcription, protein-complex formation, protein modification by adenylylation, change of intracellular localization, and proteolysis.

Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro.

PII signal transduction proteins are among the most widely distributed signaling proteins in nature, controlling nitrogen assimilation in organisms ranging from bacteria to higher plants. PII proteins integrate signals of cellular metabolic status and interact with and regulate receptors that are signal transduction enzymes or key metabolic enzymes. Prior work with Escherichia coli PII showed that all signal transduction functions of PII required ATP binding to PII and that ATP binding was synergistic with the binding of alpha-ketoglutarate to PII. Furthermore, alpha-ketoglutarate, a cellular signal of nitrogen and carbon status, was observed to strongly regulate PII functions. Here, we show that in reconstituted signal transduction systems, ADP had a dramatic effect on PII regulation of two E. coli PII receptors, ATase, and NRII (NtrB), and on PII uridylylation by the signal transducing UTase/UR. ADP acted antagonistically to alpha-ketoglutarate, that is, low adenylylate energy charge acted to diminish signaling of nitrogen limitation. By individually studying the interactions that occur in the reconstituted signal transduction systems, we observed that essentially all PII and PII-UMP interactions were influenced by ADP. Our experiments also suggest that under certain conditions, the three nucleotide binding sites of the PII trimer may be occupied by combinations of ATP and ADP. In the aggregate, our results show that PII proteins, in addition to serving as sensors of alpha-ketoglutarate, have the capacity to serve as direct sensors of the adenylylate energy charge.

PII is important in regulation of nitrogen metabolism but not required for heterocyst formation in the Cyanobacterium Anabaena sp. PCC 7120.

PII is an important signal protein for regulation of nitrogen metabolism in bacteria and plants. We constructed a mutant of glnB, encoding PII, in a heterocystous cyanobacterium, Anabaena sp. PCC 7120, with a cre-loxP system. The mutant (MP2alpha) grew more slowly than the wild type under all nitrogen regimens. It excreted a large amount of ammonium when grown on nitrate due to altered activities of glutamine synthetase and nitrate reductase. MP2alpha had a low nitrogenase activity but was able to form heterocysts under diazotrophic conditions, suggesting that PII is not required for heterocyst differentiation. Analysis of the PII with mass spectroscopy found tyrosine nitration at Tyr-51 under diazotrophic conditions while no phosphorylation at Ser-49 was detected. The strains 51F and 49A, which have PII with mutations of Y51F and S49A, respectively, were constructed to analyze the functions of the two key residues on the T-loop. Like MP2alpha, they had low nitrogenase activity and grew slowly under diazotrophic conditions. 49A was also impaired in nitrate uptake and formed heterocysts in the presence of nitrate. The up-regulation of ntcA after nitrogen step-down, which was present in the wild type, was not observed in 51F and 49A. While our results showed that the Ser-49 residue is important to the function of PII in Anabaena sp. PCC 7120, evidence from the PII pattern of the wild type and 49A in non-denaturing gel electrophoresis suggested that Ser-49 is not modified. The possible physiological roles of tyrosine nitration of PII are discussed.

Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa.

The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the DeltacbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.

Nitrogen regulation in bacteria and archaea.

A wide range of Bacteria and Archaea sense cellular 2-oxoglutarate (2OG) as an indicator of nitrogen limitation. 2OG sensor proteins are varied, but most of those studied belong to the PII superfamily. Within the PII superfamily, GlnB and GlnK represent a widespread family of homotrimeric proteins (GlnB-K) that bind and respond to 2OG and ATP. In some bacterial phyla, GlnB-K proteins are covalently modified, depending on enzymes that sense cellular glutamine as an indicator of nitrogen sufficiency. GlnB-K proteins are central clearing houses of nitrogen information and bind and modulate a variety of nitrogen assimilation regulators and enzymes. NifI(1) and NifI(2) comprise a second widespread family of PII proteins (NifI) that are heteromultimeric, respond to 2OG and ATP, and bind and regulate dinitrogenase in Euryarchaeota and many Bacteria.

The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro.

Ammonium assimilation is tightly regulated in nitrogen-fixing bacteria; the target of regulation is primarily the activity of the key enzyme glutamine synthetase that is regulated by reversible covalent modification by AMP groups in reactions catalysed by the bifunctional adenylyltransferase (ATase). The properties and regulation of ATase from Escherichia coli have been studied in great detail. We have investigated the regulation of ATase from Rhodospirillum rubrum, a photosynthetic nitrogen-fixing bacterium. In this diazotroph, nitrogenase is regulated at the metabolic level in addition to the transcriptional regulation operating in all diazotrophic bacteria, which makes understanding the regulatory features of nitrogen assimilation even more interesting. We show that in R. rubrum, in contrast to the E. coli system, ATase is primarily regulated by alpha-ketoglutarate and that glutamine has no effect on neither the adenylylation nor the deadenylylation of glutamine synthetase. Furthermore, the role of the regulatory P(II) proteins is only to stimulate the adenylylation reaction, as there is no effect on the reverse reaction. We propose that in R. rubrum and possibly other diazotrophs alpha-ketoglutarate plays the central role in the regulation of ATase and thus glutamine synthetase activity.

The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel.

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the PII signal transduction protein GlnK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GlnK at a resolution of 2.5 A. This structure of PII in a complex with one of its targets reveals physiologically relevant conformations of both AmtB and GlnK. GlnK interacts with AmtB almost exclusively via a long surface loop containing Y51 (T-loop), the tip of which inserts deeply into the cytoplasmic pore exit, blocking ammonia conduction. Y51 of GlnK is also buried in the pore exit, explaining why uridylylation of this residue prevents complex formation.

Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96 A.

Ammonia conductance is highly regulated. A P(II) signal transduction protein, GlnK, is the final regulator of transmembrane ammonia conductance by the ammonia channel AmtB in Escherichia coli. The complex formed between AmtB and inhibitory GlnK at 1.96-A resolution shows that the trimeric channel is blocked directly by GlnK and how, in response to intracellular nitrogen status, the ability of GlnK to block the channel is regulated by uridylylation/deuridylylation at Y51. ATP and Mg(2+) augment the interaction of GlnK. The hydrolyzed product, adenosine 5'-diphosphate orients the surface of GlnK for AmtB blockade. 2-Oxoglutarate diminishes AmtB/GlnK association, and sites for 2-oxoglutarate are evaluated.

Global gene expression of Prochlorococcus ecotypes in response to changes in nitrogen availability.

Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium-replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways.