PII-like signaling proteins: a new paradigm in orchestrating cellular homeostasis.

Members of the PII superfamily are versatile, multitasking signaling proteins ubiquitously found in all domains of life. They adeptly monitor and synchronize the cell's carbon, nitrogen, energy, redox, and diurnal states, primarily by binding interdependently to adenyl-nucleotides, including charged nucleotides (ATP, ADP, and AMP) and second messengers such as cyclic adenosine monophosphate (cAMP), cyclic di-adenosine monophosphate (c-di-AMP), and S-adenosylmethionine-AMP (SAM-AMP). These proteins also undergo a variety of posttranslational modifications, such as phosphorylation, adenylation, uridylation, carboxylation, and disulfide bond formation, which further provide cues on the metabolic state of the cell. Serving as precise metabolic sensors, PII superfamily proteins transmit this information to diverse cellular targets, establishing dynamic regulatory assemblies that fine-tune cellular homeostasis. Recently discovered, PII-like proteins are emerging families of signaling proteins that, while related to canonical PII proteins, have evolved to fulfill a diverse range of cellular functions, many of which remain elusive. In this review, we focus on the evolution of PII-like proteins and summarize the molecular mechanisms governing the assembly dynamics of PII complexes, with a special emphasis on the PII-like protein SbtB.

Allosteric regulation of nitrate transporter NRT via the signaling protein PII.

Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.

Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites.

Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.

PII Protein-Derived FRET Sensors for Quantification and Live-Cell Imaging of 2-Oxoglutarate.

The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can't be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors' functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.

Effects of T-loop modification on the PII-signalling protein: structure of uridylylated Escherichia coli GlnB bound to ATP.

To adapt to environments with variable nitrogen sources and richness, the widely distributed homotrimeric PII signalling proteins bind their allosteric effectors ADP/ATP/2-oxoglutarate, and experience nitrogen-sensitive uridylylation of their flexible T-loops at Tyr51, regulating their interactions with effector proteins. To clarify whether uridylylation triggers a given T-loop conformation, we determined the crystal structure of the classical paradigm of PII protein, Escherichia coli GlnB (EcGlnB), in fully uridylylated form (EcGlnB-UMP3 ). This is the first structure of a postranslationally modified PII protein. This required recombinant production and purification of the uridylylating enzyme GlnD and its use for full uridylylation of large amounts of recombinantly produced pure EcGlnB. Unlike crystalline non-uridylylated EcGlnB, in which T-loops are fixed, uridylylation rendered the T-loop highly mobile because of loss of contacts mediated by Tyr51, with concomitant abolition of T-loop anchoring via Arg38 on the ATP site. This site was occupied by ATP, providing the first, long-sought snapshot of the EcGlnB-ATP complex, connecting ATP binding with T-loop changes. Inferences are made on the mechanisms of PII selectivity for ATP and of PII-UMP3 signalling, proposing a model for the architecture of the complex of EcGlnB-UMP3 with the uridylylation-sensitive PII target ATase (which adenylylates/deadenylylates glutamine synthetase [GS]) and with GS.

Unraveling of cross talk between Ca(2+) and ROS regulating enzymes in Anabaena 7120 and ntcA mutant.

In order to understand a cross talk between Ca(2+) and ROS regulating enzymes and the possible involvement of ntcA gene, Anabaena sp. PCC 7120 and its derivative ntcA mutant grown in varied levels of calcium chloride (0, 1, 10, and 100 mM) have been investigated. Scanning Electron Microscopy showed abnormal structure formation at high calcium concentration (100 mM) both in wild type and mutant. Fv /Fm values suggested that 100 mM calcium concentration was detrimental for photosynthetic apparatus. SOD, catalase, APX, GR, and peroxidase activity were found to be maximum for 100 mM and minimum for 1 mM of exogenously supplied calcium salt. NADPH contents were higher for wild type than mutant. RAPD-PCR and SDS-PAGE analysis revealed a difference in DNA as well as proteome pattern with changes in calcium chloride regime. Prominent bands of approximately 70, 33, 21, and 14 kDa expressed in the wild type served as the marker polypeptide bands under calcium supplementation. Results suggest that higher levels of calcium ion disturb the cellular homeostasis generating ROS, thereby inducing enhanced levels of antioxidative enzymes. Further, data also suggests possible involvement of ntcA gene in cross talk between calcium ion and ROS regulating enzymes.

2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state.

Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP.

From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation.

The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.

Probing the amino acids critical for protein oligomerisation and protein-nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches.

We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60-E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60-62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.

P(II) signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate.

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. We have now shown that the Escherichia coli P(II) protein, GlnK, has an ATPase activity that is inhibited by 2-OG. Hence, when a drop in the cellular 2-OG pool signals nitrogen sufficiency, 2-OG depletion of GlnK causes bound ATP to be hydrolyzed to ADP, leading to a conformational change in the protein. We propose that the role of ATP/ADP binding in E. coli GlnK is to effect a 2-OG-dependent molecular switch that drives a conformational change in the T loops of the P(II) protein. We have further shown that two other P(II) proteins, Azospirillum brasilense GlnZ and Arabidopsis thaliana P(II), have a similar ATPase activity, and we therefore suggest that this switch mechanism is likely to be a general property of most members of the P(II) protein family.

Influence of the ADP/ATP ratio, 2-oxoglutarate and divalent ions on Azospirillum brasilense PII protein signalling.

Proteins belonging to the P(II) family coordinate cellular nitrogen metabolism by direct interaction with a variety of enzymes, transcriptional regulators and transporters. The sensing function of P(II) relies on its ability to bind the nitrogen/carbon signalling molecule 2-oxoglutarate (2-OG). In Proteobacteria, P(II) is further subject to reversible uridylylation according to the intracellular levels of glutamine, which reflect the cellular nitrogen status. A number of P(II) proteins have been shown to bind ADP and ATP in a competitive manner, suggesting that P(II) might act as an energy sensor. Here, we analyse the influence of the ADP/ATP ratio, 2-OG levels and divalent metal ions on in vitro uridylylation of the Azospirillum brasilense P(II) proteins GlnB and GlnZ, and on interaction with their targets AmtB, DraG and DraT. The results support the notion that the cellular concentration of 2-OG is a key factor governing occupation of the GlnB and GlnZ nucleotide binding sites by ATP or ADP, with high 2-OG levels favouring the occupation of P(II) by ATP. Both P(II) uridylylation and interaction with target proteins responded to the ADP/ATP ratio within the expected physiological range, supporting the concept that P(II) proteins might act as cellular energy sensors.

Streptococcus mutans GlnK protein: an unusual PII family member

Streptococcus mutans is a Gram-positive bacterium present in the oral cavity, and is considered to be one of the leading causes of dental caries. S. mutans has a glnK gene, which codes for a PII-like protein that is possibly involved in the integration of carbon, nitrogen and energy metabolism in several organisms. To characterize the GlnK protein of S. mutans, the glnK gene was amplified by PCR, and cloned into the expression vectors pET29a(+) and pET28b(+). The native GlnK-Sm was purified by anion exchange (Q-Sepharose) and affinity (Hi-Trap Heparin) chromatography. The GlnK-His-Sm protein was purified using a Hi-Trap Chelating-Ni2+ column. The molecular mass of the GlnK-His-Sm proteins was 85 kDa as determined by gel filtration, indicating that this protein is a hexamer in solution. The GlnK-His-Sm protein is not uridylylated by the Escherichia coli GlnD protein. The activities of the GlnK-Sm and GlnK-His-Sm proteins were assayed in E. coli constitutively expressing the Klebsiella pneumoniae nifLA operon. In K. pneumoniae, NifL inhibits NifA activity in the presence of high ammonium levels and the GlnK protein is required to reduce the inhibition of NifL in the presence of low ammonium levels. The GlnK-Sm protein was unable to reduce NifL inhibition of NifA protein. Surprisingly, the GlnK-His-Sm protein was able to partially reduce NifL inhibition of the NifA protein under nitrogen-limiting conditions, in a manner similar to the GlnK protein of E. coli. These results suggested that S. mutans GlnK is functionally different from E. coli PII proteins.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein.

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with K(d) in the range 1.93-6.44 microM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the gamma-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.

The pivotal regulator GlnB of Escherichia coli is engaged in subtle and context-dependent control.

This study tests the purported signal amplification capability of the glutamine synthetase (GS) regulatory cascade in Escherichia coli. Intracellular concentrations of the pivotal regulatory protein GlnB were modulated by varying expression of its gene (glnB). Neither glnB expression nor P(II)* (i.e. the sum of the concentration of the P(II)-like proteins GlnB and GlnK) had control over the steady-state adenylylation level of GS when cells were grown in the presence of ammonia, in which glnK is not activated. Following the removal of ammonia, the response coefficient of the transient deadenylylation rate of GS-AMP was again zero with respect to both glnB expression and P(II)* concentration. This was at wild-type P(II)* levels. A 20% decrease in the P(II)* level resulted in the response coefficients increasing to 1, which was quite significant yet far from expected for zero-order ultrasensitivity. The transient deadenylylation rate of GS-AMP after brief incubation with ammonia was also measured in cells grown in the absence of ammonia. Here, GlnK was present and both glnB expression and P(II)* lacked control throughout. Because at wild-type levels of P(II)*, the molar ratio of P(II)*-trimer/adenylyltransferase-monomer was only slightly above 1, it is suggested that the absence of control by P(II)* is caused by saturation of adenylyltransferase by P(II)*. The difference in the control of deadenylylation by P(II)* under the two different growth conditions indicates that control of signal transduction is adjusted to the growth conditions of the cell. Adjustment of regulation rather than ultrasensitivity may be the function of signal transduction chains such as the GS cascade. We discuss how the subtle interplay between GlnB, its homologue GlnK and the adenylyltransferase may be responsible for the 'redundant', but quantitative, phenotype of GlnB.

Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio.

The PII family of signal transduction proteins is widespread amongst the three domains of life, and its members have fundamental roles in the general control of nitrogen metabolism. These proteins exert their regulatory role by direct protein-protein interaction with a multitude of cellular targets. The interactions are dependent on the binding of metabolites such as ATP, ADP and 2-oxoglutarate (2-OG), and on whether or not the PII protein is modified. In the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum three PII paralogues have been identified and termed GlnB, GlnJ and GlnK. In this report we analysed the interaction of GlnJ with known cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Our results show that the interaction of GlnJ with cellular targets is regulated in vitro by the concentrations of manganese and 2-OG and the ADP : ATP ratio. Furthermore, we show here for the first time, to our knowledge, that in the interactions of GlnJ with the three different partners, the energy signal (ADP : ATP ratio) in fact overrides the carbon/nitrogen signal (2-OG). In addition, by generating specific amino acid substitutions in GlnJ we show that the interactions with different cellular targets are differentially affected, and the possible implications of these results are discussed. Our results are important to further the understanding of the regulatory role of PII proteins in R. rubrum, a photosynthetic bacterium in which the nitrogen fixation process and its intricate control mechanisms make the regulation of nitrogen metabolism even more complex than in other studied bacteria.

Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein

Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 µM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.

Specificity and regulation of interaction between the PII and AmtB1 proteins in Rhodospirillum rubrum.

The nitrogen regulatory protein P(II) and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P(II) homolog, GlnJ, has higher affinity for an AmtB(1)-containing membrane than the other two P(II) homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB(1) complex in the presence of ATP and 2-KG, supporting a role for P(II) as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K(d) of 2-KG for GlnJ bound to AmtB(1) is 340 microM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 microM. This supports a model where multiple 2-KG and ATP molecules must bind a P(II) trimer to stimulate release of P(II) from AmtB(1), in contrast to the lower 2-KG requirement for productive uridylylation of P(II) by GlnD.

Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2-oxoglutarate dependent manner with PII and NtcA.

Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. The global nitrogen transcription factor NtcA and the signal transduction protein PII are both involved in 2-oxoglutarate sensing. PII proteins, probably the most conserved signal transduction proteins in nature, are remarkable for their ability to interact with very diverse protein targets in different systems. Despite widespread efforts to understand nitrogen signalling in cyanobacteria, the involvement of PII in the regulation of transcription activation by NtcA remains enigmatic. Here we show that PipX, a protein only present in cyanobacteria, interacts with both PII and NtcA and provides a mechanistic link between these two factors. A variety of in vivo and in vitro approaches were used to study PipX and its interactions with PII and NtcA. 2-Oxoglutarate favours complex formation between PipX and NtcA, but impairs binding to PII, suggesting that partner swapping between these nitrogen regulators is driven by the 2-oxoglutarate concentration. PipX is required for NtcA-dependent transcriptional activation in vivo, thus implying that PipX may function as a prokaryotic transcriptional coactivator.

Nla18, a key regulatory protein required for normal growth and development of Myxococcus xanthus.

NtrC-like activators regulate the transcription of a wide variety of adaptive genes in bacteria. Previously, we demonstrated that a mutation in the ntrC-like activator gene nla18 causes defects in fruiting body development in Myxococcus xanthus. In this report, we describe the effect that nla18 inactivation has on gene expression patterns during development and vegetative growth. Gene expression in nla18 mutant cells is altered in the early stages of fruiting body development. Furthermore, nla18 mutant cells are defective for two of the earliest events in development, production of the intracellular starvation signal ppGpp and production of A-signal. Taken together, these results indicate that the developmental program in nla18 mutant cells goes awry very early. Inactivation of nla18 also causes a dramatic decrease in the vegetative growth rate of M. xanthus cells. DNA microarray analysis revealed that the vegetative expression patterns of more than 700 genes are altered in nla18 mutant cells. Genes coding for putative membrane and membrane-associated proteins are among the largest classes of genes whose expression is altered by nla18 inactivation. This result is supported by our findings that the profiles of membrane proteins isolated from vegetative nla18 mutant and wild-type cells are noticeably different. In addition to genes that code for putative membrane proteins, nla18 inactivation affects the expression of many genes that are likely to be important for protein synthesis and gene regulation. Our data are consistent with a model in which Nla18 controls vegetative growth and development by activating the expression of genes involved in gene regulation, translation, and membrane structure.