NtrC Protein Research Articles

The Caulobacter NtrB-NtrC two-component system bridges nitrogen assimilation and cell development.

A suite of molecular sensory systems enables Caulobacter to control growth, development, and reproduction in response to levels of essential elements. The bacterial enhancer-binding protein (bEBP) NtrC and its cognate sensor histidine kinase, NtrB, are key regulators of nitrogen assimilation in many bacteria, but their roles in Caulobacter metabolism and development are not well defined. Notably, Caulobacter NtrC is an unconventional bEBP that lacks the σ54-interacting loop commonly known as the GAFTGA motif. Here we show that deletion of Caulobacter crescentus ntrC slows cell growth in complex medium and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glutamine synthetase expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring transcription of the glnBA operon, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nutrient limitation. We further identified dozens of direct NtrC-binding sites on the C. crescentus chromosome, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of binding sites align with those of the essential nucleoid-associated protein, GapR, or the cell cycle regulator, MucR1. NtrC is therefore predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter. IMPORTANCE Bacteria balance cellular processes with the availability of nutrients in their environment. The NtrB-NtrC two-component signaling system is responsible for controlling nitrogen assimilation in many bacteria. We have characterized the effect of ntrB and ntrC deletion on Caulobacter growth and development and uncovered a role for spontaneous IS element transposition in the rescue of transcriptional and nutritional deficiencies caused by ntrC mutation. We further defined the regulon of Caulobacter NtrC, a bacterial enhancer-binding protein, and demonstrate that it shares specific binding sites with essential proteins involved in cell cycle regulation and chromosome organization. Our work provides a comprehensive view of transcriptional regulation mediated by a distinctive NtrC protein, establishing its connection to nitrogen assimilation and developmental processes in Caulobacter.

General nitrogen regulation protein NtrC regulates the expression of nitrile hydratase and the synthesis of extracellular polysaccharide in Ensifer adhaerens CGMCC 6315

Ensifer adhaerens CGMCC 6315 contains two nitrile hydratases, CnhA and PnhA, which both convert the neonicotinoid insecticide acetamiprid to its amide metabolite. Here, we report that upregulation of PnhA and CnhA expression is related to nitrogen metabolism. Proteomic analysis showed that general nitrogen regulation protein NtrC was upregulated under nutrient deprivation. We thus constructed a ΔntrC mutant strain of E. adhaerens CGMCC 6315. In the ΔntrC strain, when cells were cultured in minimal salts medium supplemented with ammonium chloride, acetamiprid degradation and the expression of cnhA and pnhA were enhanced relative to those in wild-type E. adhaerens CGMCC 6315. In addition, NtrC negatively regulates synthesis of extracellular polysaccharides in this bacterium. The germination rate of soybean seeds was improved by inoculation with the ΔntrC strain of E. adhaerens CGMCC 6315 compared with the wild-type. This study is the first to report regulation of the expression of nitrile hydratase by NtrC, which is important for the practical remediation of pesticide pollution.

Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the “G-hinge” and the “allosteric linker.” While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12.

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote E. coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening in vitro, we identified the whole set of ‘constitutive’ promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ70), RpoS (σ38), RpoH (σ32), RpoF (σ28) or RpoE (σ24), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ54), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61–81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified in vivo under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the ‘repressive’ promoter. The presence of high-level RpoN sigma in growing E. coli K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into E. coli survival under nitrogen-depleted conditions in nature.

Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501

Pseudomonas stutzeri A1501 is a model strain used to study associative nitrogen fixation, and it possesses the nitrogen regulatory NtrC protein in the core genome. Nitrogen sources represent one of the important factors affecting the efficiency of biological nitrogen fixation in the natural environment. However, the regulation of NtrC during nitrogen metabolism in P. stutzeri A1501 has not been clarified. In this work, a phenotypic analysis of the ntrC mutant characterized the roles of NtrC in nitrogen metabolism and the oxidative stress response of P. stutzeri A1501. To systematically identify NtrC-controlled gene expression, RNA-seq was performed to further analyse the gene expression differences between the wild-type strain and the ∆ntrC mutant under nitrogen fixation conditions. A total of 1431 genes were found to be significantly altered by ntrC deletion, among which 147 associative genes had NtrC-binding sites, and the pathways for nitrogen fixation regulation, nitrogenous compound acquisition and catabolism and nitrate assimilation were discussed. Furthermore, the oxidative stress-related gene (katB), which was upregulated by ntrC deletion, was suggested to be a potential target gene of NtrC, thus highlighting the importance of NtrC in nitrogenase protection against oxygen damage. Based on these findings, we propose that NtrC is a high-ranking element in the regulatory network of P. stutzeri A1501 that controls a variety of nitrogen metabolic and oxidative stress responsive traits required for adaptation to complex rhizosphere environments.

Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222.

Paracoccus denitrificans PD1222 accumulates short-length polyhydroxyalkanoates, poly(3-hydroxybutyrate), under nitrogen-deficient conditions. Polyhydroxybutyrate metabolism requires the 3-ketoacyl-CoA thiolase PhaA, the acetoacetyl-CoA dehydrogenase/reductase PhaB and the synthase PhaC for polymerization. Additionally, P. denitrificans PD1222 grows aerobically with nitrate as sole nitrogen source. Nitrate assimilation is controlled negatively by ammonium through the two-component NtrBC system. NtrB is a sensor kinase that autophosphorylates a histidine residue under low-nitrogen concentrations and, in turn, transfers a phosphoryl group to an aspartate residue of the response regulator NtrC protein, which acts as a transcriptional activator of the P. denitrificans PD1222 nasABGHC genes. The P. denitrificans PD1222 NtrB mutant was unable to use nitrate efficiently as nitrogen source when compared to the wild-type strain, and it also overproduced poly(3-hydroxybutyrate). Acetyl-CoA concentration in the P. denitrificans PD1222 NtrB mutant strain was higher than in the wild-type strain. The expression of the phaC gene was also increased in the NtrB mutant when compared to the wild-type strain. These results suggest that accumulation of poly(3-hydroxybutyrate) in the NtrB mutant strain of PD1222 responds to the high levels of acetyl-CoA that accumulate in the cytoplasm as consequence of its inability to efficiently use nitrate as nitrogen source.

NtrC-dependent control of exopolysaccharide synthesis and motility in Burkholderia cenocepacia H111.

Burkholderia cenocepacia is a versatile opportunistic pathogen that survives in a wide variety of environments, which can be limited in nutrients such as nitrogen. We have previously shown that the sigma factor σ54 is involved in the control of nitrogen assimilation and virulence in B. cenocepacia H111. In this work, we investigated the role of the σ54 enhancer binding protein NtrC in response to nitrogen limitation and in the pathogenicity of H111. Of 95 alternative nitrogen sources tested the ntrC showed defects in the utilisation of nitrate, urea, L-citrulline, acetamide, DL-lactamide, allantoin and parabanic acid. RNA-Seq and phenotypic analyses of an ntrC mutant strain showed that NtrC positively regulates two important phenotypic traits: exopolysaccharide (EPS) production and motility. However, the ntrC mutant was not attenuated in C. elegans virulence.

A Network of “Molecular-Switches” Control the Activation of Key Bacterial Signaling Protein

Recent successes in simulating protein structure and folding dynamics have demonstrated the power of molecular dynamics to predict the long timescale behavior of proteins. Here, we extend and improve these methods to predict conformational change pathways between active and inactive state of nitrogen regulatory protein C (NtrC). By employing unbiased Markov State Model based molecular dynamics simulations, we predict a new dynamic picture of the activation of a key bacterial signaling protein NtrC, involving a complex network of molecular switches. These results are consistent with experimental observations and predict new mutants that could be used for validation of the mechanism. Moreover, these results suggest a novel mechanistic paradigm for conformational switching.

Allostery and Folding of the N-terminal Receiver Domain of Protein NtrC

The N-terminal receiver domain of protein NtrC (NtrC(r)) exhibits allosteric transitions between the inactive (unphosphorylated) and active (phosphorylated) state on the microsecond time scale. Using a coarse-grained variational model with coupled energy basins, we illustrate that significant loss of conformational flexibility is the key determinant of the inactive (I) → active (A) state transition mechanism of NtrC(r). In particular, our results reveal that the rearrangements of the native contacts involving the regulatory helix-α4 and the flexible β3-α3 loop upon activation play a crucial role in the activation mechanism. Interestingly, we find that the β3-α3 loop exhibits a gradual decrease in flexibility throughout the activation transition, while helix-α4, in contrast, becomes more rigid abruptly near the free energy barrier separating the two states. To gain further insight into role these flexible regions play in the transition mechanism, we consider folding of NtrC(r) to both states using a similar model. Our calculated folding routes suggest that helix-α4 becomes structured later when folding to the I state compared to folding of the A state, a result consistent with it is relative conformational flexibility in the two states. Finally, we find a good qualitative agreement between our predicted I → A transition mechanism and the measured backbone dynamics from nuclear magnetic resonance experiments.

Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

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. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control.

Sigma factor N, liaison to an ntrC and rpoS dependent regulatory pathway controlling acid resistance and the LEE in enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σN), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σN-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σS) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σS was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σN allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σS stability, gadE, ler or GDAR, indicating a dependence on transcription from a σN promoter(s) and not RNAP competition for the phenotype. Among a library of σN enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σS stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σN regulate GDAR and LEE expression through downregulation of σS at the post-translational level; likely by altering σS stability or activity. The regulatory interplay between NtrC, other EBPs, and σN–σS, represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.

Internucleosomal Interactions Mediated by Histone Tails Allow Distant Communication in Chromatin

Action across long distances on chromatin is a hallmark of eukaryotic transcriptional regulation. Although chromatin structure per se can support long-range interactions, the mechanisms of efficient communication between widely spaced DNA modules in chromatin remain a mystery. The molecular simulations described herein suggest that transient binary internucleosomal interactions can mediate distant communication in chromatin. Electrostatic interactions between the N-terminal tails of the core histones and DNA enhance the computed probability of juxtaposition of sites that lie far apart along the DNA sequence. Experimental analysis of the rates of communication in chromatin constructs confirms that long-distance communication occurs efficiently and independently of distance on tail-containing, but not on tailless, chromatin. Taken together, our data suggest that internucleosomal interactions involving the histone tails are essential for highly efficient, long-range communication between regulatory elements and their targets in eukaryotic genomes.

Allostery and Folding Mechanisms of the N-Terminal Receiver Domain of Protein NTRC

The nitrogen regulatory protein C (NtrC) of enteric bacteria plays a central role in the control of genes involved in nitrogen metabolism. Nuclear Magnetic Resonance (NMR) studies suggest that the N-terminal receiver domain of NtrC (NtrCr) exhibits slow conformational dynamics in the microsecond timescale. Allostery in this protein occurs by shifting the preexisting population from the inactive to active state upon phosphorylation (see Fig. 1.). Using a coarse-grained variational model, we give a site specific description of both the folding and conformational transition mechanisms of this βα-repeat protein. Differences in the folding mechanisms to the active and inactive state are consistent with a significant stabilization of the helix-α4 upon activation. Our model suggests that the allosteric conformational change of NtrCr involves a marked decrease in the flexibility of this protein upon activation without local partial unfolding. In particular, we find that in addition to the functionally important helix-α4, the β3α3 loop also plays significant role in the inactive/active conformational transition mechanism of NtrCr.View Large Image | View Hi-Res Image | Download PowerPoint SlideFig. 1. Phosphorylation induces large conformational change in the NtrCr. The unphosphorylated (inactive) structure is shown in blue and the phosphorylated (active) structure in red.

Probing microsecond time scale dynamics in proteins by methyl (1)H Carr-Purcell-Meiboom-Gill relaxation dispersion NMR measurements. Application to activation of the signaling protein NtrC(r).

To study microsecond processes by relaxation dispersion NMR spectroscopy, low power deposition and short pulses are crucial and encourage the development of experiments that employ 1H Carr−Purcell−Meiboom−Gill (CPMG) pulse trains. Herein, a method is described for the comprehensive study of microsecond to millisecond time scale dynamics of methyl groups in proteins, exploiting their high abundance and favorable relaxation properties. In our approach, protein samples are produced using [1H, 13C]-d-glucose in ∼100% D2O, which yields CHD2 methyl groups for alanine, valine, threonine, isoleucine, leucine, and methionine residues with high abundance, in an otherwise largely deuterated background. Methyl groups in such samples can be sequence-specifically assigned to near completion, using 13C TOCSY NMR spectroscopy, as was recently demonstrated (Otten, R.; et al. J. Am. Chem. Soc.2010, 132, 2952−2960). In this Article, NMR pulse schemes are presented to measure 1H CPMG relaxation dispersion profiles for CHD2 methyl groups, in a vein similar to that of backbone relaxation experiments. Because of the high deuteration level of methyl-bearing side chains, artifacts arising from proton scalar coupling during the CPMG pulse train are negligible, with the exception of Ile-δ1 and Thr-γ2 methyl groups, and a pulse scheme is described to remove the artifacts for those residues. Strong 13C scalar coupling effects, observed for several leucine residues, are removed by alternative biochemical and NMR approaches. The methodology is applied to the transcriptional activator NtrCr, for which an inactive/active state transition was previously measured and the motions in the microsecond time range were estimated through a combination of backbone 15N CPMG dispersion NMR spectroscopy and a collection of experiments to determine the exchange-free component to the transverse relaxation rate. Exchange contributions to the 1H line width were detected for 21 methyl groups, and these probes were found to collectively report on a local structural rearrangement around the phosphorylation site, with a rate constant of (15.5 ± 0.5) × 103 per second (i.e., τex = 64.7 ± 1.9 μs). The affected methyl groups indicate that, already before phosphorylation, a substantial, transient rearrangement takes place between helices 3 and 4 and strands 4 and 5. This conformational equilibrium allows the protein to gain access to the active, signaling state in the absence of covalent modification through a shift in a pre-existing dynamic equilibrium. Moreover, the conformational switching maps exactly to the regions that differ between the solution NMR structures of the fully inactive and active states. These results demonstrate that a cost-effective and quantitative study of protein methyl group dynamics by 1H CPMG relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone 15N relaxation dispersion NMR. The use of methyl groups as dynamics probes extends such applications also to larger proteins.

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.

During Transitions Proteins Make Fleeting Bonds

How do proteins efficiently and precisely shift from one conformation to another? Gardino et al. (2009) show that transient hydrogen bonds are critical to the conformational transition of the nitrogen regulatory protein NtrC between its native state and its active state.

Role of NtrC‐regulated exopolysaccharides in the biofilm formation and pathogenic interaction of Vibrio vulnificus

Vibrio vulnificus has been shown to require a global transcription factor, NtrC for mature biofilm development via controlling the biosyntheses of lipopolysaccharide and exopolysaccharide (EPS). Biofilm formation and EPS production were dramatically increased in a medium including a tricarboxylic acid cycle-intermediate as a carbon source. These phenotypes required functional NtrC and were abolished by the addition of ammonium chloride. During the initial stage of biofilm formation, both expression of the ntrC gene and the cellular content of NtrC protein increased. Thus, the regulatory roles of NtrC in EPS biosynthesis were studied with three gene clusters for EPS biosyntheses. Transcriptions of the three clusters were positively controlled by NtrC and showed maximal expression at the early stage of biofilm development. Mutants deficient in one of the genes (VV1_2661, VV2_1579 and VV1_2305) in each cluster showed decreased production of EPS, attenuated ability to form biofilm and lowered cytoadherence to human epithelial cells. However, mutations in VV2_1579 and VV1_2305 resulted in lower cytotoxicity to human cells and mortality to mice than the mutation in VV1_2661. These results demonstrate that NtrC-regulated EPS are crucial in biofilm formation of V. vulnificus, and some EPS components play important roles in interacting with hosts.

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.

Effect of anntrCmutation on amino acid or urea utilization and on nitrogenase switch-off inHerbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium that grows well with ammonium chloride or sodium nitrate as alternative single nitrogen sources but that grows more slowly with L-alanine, L-serine, L-proline, or urea. The ntrC mutant strain DCP286A was able to utilize only ammonium or urea of these nitrogen sources. The addition of 1 mmol.L-1 ammonium chloride to the nitrogen-fixing wild-type strain inhibited nitrogenase activity rapidly and completely. Urea was a less effective inhibitor; approximately 20% of nitrogenase activity remained 40 min after the addition of 1 mmol x L-1 urea. The effect of the ntrC mutation on nitrogenase inhibition (switch-off) was studied in strain DCP286A containing the constitutively expressed gene nifA of H. seropedicae. In this strain, nitrogenase inhibition by ammonium was completely abolished, but the addition of urea produced a reduction in nitrogenase activity similar to that of the wild-type strain. The results suggest that the NtrC protein is required for assimilation of nitrate and the tested amino acids by H. seropedicae. Furthermore, NtrC is also necessary for ammonium-induced switch-off of nitrogenase but is not involved in the mechanism of nitrogenase switch-off by urea.

Conformational Switching upon Phosphorylation: A Predictive Framework Based on Energy Landscape Principles

We investigate how post-translational phosphorylation modifies the global conformation of a protein by changing its free energy landscape using two test proteins, cystatin and NtrC. We first examine the changes in a free energy landscape caused by phosphorylation using a model containing information about both structural forms. For cystatin the free energy cost is fairly large indicating a low probability of sampling the phosphorylated conformation in a perfectly funneled landscape. The predicted barrier for NtrC conformational transition is several times larger than the barrier for cystatin, indicating that the switch protein NtrC most probably follows a partial unfolding mechanism to move from one basin to the other. Principal component analysis and linear response theory show how the naturally occurring conformational changes in unmodified proteins are captured and stabilized by the change of interaction potential. We also develop a partially guided structure prediction Hamiltonian which is capable of predicting the global structure of a phosphorylated protein using only knowledge of the structure of the unphosphorylated protein or vice versa. This algorithm makes use of a generic transferable long-range residue contact potential along with details of structure short range in sequence. By comparing the results obtained with this guided transferable potential to those from the native-only, perfectly funneled Hamiltonians, we show that the transferable Hamiltonian correctly captures the nature of the global conformational changes induced by phosphorylation and can sample substantially correct structures for the modified protein with high probability.