Uridylyltransferase/uridylyl-removing enzyme (UTase/UR) catalyzes uridylylation of PII and deuridylylation of PII-UMP, with both activities regulated by glutamine. In a reconstituted UTase/UR-PII cycle containing wild-type UTase/UR, the steady-state modification of PII varied from nearly complete modification to nearly complete demodification as glutamine was varied, whether the level of PII was saturating or unsaturating, but when a His-tagged version of UTase/UR was used, the robustness to variations in PII concentration was lost and the range of PII modification states in response to glutamine became smaller as the PII concentration increased. The presence of the His tag on UTase/UR did not alter PII substrate inhibition of the UT activity and had little effect on the level of the UT activity but resulted in a slight defect in UR activity. Importantly, at high PII concentrations, glutamine inhibition of the UT activity was incomplete. We hypothesized that binding of PII to the UR active site in the HD domain was responsible for PII substrate inhibition of the UT activity and, in the His-tagged enzyme, also weakened glutamine inhibition of the UT activity. Consistent with this, three different UTase/UR proteins with HD domain alterations lacked substrate inhibition of UT activity by PII; in one case, the HD alteration eliminated glutamine regulation of UT activity, while for the other two proteins, alterations of the HD domain partially compensated for the effect of the His tag in restoring glutamine regulation of UT activity. We conclude that very strong inhibition of UT activity was required for the UTase/UR-PII cycle to display robustness to the PII concentration, that in the wild-type enzyme PII brings about substrate inhibition of the UT activity by binding to the HD domain of the enzyme, and that addition of an N-terminal His tag resulted in an altered enzyme with subtle changes in the interactions between domains such that binding of PII to the HD domain interfered with glutamine regulation of the UT domain.

A reconstituted UTase/UR-PII-NRII-NRI bicyclic cascade regulated PII uridylylation and NRI phosphorylation in response to glutamine.We examined the sensitivity and robustness of the responses of the individual cycles and of the bicyclic system.The sensitivity of the glutamine response of the upstream UTase/UR-PII monocycle depended upon the PII concentration, and we show that PII exerted substrate inhibition of the UTase activity of UTase/UR, potentially contributing to this dependence of sensitivity on PII.In the downstream NRII-NRI monocycle, PII controlled NRI phosphorylation state, and the response to PII was hyperbolic at both saturating and unsaturating NRI concentration.As expected from theory, the level of NRI∼P produced by the NRII-NRI monocycle was robust to changes in the NRII or NRI concentrations when NRI was in excess over NRII, as long as the NRII concentration was above a threshold value, an example of absolute concentration robustness (ACR).Because of the parameters of the system, at physiological protein levels and ratios of NRI to NRII, the level of NRI∼P depended upon both protein concentrations.In bicyclic UTase/UR-PII-NRII-NRI systems, the NRI phosphorylation state response to glutamine was always hyperbolic, regardless of the PII concentration or sensitivity of the upstream UTase/UR-PII cycle.In these bicyclic systems, NRI phosphorylation state was only robust to variation in the PII/NRII ratio within a narrow range; when PII was in excess NRI∼P was low, and when NRII was in excess NRI phosphorylation was elevated, throughout the physiological range of glutamine concentrations.Our results show that the bicyclic system produced a graded response of NRI phosphorylation to glutamine under a range of conditions, and that under most conditions the response of NRI phosphorylation state to glutamine levels depended on the concentrations of NRI, NRII, and PII.

b Institute for Physiology, Molecular Biology, and Neuroscience, Department of Biology, University of Buenos Aires, Buenos Aires, Argentina; c Department of Mathematics, Rutgers University, New Brunswick, NJ; d Department of Internal Medicine, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI; e Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA

Glutamine synthetase (GS) activity in Escherichia coli is regulated by reversible adenylylation, brought about by a bicyclic system comprised of uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate, PII, adenylyltransferase (ATase), and its substrate, GS. The modified and unmodified forms of PII produced by the upstream UTase/UR-PII cycle regulate the downstream ATase-GS cycle. A reconstituted UTase/UR-PII-ATase-GS bicyclic system has been shown to produce a highly ultrasensitive response of GS adenylylation state to the glutamine concentration, but its composite UTase/UR-PII and ATase-GS cycles displayed moderate glutamine sensitivities when examined separately. Glutamine sensitivity of the bicyclic system was significantly reduced when the trimeric PII protein was replaced by a heterotrimeric form of PII that was functionally monomeric, and coupling between the two cycles was different in systems containing wild-type or heterotrimeric PII. Thus, the trimeric nature of PII played a role in the glutamine response of the bicyclic system. We therefore examined regulation of the individual AT (adenylylation) and AR (deadenylylation) activities of ATase by PII preparations with various levels of uridylylation. AR activity was affected in a linear fashion by PII uridylylation, but partially modified wild-type PII activated the AT much less than expected based on the extent of PII modification. Partially modified wild-type PII also bound to ATase less than expected based upon the fraction of modified subunits. Our results suggest that the AT activity is only bound and activated by completely unmodified PII and that this design is largely responsible for ultrasensitivity of the bicyclic system.

Biological signal transduction networks are commonly viewed as circuits that pass along information-in the process amplifying signals, enhancing sensitivity, or performing other signal-processing tasks-to transcriptional and other components. Here, we report on a "reverse-causality" phenomenon, which we call load-induced modulation. Through a combination of analytical and experimental tools, we discovered that signaling was modulated, in a surprising way, by downstream targets that receive the signal and, in doing so, apply what in physics is called a load. Specifically, we found that non-intuitive changes in response dynamics occurred for a covalent modification cycle when load was present. Loading altered the response time of a system, depending on whether the activity of one of the enzymes was maximal and the other was operating at its minimal rate or whether both enzymes were operating at submaximal rates. These two conditions, which we call "limit regime" and "intermediate regime," were associated with increased or decreased response times, respectively. The bandwidth, the range of frequency in which the system can process information, decreased in the presence of load, suggesting that downstream targets participate in establishing a balance between noise-filtering capabilities and a circuit's ability to process high-frequency stimulation. Nodes in a signaling network are not independent relay devices, but rather are modulated by their downstream targets.

In Escherichia coli and related bacteria, nitrogen assimilation is coordinated with carbon assimilation and cellular energy status to provide for balanced metabolism and optimal growth under a variety of conditions. Part of this regulation is accomplished by a signal transduction system that includes two bicyclic cascades of covalent modification, controlling the activity of glutamine synthetase and the transcription of nitrogen-regulated genes, respectively. Here, we review our current state of understanding of these bicyclic cascade systems, with an emphasis on the mechanisms of sensation and signal integration. We also address systems level questions relevant to sensation, focusing on how the relative levels of the proteins constituting the signal transduction system affects the sensitivity to signals, how zero-order and multistep ultrasensitivity affect the sensitivity to signals, and how downstream targets of the signalling systems may influence sensation of stimuli and signal transduction.

In prior work, we have shown that just as in many engineering systems, impedance-like effects appear at the interconnection of biomolecular systems. These effects are called retroactivity, to extend the notion of impedance to biological systems. Signaling components, such as covalent modification cycles, play a central role in the transmission of signals within a cell and from outside the cell. They are typically found in highly interconnected architectures in which a component has several downstream clients. In this paper, we show that retroactivity from downstream targets decreases the sensitivity of response to an input stimulus.

We used a model system of purified components to explore the effects of a downstream target on the signaling properties of a covalent modification cycle, an example of retroactivity. In the experimental system used, a bifunctional enzyme catalyzed the modification and demodification of its substrate protein, with both activities regulated by a small molecule stimulus. Here we examined how a downstream target for one or both forms of the substrate of the covalent modification cycle affected the steady-state output of the system, the sensitivity of the response to the stimulus, and the concentration of the stimulus required to provide the half-maximal response (S50). When both the modified and unmodified forms of the substrate protein were sequestered by the downstream target, the sensitivity of the response was dramatically decreased, but the S50 was only modestly affected. Conversely, when the downstream target only sequestered the unmodified form of the substrate protein, significant effects were observed on both system sensitivity and S50. Behaviors of the experimental systems were well approximated both by simple models allowing analytical solutions and by a detailed model based on the known interactions and enzymatic activities. Modeling and experimentation indicated that retroactivity may result in subsensitive responses, even if the covalent modification cycle displays significant ultrasensitivity in the absence of retroactivity. Thus, we provide examples of how a downstream target can alter the signaling properties of an upstream signal transduction covalent modification cycle.

PII proteins are sensors of alpha-ketoglutarate and adenylylate energy charge that regulate signal transduction proteins, metabolic enzymes, and permeases involved in nitrogen assimilation. Here, purified Escherichia coli PII and two of its receptors, ATase and NRII, were used to study the mechanisms of sensation by PII. We assembled heterotrimeric forms of PII from wild-type and mutant subunits, which allowed us to assess the role of the three binding sites for alpha-ketoglutarate and adenylylate nucleotide in the PII trimer. Signaling of alpha-ketoglutarate and adenylylate energy charge by these heterotrimeric PII proteins required multiple binding sites for these effectors, and the ligand-binding sites on different subunits could influence the function of a single subunit interacting with a receptor, implying communication between PII subunits. Wild-type and heterotrimeric forms of PII were also used to examine the effects of alpha-ketoglutarate and ADP on PII activation of the adenylyltransferase (AT) activity of ATase. Previous work showed that when ATP was the sole adenylylate nucleotide, alpha-ketoglutarate controlled the extent of PII activation but did not alter the PII activation constant (K(act)). We show that ADP affected both the PII K(act) and the extent of activation by PII. When ATP was present, ADP dramatically reduced the K(act) for wild-type PII, and this effect was antagonized by alpha-ketoglutarate. Consequently, when ATP was present, the antagonism between ADP and alpha-ketoglutarate allowed each of these effectors to influence the PII K(act) for activation of ATase. A study of heterotrimeric forms of PII suggested that the major part of the ability of ADP to improve the binding of PII to ATase required multiple nucleotide binding sites and intersubunit communication. We also used nondenaturing gel electrophoresis to investigate the effect of ADP and alpha-ketoglutarate on the binding of PII to ATase and NRII. These studies showed that ATase and NRII differ in their requirements for interaction with PII, and that under the appropriate conditions, the antagonism between alpha-ketoglutarate and ADP allowed each of these effectors to influence the binding of PII to receptors.

ATase brings about the short-term regulation of glutamine synthetase (GS) by catalyzing the adenylylation and deadenylylation of GS in response to signals of cellular nitrogen status and energy.The adenylyltransferase (AT) activity of ATase is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by PII-UMP.Conversely, the adenylyl-removing (AR) activity of ATase is activated by PII-UMP and inhibited by unmodified PII and by glutamine.Here, we show that the enzyme can be reconstituted from two purified polypeptides that comprise the N-terminal two-thirds of the protein and the C-terminal one-third of the protein.Properties of the reconstituted enzyme support recent hypotheses for the sites of regulatory interactions and mechanisms for intramolecular signal transduction.Specifically, our results are consistent with the protein activators (PII and PII-UMP) binding to the enzyme domain with the opposing activity, with intramolecular signal transduction by direct interactions between the N-terminal AR catalytic domain and the C-terminal AT catalytic domain.Similarly, glutamine inhibition of the AR activity involved intramolecular signaling between the AT and AR domains.Finally, our results are consistent with the hypothesis that the AR activity of the N-terminal domain required activation by the opposing C-terminal (AT) domain.

PII signal transduction proteins are among the most widely distributed signaling proteins in nature; these proteins are direct sensors of alpha-ketoglutarate and adenylylate energy charge and control receptors that are signal transduction proteins, metabolic enzymes, or permeases involved in nitrogen metabolism. Prior studies showed that alpha-ketoglutarate regulated the ability of PII to control the activities of glutamine synthetase adenylyltransferase (ATase) but did not affect the ability of PII to bind to ATase. Here, we show that a similar pattern of alpha-ketoglutarate regulation was obtained with another PII receptor, the two-component system transmitter protein NRII (NtrB). Although alpha-ketoglutarate was required for the binding of PII to NRII, PII bound to NRII equally well as the concentration of alpha-ketoglutarate was varied through its physiological range. Variation of the concentration of alpha-ketoglutarate through its physiological range provided dramatic regulation of the ability of PII to activate the phosphatase activity of NRII and controlled the ability of PII to inhibit the autophosphorylation of NRII. Thus, PII control of NRII activities could be dissected into distinct binding and regulation steps, and when present in its physiological concentration range, alpha-ketoglutarate apparently played a role in only the latter step.

Glutamine synthetase adenylyltranferase (ATase, EC 2.7.7.49) catalyzes the adenylylation and deadenylylation of glutamine synthetase (GS), regulating GS activity. The adenylyltransferase (AT) reaction is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by the uridylylated form of PII, PII-UMP. Conversely, the adenylyl-removing (AR) reaction is activated by PII-UMP and is inhibited by glutamine and by PII. Both AT and AR reactions are regulated by alpha-ketoglutarate, which binds to PII and PII-UMP. Here, we present a kinetic analysis of the AT and AR activities and their regulation. Both AT and AR reactions used a sequential mechanism of rapid equilibrium random binding of substrates and products. Activators and inhibitors had little effect on the binding of substrates, instead exerting their effects on catalysis. Our results were consistent with PII, PII-UMP, and glutamine shifting the enzyme among at least six different enzyme forms, two of which were inactive, one of which exhibited AR activity, and three of which exhibited AT activity. In addition to a site for glutamine, the enzyme appeared to contain two distinct sites for PII and PII-UMP. The PII, PII-UMP, and glutamine sites were in communication so that the apparent activation and inhibition constants for regulators depended upon each other. The binding of PII was favored by glutamine and its level reduced by PII-UMP, whereas glutamine and PII-UMP competed for the enzyme. alpha-Ketoglutarate, which acts exclusively through its binding to PII and PII-UMP, did not alter the binding of PII or PII-UMP to the enzyme. Rather, alpha-ketoglutarate dramatically affected the extent of activation or inhibition of the enzyme by PII or PII-UMP. A working hypothesis for the regulation of the AT and AR activities, consistent with all data, is presented.

Glutamine synthetase adenylyltransferase (ATase) regulates the activity of glutamine synthetase by adenylylation and deadenylylation in response to signals of nitrogen and carbon status: glutamine, alpha-ketoglutarate, and the uridylylated and unmodified forms of the PII signal transduction protein. ATase consists of two conserved nucleotidyltransferase (NT) domains linked by a central region of approximately 200 amino acids. Here, we study the activities and regulation of mutated and truncated forms of ATase. Our results indicate the following. (i) The N-terminal NT domain contained the adenylyl-removing (AR) active site, and the C-terminal NT domain contained the adenylyltransferase (AT) active site. (ii) The enzyme contained a glutamine binding site, and glutamine increased the affinity for PII. (iii) The enzyme appeared to contain multiple sites for the binding of PII and PII-UMP. (iv) Truncated versions of ATase missing the C-terminal (NT) domain lacked both AT and AR activity, suggesting a role for the C-terminal NT domain in both activities. (v) The purified C-terminal NT domain and larger polypeptides containing this domain had significant basal AT activity, which was stimulated by glutamine. These polypeptides were indifferent to PII and PII-UMP, or their ATase activity was inhibited by either PII or PII-UMP. (vi) Certain point mutations in the central region or an internal deletion removing most of this part of the protein eliminated the AR activity and eliminated activation of the AT activity by PII, while not eliminating the binding of PII or PII-UMP. That is, these mutations in the central region appeared to destroy the communication between the PII and PII-UMP binding sites and the AT and AR active sites. (vii) Certain mutations in the central region of ATase appeared to dramatically improve the binding of glutamine to the enzyme. (viii) While the isolated AT and AR domains of ATase bound poorly to PII and PII-UMP, these domains bound PII and PII-UMP significantly better when linked to the central region of ATase. Together, our results indicate a highly coordinated enzyme, in which the AT and AR domains participate in each other's regulation and distant regulatory sites are in communication with each other. A model for the regulation of ATase by glutamine, PII, and PII-UMP consistent with all data is presented.

PII proteins are small homotrimeric signal transduction proteins that regulate the activities of metabolic enzymes and permeases, and control the activities of signal transduction enzymes. The protein family shows high conservation, with examples in eukaryota (plants and eukaryotic algae), archaea, and bacteria. This distribution indicates that PII is one of the most ancient signalling proteins known.

The NRI/NRII two-component system controls the expression of nitrogen regulated (Ntr) genes in response to signals of carbon and nitrogen status. NRII dimers consist of three types of protein domains: N-terminal domains involved in intramolecular signal transduction; a central domain mediating dimerization involved in kinase, phosphotransfer, and phosphatase catalytic activities; and C-terminal ATP-binding domains. Data indicate that the kinase and phosphatase activities of the central domain of NRII are regulated by the binding of the PII protein to the C-terminal ATP-binding domains of NRII and the N-terminal domains of NRII are involved in stabilizing the "phosphatase" conformation of the NRII central domain. The two subunits of the NRII dimer act in a highly concerted manner during the autophosphorylation reaction. An "alternating sites" hypothesis is used to explain the autophosphorylation mechanism of NRII and the regulation of NRII activities by PII.

Based on the portrait method proposed by Hao et al. (Chaos Solitons Fractals 11 (6) (2000) 825), four parameters (distance, correlation coefficient, entropy and mutual information) are introduced to provide exact measures of the difference between a real genome and the “white noise” genome of it. Here the “white noise” genome of a real genome means a random sequence based on the frequencies of four kinds of nucleotides appearing in the real genome. These parameters can also be used to discuss the classification and evolution among organisms. The evolution trees of some bacteria based on these parameters are given.

Publisher Summary To maintain balanced metabolism, Escherichia coli must coordinate the assimilation of nitrogen with the assimilation of carbon and other essential nutrients. This coordination is accomplished in part by a signal transduction system that measures the signals of carbon and nitrogen status and regulates the activity of glutamine synthetase (GS) and the transcription of nitrogen-regulated (Ntr) genes, whose products facilitate the use of poor nitrogen sources. The key sensory components of this signal transduction system are the uridylyltransferase/ uridylyl-removing enzyme (UTase/UR), PII protein, and adenylyltransferase (ATase) that regulates GS by reversible adenylylation. This chapter discusses the current state of understanding of these signal-transducing proteins and the mechanisms by which they detect and transduce the signals of nitrogen and carbon status. It also discusses the physiology of the response to nitrogen and carbon availability and presents an overview of the signal transduction system.

The PII signal transduction protein regulates the transcription of nitrogen-regulated genes by controlling the kinase and phosphatase activities of NRII. We used a cross-linking approach to study the interaction of the T-loop of the PII protein with NRII. Cross-linking of PII to NRII required ATP and 2-ketoglutarate, allosteric effectors known to control PII activity, and was not affected by the presence of excess nonspecific proteins such as bovine serum albumin. The purified cross-linked species appeared to consist mainly of PII trimers in which one of the three subunits was cross-linked to a single subunit of the NRII dimer; this complex had the phosphatase activity characteristic of the un-cross-linked PII-NRII complex, and had significant phosphatase activity in the absence of 2-ketoglutarate, suggesting that once PII was tethered to NRII the active conformation was stabilized. Studies with truncated forms of NRII indicated that the purified N-terminal "sensory" domain of NRII was not cross-linked to PII, nor was a polypeptide consisting of NRII residues 1-189. In contrast, polypeptides containing the kinase domain of the transmitter module of NRII (residues 190-349) were cross-linked to PII in an ATP- and 2-ketoglutarate-dependent reaction. These results indicate that PII controls NRII by interaction with the conserved kinase domain of the transmitter module.

The dimeric two-component system transmitter protein NRII (NtrB) of Escherichia coli, product of glnL (ntrB), controls transcription of nitrogen-regulated genes by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI (NtrC). Previous studies showed that the PII signal transduction protein inhibits the kinase activity of NRII and activates its phosphatase activity. We observed that PII greatly stimulated the NRII phosphatase activity under conditions where the cleavage of ATP was prevented, indicating that the phosphatase activity did not result simply from prevention of the antagonistic NRII kinase activity by PII. Rather, PII was an activator of the phosphatase activity. To study this regulation, we examined the dimerization and enzymatic activities of NRII and various polypeptides derived from NRII, and their regulation by PII. Our results were consistent with the hypothesis that NRII consists of three domains: an N-terminal domain found only in NRII proteins and two domains formed by the conserved transmitter module of NRII, the phosphotransferase/phosphatase/dimerization (central) domain and the kinase domain. All three domains were involved in regulating the kinase and phosphatase activities of NRII. The N-terminal domain was involved in intramolecular signal transduction, and controlled access to the NRII active site for the isolated dimeric central domain added in trans. The central domain was responsible for dimerization and the phosphotransferase and phosphatase activities of NRII, but the latter activity was weak in the isolated domain and was not regulated by PII. The C-terminal kinase domain was responsible for the kinase activity. The PII protein appeared to interact with the isolated transmitter module of NRII, and not with the N-terminal domain as previously thought, since PII dramatically increased the stoichiometry of autophosphorylation of the isolated transmitter module. However, the phosphatase activity of the transmitter module of NRII was low even in the presence of PII, suggesting that the N-terminal domain was necessary for the central domain to assume the conformation necessary for potent phosphatase activity. Also, PII significantly reduced the rate of transphosphorylation of the isolated central domain by the isolated kinase domain, suggesting that PII interacts directly with the kinase domain. We hypothesize that the binding of PII to the kinase domain of NRII results in an altered conformation that is transmitted to the central and N-terminal domains; this causes the central domain to assume the conformation with potent phosphatase activity.

Autophosphorylation of the homodimeric two-component system transmitter protein nitrogen regulator II (NRII; also NtrB) of Escherichia coli is the first step in the activation of nitrogen-regulated (Ntr) gene transcription. We show that the autophosphorylation of NRII was asymmetric, with phosphorylation of the first and second subunits of the dimer displaying different equilibria (under our experimental conditions K(1) approximately 0. 345, K(2) approximately 0.0044). Phosphorylation of both subunits of NRII was rapid, but the very rapid reversal of the phosphorylation of the second subunit was responsible for the equilibrium position of the reaction. Complete phosphorylation of NRII was only observed under conditions where ADP, a product of the autophosphorylation reaction, was removed by an enzymatic system. Purified, doubly phosphorylated NRII (NRII approximately P(2)) was stable in the absence of nucleotides at 0 degrees C but was dephosphorylated to the hemiphosphorylated form at 37 degrees C. In the presence of a low concentration of ADP, half of the phosphoryl groups from NRII approximately P(2) were rapidly dephosphorylated, while the remaining phosphoryl groups were slowly dephosphorylated. Experiments with heterodimers containing wild-type and mutant, nonphosphorylatable subunits suggested that the asymmetry of NRII autophosphorylation was not preexisting but resulted from the autophosphorylation of one subunit.