A source of ultrasensitivity in the glutamine response of the bicyclic cascade system controlling glutamine synthetase adenylylation state and activity in Escherichia coli

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.     

Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.     

In situ inactivation of E. coli uridylylation cycle is independent of the state of covalent modification of the components of the glutamine synthetase cascade

Permeabilization of nitrogen-starved cells of Escherichia coli W with Lubrol WX leads to a selective inactivation of the uridylyl-removing uridylyltransferase (UR/ UTase) enzyme of the glutamine synthetase (GS) cascade system; whereas similar treatment does not affect activity of UR/UTase in cells grown under conditions of nitrogen excess (10 mm glutamine) (Mura, U., and Stadtman, E. R. (1981) J. Biol. Chem.256, 13014–13021). The possibility that susceptibility to Lubrol inactivation is related to differences in the state of adenylylation of GS and/or in the state of uridylylation of the P_II protein was investigated. Permeabilized cells from nitrogen sufficient as well as from nitrogen-limited growth medium were exposed to Lubrol after prior incubation under conditions that lead to high or low states of GS adenylylation and high or low P_IID/P_IIA ratios. Integrity of UR/UTase was monitored by measuring the capacity of UTP to stimulate the deadenylylation of GS in situ. The results showed that the inactivation of UR/UTase by Lubrol is not affected by the states of GS adenylylation or P_II uridylylation.     

Glutamine synthetase of Klebsiella aerogenes: genetic and physiological properties of mutants in the adenylylation system.

Mutations resulting in defects in the adenylylation system of glutamine synthetase (GS) affect the expression of glnA, the structural gene for GS. Mutants with lesions in glnB are glutamine auxotrophs and contain repressed levels of highly adenylylated GS. Glutamine-independent revertants of the glnB3 mutant have acquired an additional mutation at the glnE site. The glnE54 mutant is incapable of adenylylating GS and produces high levels of enzyme, even when ammonia is present in the growth medium. The fact that mutations in glnB and glnE simultaneously disturb both the normal adenylylation and repression patterns of GS in Klebsiella aerogenes indicates that the adenylylation system, or adenylylation state, of GS is critical for the regulation of synthesis of GS.     

Robustness in Escherichia coli Glutamate and Glutamine Synthesis Studied by a Kinetic Model

Metabolic control of glutamine and glutamate synthesis from ammonia and oxoglutarate in Escherichia coli is tight and complex. In this work, the role of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) regulation in this control was studied. Both enzymes form a linear pathway, which can also have a cyclic topology if glutamate–oxoglutarate amino transferase (GOGAT) activity is included. We modelled the metabolic pathways in the linear or cyclic topologies using a coupled nonlinear differential equations system. To simulate GS regulation by covalent modification, we introduced a relationship that took into account the levels of oxoglutarate and glutamine as signal inputs, as well as the ultrasensitive response of enzyme adenylylation. Thus, by including this relationship or not, we were able to model the system with or without GS regulation. In addition, GS and GDH activities were changed manually. The response of the model in different stationary states, or under the influence of N-input exhaustion or oscillation, was analyzed in both pathway topologies. Our results indicate a metabolic control coefficient for GDH ranging from 0.94 in the linear pathway with GS regulation to 0.24 in the cyclic pathway without regulation, employing a default GDH concentration of 8 μM. Thus, in these conditions, GDH seemed to have a high degree of control in the linear pathway while having limited influence in the cyclic one. When GS was regulated, system responses to N-input perturbations were more sensitive, especially in the cyclic pathway. Furthermore, we found that effects of regulation against perturbations depended on the relative values of the glutamine and glutamate output first-order kinetic constants, which we named k ₆ and k ₇, respectively. Effects of regulation grew exponentially with a factor around 2, with linear increases of (k ₇???k ₆). These trends were sustained but with lower differences at higher GS concentration. Hence, GS regulation seemed important for metabolic stability in a changing environment, depending on the cell’s metabolic status.     

Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam

Selected pathovars of Pseudomonas syringae produce an extracellular phytotoxin, tabtoxinine-beta-lactam, that irreversibly inhibits its known physiological target, glutamine synthetase (GS). Pseudomonas syringae subsp. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection mechanism(s) used by these pathovars, we have determined that GS becomes adenylylated after toxin production is initiated and that the serine released from the zinc-activated hydrolysis of tabtoxin is a factor in the initiation of this adenylylation. The adenylylation state of this GS was estimated to range from E5.0-7.5. The irreversible inactivation by tabtoxinine-beta-lactam of unadenylylated and adenylylated glutamine synthetase purified from P. syringae subsp. "tabaci" was investigated. Adenylylated GS was inactivated by tabtoxinine-beta-lactam at a slower rate than was unadenylylated enzyme. Adenylylated GS (E7.5-10.5) was significantly protected from this inactivation in the presence of the enzyme effectors, AMP, Ala, Gly, His, and Ser. Thus, the combination of the adenylylation of GS after toxin production is initiated and the presence of the enzyme effectors in vivo could provide part of the self-protection mechanism used by subsp. "tabaci".     

The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction.

Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C). We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII. The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.     

Reversible adenylylation of glutamine synthetase is dynamically counterbalanced during steady-state growth of Escherichia coli

Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by small effectors, most notably glutamine and 2-oxoglutarate. In vivo, adenylyltransferase (AT) activity is critical for growth adaptation when cells are shifted from nitrogen-limiting to nitrogen-excess conditions and a rapid decrease of GS activity by adenylylation is needed. Here, we show that the adenylyl-removing (AR) activity of ATase is required to counterbalance its AT activity during steady-state growth under both nitrogen-excess and nitrogen-limiting conditions. This conclusion was established by studying AR⁻/AT⁺ mutants, which surprisingly displayed steady-state growth defects in nitrogen-excess conditions due to excessive GS adenylylation. Moreover, GS was abnormally adenylylated in the AR⁻ mutants even under nitrogen-limiting conditions, whereas there was little GS adenylylation in wild-type strains. Despite the importance of AR activity, we establish that AT activity is significantly regulated in vivo, mainly by the cellular glutamine concentration. There is good general agreement between quantitative estimates of AT regulation in vivo and results derived from previous in vitro studies except at very low AT activities. We propose additional mechanisms for the low AT activities in vivo. The results suggest that dynamic counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes such as GS, whose physiological output allows adaptation to environmental fluctuations.     

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.     

Isolation and crystallization of unadenylylated glutamine synthetase from Salmonella typhimurium

The enzyme glutamine synthetase (GS) has been isolated from a mutant strain of Salmonella typhimurium, constructed by Kustu, which lacks the enzymatic activity for adenylylation of glutamine synthetase. Thus the purified GS is uniformly unadenylylated, as confirmed by gel electrophoresis and enzyme assays. It crystallizes readily in many morphologies, at least six of which are distinct polymorphs. The most favorable crystal form for structural studies belongs to space group C2, with unit cell dimensions a = 235.5 A, b = 134.5 A, c = 200.1 A, beta = 102.8 degrees, and with one GS molecule per asymmetric unit. The crystals diffract to about 2.8 A resolution in rotation X-ray photographs and thus appear suitable for structural studies at moderate resolution. These crystals are isomorphous with crystalline GS from Escherichia coli in both adenylylated and unadenylylated states, suggesting that the enzymes from the two bacteria are similar molecules, and that adenylylation does not greatly affect the conformation of the molecule.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Covalent modification of bacterial glutamine synthetase: physiological significance

Summary Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH₄ ⁺). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to 10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH₄ ⁺ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH₄ ⁺-excess conditions and thereby to allow rapid growth.     

Mutations that alter the covalent modification of glutamine synthetase in Salmonella typhimurium.

glnD and glnE mutant strains of Salmonella typhimurium lack three of the four activities required for reversible covalent modification of glutamine synthetase (GS; EC 6.3.1.2). The glnD strains, which are unable to deadenylylate GS and therefore accumulate the adenylylated or less active form of the enzyme, were isolated as glutamine bradytrophs. They lack the activity of PIIA uridylyl-transferase, one of the proteins required for deadenylylation of GS; in addition, they lack PIID uridylyl-removing activity. Mutations in glnD are suppressed by second-site mutations in glnE that eliminate the activity of GS adenylyltransferase (EC 2.7.7.42) and thus prevent adenylylation of GS. The glnD and glnE strains have one-third to one-half as much total GS as the wild-type strain when they are grown in a medium containing a high concentration of NH4+. The wild-type strain derepresses synthesis of GS fourfold in response to nitrogen limitation; glnD and glnE strains derepress synthesis of the enzyme fourfold and sevenfold, respectively. Thus, mutations that alter covalent modification of GS in Salmonella do not significantly affect derepression of its synthesis. The glnD gene lies at 7 min on the Salmonella chromosome and is 50% linked to pyrH by P22-mediated transduction.     

Glutamine synthetase adenylylation in permeabilized cells of Escherichia coli

Following a freeze-thaw cycle, treatment of Escherichia coli with the nonionic detergent, Lubrol WX, renders the cells permeable to small molecules but not to cytosolic proteins. After such treatment, the permeabilized cell suspensions can be assayed directly by standard procedures both for intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number, n, of adenylylated subunits/dodecameric molecule). Permeabilization of cells from cultures containing an adequate supply of glutamine as the sole nitrogen source led to complete retention of all protein components of the bicyclic cascade that regulates the interconversion of glutamine synthetase between adenylylated and unadenylylated forms; similar treatment of glutamine-starved cells leads to selective inactivation, only, of the uridylyltransferase. When suspended in buffers containing ATP and glutamine, the value of n in permeabilized cells increased to high values (n = 11), whereas in the presence of alpha-ketoglutarate, Pi, and ATP, the value of n decreased to approximately 2.0. Time-dependent changes in n that occur during incubations of permeabilized cells in buffers containing these effectors can be arrested either by sonication at 0-4 degrees C or by the addition of cetyltrimethylammonium bromide (to inactivate adenylyltransferase). It is thus evident that Lubrol-treated cells may be used to investigate the regulation of glutamine synthetase adenylylation in situ.     

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.     

Escherichia coli glutamine synthetase adenylyltransferase (ATase, EC 2.7.7.49): kinetic characterization of regulation by PII, PII-UMP, glutamine, and alpha-ketoglutarate

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.     

Evidence for a glutamine synthetase-chromatophore association in the phototroph Rhodospirillum rubrum: purification, properties, and regulation of the enzyme.

The characteristics of soluble and membrane-bound glutamine synthetase (GS) from Rhodospirillum rubrum were compared with those of the enzyme located in situ (measured in detergent-treated cells). The results suggest that in vivo GS may be associated with, or bound to, the chromatophore membranes. GS was found to reversibly associate and dissociate from purified chromatophores as a function of the ionic strength of the buffer or the Mg2+ concentration. Solubilized GS was purified to homogeneity and found to be similar to the GS of enteric bacteria in that its molecular weight was about 600,000 and it had one type of subunit of 51,000 molecular weight. Removal of GS from the membrane had no effect on the Km values for the substrates of the biosynthetic reaction, but it did have a substantial effect on both its Mg2+ requirement (the Km increased 10-fold) and the sensitivity of the gamma-glutamyl transferase reaction to the inhibitor methionine sulfoximine (the I0.5 decreased from 1,500 to 60 microM). Both observations suggest that the active site of GS is influenced by its association with the membrane. GS activity was shown to respond to NH4+, phosphodiesterase, Mg2+, and adenylylation cofactors in a manner identical to that of the GS of the coliform bacteria, suggesting that the former may also respond to adenylylation and deadenylylation. Finally, R. rubrum GS was also inhibited by NH4+ by a newly observed, as yet undefined, system.     

Attenuation of noise in ultrasensitive signaling cascades

Ultrasensitive cascades often implement thresholding operations in cell signaling and gene regulatory networks, converting graded input signals into discrete all-or-none outputs. However, the biochemical and genetic reactions involved in such cascades are subject to random fluctuations, leading to noise in output signal levels. Here we prove that cascades operating near saturation have output signal fluctuations that are bounded in magnitude, even as the number of noisy cascade stages becomes large. We show that these fluctuation-bounded cascades can be used to attenuate the noise in an input signal, and we find the optimal cascade length required to achieve the best possible noise reduction. Cascades with ultrasensitive transfer functions naturally operate near saturation, and can be made to simultaneously implement thresholding and noise reduction. They are therefore ideally suited to mediate signal transfer in both natural and artificial biological networks.