Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis

We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GS(E)) and Klebsiella aerogenes (GS(K)). In gels containing sodium dodecyl sulfate (SDS), we found that GS(K) had a mobility which differed significantly from that of GS(E). In addition, for both GS(K) and GS(E), adenylylated subunits (GS(K)-adenosine 5'-monophosphate [AMP] and GS(E)-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GS(K)-AMP < GS(K) < GS(E)-AMP < GS(E). We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GS(K), and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GS(K) or GS(K)-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele.     

Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique.

Ammonia-nitrogen-limited continuous cultures of Escherichia coli and Klebsiella aerogenes contain induced levels of glutamine synthetase that is deadenylyated (i.e., fully active). In the presence of excess ammonia or glutamate in glucose-limited cultures of E. coli, glutamine synthetase is repressed and adenylylated (inactive). The average state of adenylylation (n) is a linear function of the specific growth rate. At low specific growth rates, glutamine synthetase is adenylylated; as the specific growth rate increases, n decreases, approaching 0 to 2 at rapid growth rates. The average state of adenylylation correlates well with the intracellular concentrations and ratios of alpha-ketoglutarate and glutamine, which are key effectors in the adenylylation-deadenylylation systems. E. coli and K. aerogenes differ markedly in their growth yields, growth rates, and enzymatic composition during nitrogen limitation. The data suggest that, unlike K. aerogenes, E. coli W uses glutamate dehydrogenase to incorporate ammonia during nitrogen limitation. In E. coli, glutamate dehydrogenase is progressively induced during nitrogen limitation when mu (growth rate) approaches mumax. In contrast, in K. aerogenes glutamate dehydrogenase is repressed during nitrogen limitation, whereas glutamate synthase, an alternative supplier of glutamate to the cell, is induced. Data are presented that support the regulatory schemes proposed for the control of glutamine synthetase activity by induction-repression phenomena and adenylylation-deadenylylation reaction. We propose that the intracellular ratio of alpha-ketoglutarate to glutamine may be the most important physiological parameter in determining the activity of glutamine synthetase.     

Distinctive properties of glutamine synthetase from the cyanobacterium Anacystis nidulans.

The intracellular levels of glutamine synthetase (GS) in Anacystis nidulans grown under different conditions were determined using a whole-cell assay. Nitrate-grown cells have 64% more GS than cells grown in ammonium sulfate. Nitrogen starvation does not affect GS levels appreciably. Incubation of nitrate-grown cells with ammonium sulfate does not change the ratio of gamma-glutamyl transferase activities stimulated by Mg2+ and Mn2+ ions. An in vitro test of adenylylation indicates that algae do not have an endogenous adenylyl transferase (ATase) and that algal GS is not adenylylatable by the Klebsiella aerogenes ATase. Some characteristics of the GS-membrane complex were determined by centrifugation of the complex under varying conditions of pH and ionic strength. In this way, it was shown that acid pH (4.5) stabilizes the complex and high ionic strength tends to solubilize the enzyme. A simple partial purification of GS (89-fold) was developed based on the sedimentation properties of GS.     

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.     

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.     

Immunoprotective extracellular polysaccharides of Klebsiella aerogenes capsular type K1, expressed in Escherichia coli

Abstract A gene library of genomic DNA Klebsiella aerogenes of capsular serotype K1 was constructed in E. coli LE392 using the cosmid pMMB33. Culture filtrates of E. coli recombinants were screened by ELISA for extracellular polysaccharides specific for K. aerogenes K1. Extracellular polysaccharide extracts from K. aerogenes K1 and 3% of the E. coli recombinants contained immunoprotective extracellular polysaccharides (IEP) with similar chemical and immunological properties as shown by gel filtration through Sephacryl 1000, double immunodiffusion and mouse protection tests. IEPs contained no detectable protein, had molecular weights of several hundred million and protected mice against lethal autologous K. aerogenes K1 challenge at a dosage of 10 nanograms per mouse.     

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.     

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.     

Purification and characterization of glutamine synthetase from the archaebacterium Methanobacterium ivanovi.

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the obligate anaerobic archaebacterium Methanobacterium ivanovi. The 130-fold-purified enzyme was obtained by heat treatment, ion-exchange chromatography, and gel filtration. Like all other eubacterial GSs known so far, the GS of M. ivanovi was found to be a dodecamer of about 600,000 daltons composed of a single type of subunit. The enzyme was stable at 63 degrees C for 10 min and was not sensitive to oxygen. The isoelectric point was 4.6, and the optimum pH of gamma-glutamyltransferase activity was 8.0. The Km values for hydroxylamine, glutamine, and ADP in the transferase reaction were 6.8, 22.7, and 0.35 mM, respectively. L-Methionine-DL-sulfoximine strongly inhibited the activity. Like the GS from gram-positive bacteria, Anabaena sp., several yeasts, and mammals, the enzyme from M. ivanovi was not regulated by adenylylation as demonstrated by snake venom phosphodiesterase treatment. Inhibition of the transferase activity by L-alanine, glycine, L-histidine, and L-tryptophan was observed. L-Glutamine alone or in the presence of AMP did not inhibit the GS synthetic activity. The GS of Methanobacterium ivanovi did not cross-react with a variety of antisera against GS from Escherichia coli, Anabaena strain 7120, or Bacillus megaterium. Archaebacterial GS appears to be structurally and functionally similar to eubacterial GS in gram-positive bacteria.     

Immunoprotective extracellular polysaccharides of Klebsiella aerogenes capsular type K1, expressed in Escherichia coli

A gene library of genomic DNA Klebsiella aerogenes of capsular serotype K1 was constructed in E. coli LE392 using the cosmid pMMB33. Culture filtrates of E. coli recombinants were screened by ELISA for extracellular polysaccharides specific for K. aerogenes K1. Extracellular polysaccharide extracts from K. aerogenes K1 and 3% of the E. coli recombinants contained immunoprotective extracellular polysaccharides (IEP) with similar chemical and immunological properties as shown by gel filtration through Sephacryl 1000, double immunodiffusion and mouse protection tests. IEPs contained no detectable protein, had molecular weights of several hundred million and protected mice against lethal autologous K. aerogenes K1 challenge at a dosage of 10 nanograms per mouse.     

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.     

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.     

Inhibition of the β-lactamases of Escherichia coli and Klebsiella aerogenes by semi-synthetic penicillins

1. A new automated micro-iodometric method is described for screening compounds for inhibitory action against beta-lactamase enzymes. 2. Over 1000 semi-synthetic penicillins were tested for inhibitory activity against the beta-lactamase of Escherichia coli B11 and 18 showed a fractional inhibition similar to or higher than that of methicillin. 3. The best inhibitors were alkoxy- and halogen-substituted phenyl-, naphthyl- or quinolyl-penicillins. 2-Isopropoxy-1-naphthylpenicillin (BRL 1437) was clearly the best and had a K(i) value about 1% of that of methicillin. 4. The inhibition of the beta-lactamase of E. coli B11 by BRL 1437 was shown to be reversible and competitive. The K(i) was 0.004mum and K(i)/K(m) with ampicillin and p-hydroxyampicillin (BRL 2333) was about 0.0001. The K(m) and V(max.) values were determined for the beta-lactamases of E. coli B11 and Klebsiella aerogenes A against a variety of penicillins. Cell-bound and solubilized enzymes gave similar K(i) and K(m) values. 5. BRL 1437 was superior to cloxacillin and methicillin for inhibition of the beta-lactamase of live, fully grown cultures of several strains of E. coli and K. aerogenes. Of a group of inhibitors BRL 1437 was the most stable to the beta-lactamase of E. coli B11.     

Allosteric interactions and bifunctionality make the response of glutamine synthetase cascade system of Escherichia coli robust and ultrasensitive

Glutamine synthetase (GS) regulation in Escherichia coli by reversible covalent modification cycles is a prototype of signal transduction by enzyme cascades. Such enzyme cascades are known to exhibit ultrasensitive response to primary stimuli and act as signal integration systems. Here, we have quantified GS bicyclic cascade based on steady state analysis by evaluating Hill coefficient. We demonstrate that adenylylation of GS with glutamine as input is insensitive to total enzyme concentrations of GS, uridylyltransferase/uridylyl-removing enzyme, regulatory protein PII, and adenylyltransferase/adenylyl-removing enzyme. This robust response of GS adenylylation is also observed for change in system parameters. From numerical analyses, we show that the robust ultrasensitive response of bicyclic cascade is because of allosteric interactions of glutamine and 2-ketoglutarate, bifunctionality of converter enzymes, and closed loop bicyclic cascade structure. By system level quantification of the GS bicyclic cascade, we conclude that such a robust response may help the cell in adapting to different carbon and nitrogen status.     

Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form.

Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest. The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay. There is no difference in pH optimum between the forms in the biosynthetic assay. In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+). It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay. The two forms of Rsp. rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors. In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition. In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive. The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation. It is also shown that the low-activity form of Rsp. rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine.     

Time-resolved fluorescence and computational studies of adenylylated glutamine synthetase: analysis of intersubunit interactions.

Adenylylation of Tyr-397 of each subunit of Escherichia coli glutamine synthetase (GS) down-regulates enzymatic activity in vivo. The overall structure of the enzyme consists of 12 subunits arranged as two hexamers, face to face. Research reported in this paper addresses the question of whether the covalently attached adenylyl group interacts with neighboring amino acid residues to produce the regulatory phenomenon. Wild-type GS has two Trp residues (positions 57 and 158) and the adenylylation site lies within 7-8 A of the Trp-57 loop in the adjacent subunit of the same hexameric ring; Trp-158 is about 35 A from the site of adenylylation. Fluorescence lifetimes and quantum yields have been determined for two fluorophores with wild-type and mutant GS. One fluorophore is epsilon-AMP adenylylated GS (at Tyr-397), and the other fluorophore is the intrinsic protein residue Trp-57. These experiments were conducted in order to detect possible intersubunit interactions between adenylyl groups and the neighboring Trp-57 to search for a role for the Trp-57 loop in the regulation of GS. The fluorescence due to epsilon-AMP of two adenylylated enzymes, wild-type GS and the W158F mutant, exhibits heterogeneous decay kinetics; the data adequately fit to a double exponential decay model with recovered average lifetime values of 18.2 and 2.1 ns, respectively. The pre-exponential factors range from 0.66 to 0.73 for the long lifetime component, at five emission wavelengths. The W57L-epsilon-AMP enzyme yields longer average lifetime values of 19.5 and 2.4 ns, and the pre-exponential factors range from 0.82 to 0.85 for the long lifetime component. An additional residue in the Trp-57 loop, Lys-58, has been altered and the K58C mutant enzyme has been adenylylated with epsilon-AMP on Tyr-397. Lys-58 is near the ATP binding site and may represent a link by which the adenylyl group controls the activity of GS. The fluorescence of epsilon-AMP-adenylylated K58C mutant GS is best described by a triple exponential decay with average recovered lifetime values of 19.9, 4.6, and 0.58 ns, with the largest fraction being the median lifetime component. Relative quantum yields of epsilon-AMP-Tyr-397 were measured in order to determine if static quenching occurs from adenine-indole stacking in the wild-type GS. The relative quantum yield of the epsilon-AMP-adenylylated W57L mutant is larger than the wild-type protein by the amount predicted from the difference in lifetime values: thus, no static quenching is evident.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of glutamine synthetase from a variety of bacteria

We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.     

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.