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.     

Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice

Mycobacterium tuberculosis and Mycobacterium bovis cause tuberculosis, which is responsible for the deaths of more people each year than any other bacterial infectious disease. Disseminated disease with Mycobacterium bovis BCG, the only currently available vaccine against tuberculosis, occurs in immunocompetent and immunodeficient individuals. Although mycobacteria are obligate aerobes, they are thought to face an anaerobic environment during infection, notably inside abscesses and granulomas. The purpose of this study was to define a metabolic pathway that could allow mycobacteria to exist under these conditions. Recently, the complete genome of M. tuberculosis has been sequenced, and genes homologous to an anaerobic nitrate reductase (narGHJI), an enzyme allowing nitrate respiration when oxygen is absent, were found. Here, we show that the narGHJI cluster of M. tuberculosis is functional as it conferred anaerobic nitrate reductase activity to Mycobacterium smegmatis. A narG mutant of M. bovis BCG was generated by targeted gene deletion. The mutant lacked the ability to reduce nitrate under anaerobic conditions. Both mutant and M. bovis BCG wild type grew equally well under aerobic conditions in vitro. Histology of immunodeficient mice (SCID) infected with M. bovis BCG wild type revealed large granulomas teeming with acid-fast bacilli; all mice showed signs of clinical disease after 50 days and succumbed after 80 days. In contrast, mice infected with the mutant had smaller granulomas containing fewer bacteria; these mice showed no signs of clinical disease after more than 200 days. Thus, it seems that nitrate respiration contributes significantly to virulence of M. bovis BCG in immunodeficient SCID mice.     

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.     

Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads

A two-step protocol has been developed for isolation of plasmids from recombinant mycobacteria via Escherichia coli. First either mycobacterial primary transformants or propagated cultures were lysed in a mini-bead beater using zirconia beads and the lysate thus obtained was used to transform E. coli recA mutant cells. Secondly, plasmid DNA was isolated from recombinant E. coli cells and analysed. Bead beating times of 2 min for Mycobacterium smegmatis, a rapid grower, and 4 min for M. bovis BCG, a slow grower, were found to be optimal for recovery of plasmid DNA. This protocol was also amenable to other mycobacterial species such as M. avium, M. fortuitum and M. tuberculosis H37Ra. Plasmid recovery from the recombinant M. bovis BCG using this protocol is approximately 300-fold higher than that reported for the electroduction method.     

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.     

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.     

Role of protein kinase G in growth and glutamine metabolism of Mycobacterium bovis BCG

The survival of pathogenic mycobacteria in macrophages requires the eukaryotic enzyme-like serine/threonine protein kinase G. This kinase with unknown specificity is secreted into the cytosol of infected macrophages and inhibits phagosome-lysosome fusion. The pknG gene is the terminal gene in a putative operon containing glnH, encoding a protein potentially involved in glutamine uptake. Here, we report that the deletion of pknG did not affect either glutamine uptake or intracellular glutamine concentrations. In vitro growth of Mycobacterium bovis BCG lacking pknG was identical to that of the wild type. We conclude that in M. bovis BCG, glutamine metabolism is not regulated by protein kinase G.     

Manipulation of immune responses to Mycobacterium bovis by vaccination with IL-2- and IL-18-secreting recombinant bacillus Calmette Guerin

Bacillus Calmette Guerin (BCG) has been reported to show variable efficacy as a vaccine against tuberculosis. We demonstrated that the secretion of biologically active IL-2 (rBCG/IL-2),but not IL-18 (rBCG/IL-18), by BCG improves its ability to induce and maintain a strong type 1 immune response in BALB/c mice. rBCG/IL-2 induced significantly higher Ag-specific proliferative responses, high IFN-gamma production and serum titres of IgG2a 16 weeks after vaccination. This immune profile was correlated to an increased rate of clearance of non-pathogenic mycobacteria (live BCG delivered intranasally). Surprisingly, however,this strong type 1 immune profile induced no greater protective immunity against aerosol challenge with virulent Mycobacterium bovis than that induced by normal BCG (nBCG). By comparison,vaccination with rBCG/IL-18 was found to induce significantly less IFN-gamma production in splenic lymphocytes than nBCG.This impaired induction of IFN-gamma was correlated to a significantly lower protective efficacy against M. bovis challenge, as compared to nBCG. The data suggest that manipulation of the immune response to tuberculosis and tuberculosis vaccines will require a more complete understanding of the factors that are important in generating a protective immune response.     

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.     

A Point Mutation in cycA Partially Contributes to the D-cycloserine Resistance Trait of Mycobacterium bovis BCG Vaccine Strains

In mycobacteria, CycA a D-serine, L- and D-alanine, and glycine transporter also functions in the uptake of D-cycloserine, an important second-line anti-tubercular drug. A single nucleotide polymorphism identified in the cycA gene of BCG was hypothesized to contribute to the increased resistance of Mycobacterium bovis bacillus Calmette-Guérin (BCG) to D-cycloserine compared to wild-type Mycobacterium tuberculosis or Mycobacterium bovis. Working along these lines, a merodiploid strain of BCG expressing Mycobacterium tuberculosis CycA was generated and found to exhibit increased susceptibility to D-cycloserine albeit not to the same extent as wild-type Mycobacterium tuberculosis or Mycobacterium bovis. In addition, recombinant Mycobacterium smegmatis strains expressing either Mycobacterium tuberculosis or Mycobacterium bovis CycA but not BCG CycA were rendered more susceptible to D-cycloserine. These findings support the notion that CycA-mediated uptake in BCG is impaired as a result of a single nucleotide polymorphism; however, the partial contribution of this impairment to D-cycloserine resistance suggests the involvement of additional genetic lesions in this phenotype.     

On the killing of mycobacteria by macrophages

Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis , the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.     

Impaired generation of reactive oxygen species during differentiation of dendritic cells (DCs) by Mycobacterium tuberculosis secretory antigen (MTSA) and subsequent activation of MTSA-DCs by mycobacteria results in increased intracellular survival

We investigated the role of reactive oxygen species (ROS) in dendritic cell (DC) differentiation by 10-kDa Mycobacterium tuberculosis secretory Ag (MTSA) and survival of mycobacteria therein. Compared with GM-CSF, MTSA induced lower ROS production during DC differentiation from precursors. This result correlated with higher superoxide dismutase 1 expression in MTSA stimulated precursors as compared with GM-CSF stimulation. Furthermore, a negative regulation of protein kinase C (PKC) activation by ROS was observed during DC differentiation. ROS inhibited the rapid and increased phosphorylation of PKCalpha observed during DC differentiation by MTSA. In contrast, ROS inhibition increased the weak and delayed PKCalpha phosphorylation by GM-CSF. Similar to DC differentiation, upon activation with either M. tuberculosis cell extract (CE) or live Mycobacterium bovis bacillus Calmette-Guérin (BCG), DCs differentiated with MTSA (MTSA-DCs) generated lower ROS levels when compared with DCs differentiated with GM-CSF (GM-CSF-DCs). Likewise, a negative regulation of PKCalpha phosphorylation by ROS was once again observed in DCs activated with either M. tuberculosis CE or live M. bovis BCG. However, a reciprocal positive regulation between ROS and calcium was observed. Compared with MTSA-DCs, stimulation of GM-CSF-DCs with M. tuberculosis CE induced a 2-fold higher ROS-dependent calcium influx. However, pretreatment of MTSA-DCs with H(2)O(2) increased calcium mobilization. Finally, lower ROS levels in MTSA-DCs correlated with increased intracellular survival of M. bovis BCG when compared with survival in GM-CSF-DCs. Although inhibiting ROS in GM-CSF-DCs increased M. bovis BCG survival, H(2)O(2) treatment of MTSA-DCs decreased survival of M. bovis BCG. Overall our results suggest that DCs differentiated with Ags such as MTSA may provide a niche for survival and/or growth of mycobacteria following sequestration of ROS.     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

New methods for the colorimetric assay of PIID regulatory protein, uridylyltransferase, and uridylyl-removing enzyme in glutamine synthetase cascade

Adenylylation and deadenylylation of glutamine synthetase (GS) are catalyzed by the same adenylyltransferase (ATase). The ability of ATase to catalyze adenylylation is markedly stimulated by the unmodified form of a regulatory protein, P_IIA, whereas its capacity to catalyze deadenylylation is stimulated by the uridylylated form (P_IID) of the regulatory protein. Interconversion between P_IIA and P_IID is catalyzed by uridylyltransferase (UTase) and uridylylremoving enzyme (UR). New colorimetric methods were developed for the assays of P_IID, UTase, and UR activities. The P_IID activity is monitored by its unique ability to stimulate the ATase catalyzed formation of unadenylylated subunits from adenylylated GS. The inerease of unadenylylated subunits is determined by measuring the γ-glutamyltransferase activity of GS under conditions where the activity of an unadenylylated subunit is about 15 times greater than that of an adenylylated subunit (i.e., at pH 8.0 in the presence of Mn²⁺). Assays for UTase and UR enzyme are derived by coupling the P_IID assay to the UTase and UR reactions. For the UTase reaction, the formation of P_IID from P_IIA is measured, whereas the decrease in P_IID is followed for the UR assay. These assays have been applied to follow the activities of these proteins during their purification procedures, to the mechanistic studies on the deadenylylation reaction, and to determine the activities of these proteins in mutants produced during the genetic study of glutamine synthetase cascade. The problems evolved from these assays are discussed.     

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".     

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)