Expression of glutamine synthetase in mutant strains of the cyanobacterium Anabaena variabilis which liberate ammonia

Abstract The nucleoside analogue ganciclovir has clinical efficacy in the treatment of serious infections with human cytomegalovirus (CMV) in AIDS patients. The mechanism of action of the drug against CMV is different from that described for herpes simplex viruses (HSVs) as the crucial formation of the monophosphate derivative appears to be carried out by cellular rather than virus-coded enzymes. Adenovirus infections also induce the expression of cellular genes including kinase activity and a novel DNA polymerase and the results reported here show that these viruses are sensitive to ganciclovir. The 50% effective dose (ED₅₀) range for known serotypes and one clinical isolate was 4.5−33 μM. By comparison with the sensitivity of CMV in vitro and the known clinical response of infections with this virus to ganciclovir, our results suggest that this drug or its analogous may form the basis of chemotherapy for adenovirus infections.     

Biochemical characterization of glutamine synthetase from the diazotrophic cyanobacterium,Anabaena doliolum

In cyanobacteria, the glutamine synthetase-L-glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) pathway is the major ammonia-assimilating route. The GS ofAnabaena doliolum was synthesized more under N₂-fixing conditions, followed by ammonium, nitrate, and nitrite as nitrogen sources. The activities of both the glutamine synthetase, Mg²⁺-dependent biosynthetic and Mn²⁺-dependent -glutamyl transferase were optimum at pH 7. The active site of the enzyme bears sulfhydryl (-SH) groups; this was confirmed with the-SH group inhibitors, para-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM). The biosynthetic and -glutamyl transferase activities showed specificity for the divalent cations, Mg²⁺ and Mn²⁺, respectively. The other divalent cations Co²⁺, Cu²⁺, and Ni²⁺ were poor substitutes. This enzyme also required these divalent cations to stabilize its structure and function under extreme conditions such as high and low temperatures and urea denaturation. The glutamate analogl-methionine-d,l-sulfoximine, inactivated the enzyme, whereas the GOGAT inhibitor, azaserine, had no effect on the enzyme activity. The GS enzyme required de novo protein synthesis.     

The activation of glutamine synthetase from the cyanobacterium Anabaena cylindrica by thioredoxin

Abstract The present communication defines the conditions under which thioredoxin activates glutamine synthetase from Anabaena cylindrica . Effects are obtained at pH values around neutrality, and the activation is affected by Mg²⁺ in the assays. The thioredoxin systems from A. cylindrica and spinach are functionally interchangeable in the activation of glutamine synthetase. The enzyme is efficiently activated by thioredoxin_m and also by thioredoxin_f, but at much higher concentrations. Thioredoxin_m has previously been shown to activate NADPH-dependent malate dehydrogenase and isocitrate dehydrogenase from cyanobacteria. It is speculated that thioredoxin_m plays a role in the differentiation of vegetative cells to heterocysts.     

Kinetic and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120

A number of biochemical parameters of glutamine synthetase (EC 6.3.1.2) isolated from the cyanobacterium Anabaena 7120 were determined. Apparent Michaelis constants for glutamate and ATP were found to be 2.1 and 0.32 mM, respectively; that for ammonia was found to be below 20 microM, significantly lower than that reported for glutamine synthetases from other species. Serine, alanine, glycine, cysteine, aspartic acid, methionine sulfone, and methionine sulfoximine were found to inhibit the enzyme. The enzyme is controlled neither by adenylylation nor by feedback inhibition by glutamine, mechanisms found in some other prokaryotes. It must therefore be regulated by a different mechanism, possibly a combination of feedback by alanine, serine, and glycine, metabolites which are especially effective in inhibiting Anabaena glutamine synthetase.     

Effect of glutamine on growth and heterocyst differentiation in the cyanobacterium Anabaena variabilis.

Mutants of the cyanobacterium Anabaena variabilis that were capable of increased uptake of glutamine, as compared with that in the parental strains, were isolated. Growth of these mutants and their parental strains was measured in media containing N2, ammonia, or glutamine as a source of nitrogen. All strains grew well with any one of these sources of fixed nitrogen. Much of the glutamine taken up by the cells was converted to glutamate. The concentrations of glutamine, glutamate, arginine, ornithine, and citrulline in free amino acid pools in glutamine-grown cells were high compared with the concentrations of these amino acids in ammonia-grown or N2-grown cells. All strains capable of heterocyst differentiation, including a strain which produced nonfunctional heterocysts, grew and formed heterocysts in the presence of glutamine. However, nitrogenase activity was repressed in glutamine-grown cells. Glutamine may not be the molecule directly responsible for repression of the differentiation of heterocysts.     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.     

Photophobic responses of the cyanobacterium Anabaena variabilis

The photophobic responses in the Cyanobacterium Anabaena variabilis which belongs to the Nostocaceae have been studied with aid of a population method as well as by single trichome observations. In white light experiments both step-up and step-down photophobic responses were observed. The wavelength dependence was examined at a constant fluence rate. The photophobically active light is absorbed by the photosynthetic pigments, mainly by the phycobiliproteins and chlorohyll a. Above 690 nm only negative reactions were observed, i.e. the trichomes left the light trap. In white light experiments DCMU strongly inhibited the photophobic responses, whereas photokinesis was not affected to the same extent indicating that the reaction is coupled with the non cyclic photosynthetic electron transport. DBMIB impaired the photophobic behaviour only slightly. It seems that the photophobic responses of A. variabilis are controlled by a similar mechanism as in Phormidium uncinatum (Oscillatoriaceae) although the two families and, hence, the two species differ in their movement mechanism as well as in their photoactic behaviour.     

Transport of leucine in the cyanobacterium Anabaena variabilis

The amino acid leucine was transported by the cyanobacterium Anabaena variabilis. The K _m for transport was 10.8 M; the V _max was 8.7 nmoles min^–1 mg^–1 chlorophyll a. Transport of leucine was energy dependent: uptake of leucine was inhibited in the dark, and by DCMU and cyanide. Transport was neither dependent on nor enhanced by Na⁺. Prior growth of cells with leucine did not repress transport of [¹⁴C]-leucine. Alanine, glycine, valine, and methionine were strong competitive inhibitors of leucine uptake; serine, threonine, isoleucine, norleucine, and d-alanine competitively inhibited to a lesser degree. Other amino acids or amino acid analogues, including d-leucine, -aminoisobutyrate, and d-serine did not inhibit the transport of leucine.Abbreviations Chl a chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TES N-tris(hydroxymethyl)-2-aminoethane-sulfonic acid - TCA trichloroacetic acid - Tris N-tris(hydroxymethyl)aminoethane     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.     

Immuno-gold localization of glutamine synthetase in a nitrogen-fixing cyanobacterium (Anabaena cylindrica)

Localization of glutamine synthetase in thin sections of nitrogen-fixing Anabaena cylindrica was performed using immuno-gold/transmission electronmicroscopy. The enzyme was present in all of the three cell types possible; vegetative cells, heterocysts and akinetes. The specific gold label was always more pronounced in heterocysts compared with vegetative cells, and showed a uniform distribution in all three types. No specific label was associated with subcellular inclusions such as carboxysomes, cyanophycin granules and polyphosphate granules. When anti-glutamine synthetase antiserum was omitted, no label was observed.Abbreviation GS glutamine synthetase     

New low-copy plasmid in cyanobacterium Anabaena variabilis

Complete genome sequencing was performed for Anabaena variabilis ATCC 29413 from the collection of the Chair of Genetics, Department of Biology, Moscow State University, Russia. In addition to known plasmids A, B, and C, a new circular low-copy plasmid was detected and named D. It was also sequenced completely and found to have 27051 bp. The plasmid contained the parA and parB genes of the partition system, two genes that encode replication proteins, a gene for site-specific recombinase, a type-I restriction-modification system, and several genes with unknown functions. Analysis by PCR revealed the presence of plasmid D in two epiphytic strains from Vietnam, i.e., Anabaena sp. 182 and Anabaena sp. 281, as well as in Anabaena sp. V5 and A. azollae (Newton’s isolate).     

Some properties of glutamine synthetase from Anabaena cylindrica

Some properties of the biosynthetic and -glutamyltransferase activities of glutamine synthetase (EC 6.3.1.2) from Anabaena cylindrica are described, including requirement for divalent cations, pH optimum and K_m for substrates. The -glutamyl-transferase reaction was inhibited by L-glutamate, ammonia and ATP. The inhibition by L-glutamate and ammonia was competitive for L-glutamine and non-competitive for hydroxylamine. Both the biosynthetic and the -glutamyltransferase activities of the desalted enzyme were much more sensitive to inactivation by treatments such as urea, hydroxylamine and incubation at 50° C than the preparation which contained a divalent cation. The effects of some substrates of these reactions on protection against thermal denaturation and hydroxylamine were examined. An interpretation of these results in terms of the sequence of binding of substrates both in the biosynthetic and the -glutamyltransferase reactions are discussed.     

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.     

Regulation of uridylic acid biosynthesis in the cyanobacterium Anabaena variabilis.

The pathway of uridylic acid biosynthesis established by Leiberman, Kornberg, and Simms has been shown to be operative in the filamentous cyanobacterium Anabaena variabilis. The only enzyme of uridylic acid biosynthesis found to be lacking in two uracil-requiring strains of A. variabilis was aspartate transcarbamylase, the first enzyme in the pathway of de novo biosynthesis of uridvlic acid. Neither uracil-limited growth of a uracil-requiring mutant nor growth of the wild type in high concentrations of uracil resulted in substantial changes in the specific activities of enzymes of uridylic acid biosynthesis. It is therefore concluded that A. variabilis does not regulate all enzymes of this pathway by means of repression. However, control of the flow of intermediates through this pathway is possible by feedback inhibition of aspartate transcarbamylase by a variety of nucleotides.     

Induction of nitrate assimilation in the cyanobacterium Anabaena variabilis

Filaments of Anabaena variabilis Kütz strain ATCC 29413 grown in the absence of nitrate contain nitrate reductase that is active in permeabilized filaments, but not in intact, living filaments until they have been incubated for about 40 min in the presence of nitrate. The delayed acquisition of the ability to reduce nitrate is insensitive to chloramphenicol. Thus, switching on of enzyme activity in the presence of nitrate does not involve protein synthesis and nitrate reductase activity is not regulated by the amount of enzyme present.     

Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis.

Cells of the cyanobacterium Anabaena variabilis starved for phosphate for 3 days took up phosphate at about 100 times the rate of unstarved cells. Kinetic data suggested that a new transport system had been induced by starvation for phosphate. The inducible phosphate transport system was quickly repressed by addition of Pi. Phosphate-starved cells were more sensitive to the toxic effects of arsenate than were unstarved cells, but phosphate could alleviate some of the toxicity. Arsenate was a noncompetitive inhibitor of phosphate transport; however, the apparent Ki values were high, particularly for phosphate-replete cells. Preincubation of phosphate-starved cells with arsenate caused subsequent inhibition of phosphate transport, suggesting that intracellular arsenate inhibited phosphate transport. This effect was not seen in phosphate-replete cells.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine