Mutations in Bacillus subtilis glutamine synthetase that block its interaction with transcription factor TnrA

In Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein- protein interaction with glutamine synthetase. During growth with excess nitrogen, the feedback-inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA. Missense mutations in glutamine synthetase that constitutively express the TnrA-regulated amtB gene were characterized. Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA-binding activity of TnrA. Two of the mutant proteins exhibited enzymatic properties similar to those of wild-type glutamine synthetase. A model of B. subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme. Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site. These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity.     

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica

The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO₃⁻-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH₄⁺ have high NADPH-GDH activity, the levels of which increase with increasing NH₄⁺ supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH₄⁺-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH₄⁺.     

Metabolic regulation of ammonia emission in different senescence phenotypes of Nicotiana tabacum

In order to reveal the character of ammonia emission in senescent tobacco (Nicotiana tabacum), the content of NH₄⁺, total nitrogen, and soluble protein, and the activities of nitrogen metabolism-related enzymes were measured in leaves of a quick-leaf-senescence phenotype ZY90 and a slow-leaf-senescence phenotype NC89. Compared with NC89, ZY90 had a higher NH₄⁺ accumulation, a lower glutamine synthetase activity, and a significantly higher stomatal ammonia compensation point, and ammonia emission during 40 to 60 d after leaf emergence. During senescence, the quick-leafsenescence phenotype was characterized by nitrogen re-transfer by ammonia emmission, whereas the slow-leafsenescence phenotype by nitrogen re-assimilation. The ammonia emission was primarily regulated by glutamine synthetase activity, apoplastic pH, and NH₄⁺ content.     

Organ-specific changes in the activity and subunit composition of glutamine-synthetase isoforms of barley (Hordeum vulgare L.) after growth on different levels of NH+ 4

One cytosolic glutamine synthetase (GS, EC 6.3.1.2) isoform (GS 1a) was active in the germinating seeds of barley (Hordeum vulgare L.). A second cytosolic GS isoform (GS 1b) was separated from the leaves as well as the roots of 10-d-old seedlings. The chloroplastic isoform (GS 2) was present and active only in the leaves. The three GS isoforms were active in N-supplied (NH⁺ ₄ or NO ₃ ^– ) as well as in N-free-grown seedlings. This indicates (i) that a supply of nitrogen to the germinating seeds was not necessary for the induction of the GS isoforms and (ii) that no nitrogen-specific isoforms appeared during growth of seedlings with different nitrogen sources. The activity of GS, however, depended on the seedlings' nitrogen source: the specific activity was much higher in the leaves and much lower in the roots of NH⁺ ₄-grown barley than in the respective organs of NO ₃ ^– -fed or N free-grown plants. With increasing concentrations of NH⁺ ₄ (supplied hydroponically during growth), the specific activity of GS 1b increased in the leaves, but decreased in the roots. The activity of GS 2 (leaf) also increased with increasing NH⁺ ₄ supply, whereas GS 1a activity (leaf and root) was not affected. The changes in the activities of GS 1b and GS 2 were correlated with changes in the subunit compositions of the active holoenzymes: growth at increased levels of external NH⁺ ₄ resulted in an increased abundance of one of the four GS subunits, and of two of the five GS 1b subunits in the leaves. In the roots, however, the abundance of these two GS 1b subunits was decreased under the same growth conditions, indicating an organ-specific difference either in the expression of the genes coding for the respective GS 1b subunits or in the assembly of the GS 1b holoenzymes. Furthermore, growth at different levels of NH⁺ ₄ resulted in changes in the substrate affinities of the isoforms GS 1b (root and leaf) and GS 2 (leaf), presumably due to the changes in the subunit compositions of the active holoenzymes.Abbreviations FPLC fast protein liquid chromatography - GHA -glutamyl hydroxamate - GS glutamine synthetase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.     

Glutamine synthetase of Chlamydomonas: Rapid reversible deactivation

A 70% reduction in glutamine synthetase (GS) activity was observed within 5 min when 5 mM NH₃ and darkness was applied to steady-state cells of Chlamydomonas utilising NO₃. The enzyme was reactivated in vivo by reillumination of the culture and in vitro by treatment with thiol reagents. The activity modulations affected the synthetase and transferase activities similarly and were not influenced by protein synthesis inhibitors. Deactivation of GS was also observed when steady-state cells were treated with an uncoupler of phosphorylation, carbonylcyanide m-chlorophenylhydrazone (CCCP) or inhibitors of the electron transport chain but under these conditions the activity modulation affected over 90% of the activity and was irreversible. The mechanism of the physiological deactivation of GS is discussed in relation to both the in vivo and in vitro findings.Abbreviations GS glutamine synthetase (EC 6.3.1.2.) - GSs glutamine synthetase, synthetase activity - GSt glutamine synthetase, transferase activity - CAP chloramphenicol - CCCP carbonylcyanide m-chlorophenyl hydrazone - CHX cycloheximide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DSPD disalicylidene propanediamine - DTT dithiothreitol - GSH reduced glutathione     

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.