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Keywords:

  • Glutamine synthetase;
  • Nitrogen fixation;
  • Ammonium assimilation;
  • Acetobacter diazotrophicus

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Glutamine synthetase from Acetobacter diazotrophicus, an endophyte originally isolated from sugarcane, was studied as a step in the identification of mechanisms underlying the role of A. diazotrophicus as a major supplier of fixed nitrogen to its host plant. The enzyme was purified and partially characterized. It was also shown that the enzyme is regulated by adenylylation in response to the nitrogen source. Interestingly, there is no upregulation of the synthesis of the enzyme under diazotrophic conditions, which is in contrast to the situation in enterics, e.g. Klebsiella pneumoniae.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Acetobacter diazotrophicus is one of a number of diazotrophs isolated from sugarcane [1,2] and it has also been isolated from other economically important plants [3]. A. diazotrophicus was earlier shown to play a major role as supplier of fixed nitrogen to sugarcane [4]. This report was given strong support by the recent demonstration that sugarcane plantlets inoculated with A. diazotrophicus showed significant growth stimulation compared to those inoculated with a nif strain of A. diazotrophicus or uninoculated plants [3].

Investigations of the physiology of A. diazotrophicus have shown that this diazotroph exhibits some unique characteristics such as growth and nitrogenase activity at low pH, no effect of nitrate on nitrogen fixation, growth in the presence of high sucrose concentrations, and nitrogen fixation under aerobic conditions [5–7]. As in most other diazotrophs assimilation of ammonium ions is believed to occur mainly through the glutamine synthetase–glutamate synthase pathway under nitrogen fixing conditions [5,7]. Genetic studies have established the presence of a number of nif genes including the structural genes nifHDK and the regulatory gene nifA[8]. Furthermore, genes encoding proteins involved in the regulation of nitrogen metabolism in general and ammonium assimilation in particular, e.g. glnB, glnD and ntrBC, have also been demonstrated [9,10]. It is thus most likely that the systems controlling these processes in other diazotrophs are also operating in A. diazotrophicus, although the details have not been elucidated. In the light of the proposed role of A. diazotrophicus as a major supplier of fixed nitrogen to sugarcane and possibly other plants, it is of great importance to clarify the mechanisms by which the fixed nitrogen is exported to the plant and how these processes are regulated. As glutamine synthetase has been shown to play a central role in nitrogen metabolism in most diazotrophs, it is an obvious target for studies aiming at understanding the processes leading to export of fixed nitrogen. We have studied glutamine synthetase in A. diazotrophicus and report here on its characteristics and its activities from cells grown with high concentrations of ammonium ions or diazotrophically.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Cells and growth conditions

A. diazotrophicus PAL 5 (provided by Dr. Döbereiner) was grown in a medium containing (g l−1): citric acid (4.4), K2HPO4 (10), CaCl2 (0.02), NaCl (0.15), MgSO4 (0.4) and sucrose (20). FeCl3 (80 μM) and Na2MoO4 (30 μM) were also included, and pH was adjusted to 5.5. N+ medium contained 30 mM NH4Cl as nitrogen source. For diazotrophic growth (N) a 1% inoculum from an N+ culture was transferred to medium containing 1 mM NH4Cl as a starter as described in [11]. Cells were in general grown to OD600 around 0.8 and either used directly for in vivo experiments or harvested.

2.2Purification of glutamine synthetase

Cell extracts were produced by passing cells suspended in buffer (100 mM Tris pH 7.1, 50 mM NaCl, 2 mM MnCl2 and 2 mM sodium dithionite) through a Ribi cell fractionator. Extracts were centrifuged at 10 000×g for 15 min, and the supernatant further centrifuged at 100 000×g for 60 min. The supernatant from the latter centrifugation was used for purification of glutamine synthetase, according to the protocol for glutamine synthetase from Rhodospirillum rubrum[12], with some modifications. The supernatant was loaded onto a DEAE-Sepharose column (10×3 cm), and after washing with buffer, glutamine synthetase was eluted with buffer containing 0.15 M NaCl. Fractions containing glutamine synthetase activity were pooled and loaded onto a Blue Sepharose column (2×0.5 cm). After washing with buffer containing 0.15 M NaCl, glutamine synthetase was eluted with buffer containing 0.15 M NaCl and 5 mM ADP. The fractions containing the highest activity were pooled and used in the experiments.

2.3Enzyme activity determinations

Nitrogenase activity was determined as acetylene reduction in air [11] and glutamine synthetase activity either as the γ-glutamyl transferase activity or in a biosynthetic assay [12]. Using the Escherichia coli procedure to determine if the enzyme was adenylylated, the transferase assay was run in the absence and presence of 60 mM Mg2+. The adenylylated glutamine synthetase subunit is inhibited by this addition, whereas the unadenylylated is not [13].

To further study the adenylylation, purified glutamine synthetase (0.7 μg), from N or N+ cells, was treated with snake venom phosphodiesterase (10 μg) according to the manufacturer's instructions (Boehringer Mannheim). This enzyme catalyzes the removal of AMP from glutamine synthetase subunits by hydrolysis as originally shown for the E. coli enzyme [14]. Samples were taken at 0 and 60 min for activity measurements and Western blot analysis.

2.4SDS–PAGE, Western blot, and protein determination

SDS–polyacrylamide electrophoresis was run according to Laemmli [15] and electrophoretic blotting was done as described by Towbin [16]. The antibodies used for immunological detection were raised against dinitrogenase reductase and glutamine synthetase respectively, both from R. rubrum[17]. Protein concentrations were determined by the Bradford method [18] with bovine serum albumin as standard.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

In order to demonstrate possible differences in glutamine synthetase from A. diazotrophicus grown with or without fixed nitrogen in the growth medium, the enzyme was purified to near homogeneity from cells grown diazotrophically or with 30 mM NH4Cl. Results from a typical purification are shown in Table 1. The specific activity of the enzyme purified from cells grown diazotrophically was lower in the transferase assay than that from cells grown with ammonium ions, but was not affected by the presence of Mg2+. This indicates that the enzyme from N cells was not adenylylated. However, the activity of glutamine synthetase from N+ grown cells decreased to about 50% when 60 mM Mg2+ was included in the assay, suggesting that glutamine synthetase in A. diazotrophicus is regulated by adenylylation as in enterics, e.g. E. coli[13]. The molecular mass of the glutamine synthetase subunit was determined to be 50 kDa by SDS–PAGE and the total molecular mass of the enzyme was estimated to be 500 kDa by gel filtration (data not shown). The pH optimum in the transferase assay for both forms purified was determined to be 7.6 (data not shown).

Table 1.  Glutamine synthetase specific activity (nmole γ-glutamyl hydroxamate min−1 mg−1) in extracts and of the purified enzyme from A. diazotrophicus grown with 30 mM NH4Cl or diazotrophically
 N+ cellsN cells
 −60 mM Mg2++60 mM Mg2+−60 mM Mg2++60 mM Mg2+
  1. Assays were run in duplicate and results are shown with standard deviation.

Cell free extract16±0.59±0.49±0.410±0.9
Purified245±7107±7195±8211±8

The results shown in Table 1 are contradictory to those reported by Alvarez and Drets [5] who found an increase in glutamine synthetase activity under diazotrophic conditions, an effect also observed in other diazotrophs, e.g. Klebsiella pneumoniae[19] and R. rubrum[20]. In order to verify our activity determinations, the amount of glutamine synthetase protein in the extracts was investigated using Western blot analysis with antibodies against glutamine synthetase from R. rubrum. The results are shown in Fig. 1. The relative amount of glutamine synthetase in the N+ extract was about 70% higher than in the N extract as estimated by laser densitometry scanning. To confirm the N-status of the cells used, e.g. that the N+ cells were not derepressed with respect to nitrogenase, i.e. were not diazotrophic, Western blot analysis with antibodies against dinitrogenase reductase from R. rubrum was performed. Lanes 3 and 4 in Fig. 1 show that in the N cells dinitrogenase reductase was synthesized but absent in the N+ cells, in agreement with measurements of nitrogenase activities, confirming that the N cells were in fact derepressed and the N+ cells were not. Further support for the suggestion that the level of glutamine synthetase is not lower under non-diazotrophic conditions is provided in the experiment shown in Fig. 2. NH4Cl (30 mM) and/or the protein synthesis inhibitor tetracycline were added to a diazotrophically growing culture of A. diazotrophicus and samples were taken for Western blot analysis after 1 and 6.5 h. As shown there was no decrease in the amount of glutamine synthetase after addition of ammonium ions and/or tetracycline. If the synthesis of the enzyme was upregulated under nitrogen fixing conditions and if there was an effect on the turnover rate of glutamine synthetase, the amount of enzyme would decrease when changing the conditions from diazotrophic to non-diazotrophic. In fact comparison of lanes 6 (+NH4Cl) and 8 (+NH4Cl and tetracycline) in Fig. 2 indicates that the concentration of glutamine synthetase increased slightly when the protein synthesis inhibitor was not included during 6.5 h after the change from diazotrophic to non-diazotrophic conditions.

image

Figure 1. Western blot extracts from cells grown diazotrophically or in the presence of 30 mM NH4Cl. Lanes 1 and 3 are extracts from N cells; lanes 2 and 4 from N+ cells. Lanes 1 and 2 were probed with antibodies against glutamine synthetase and lanes 3 and 4 with antibodies against dinitrogenase reductase.

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image

Figure 2. Effect on glutamine synthetase of addition of NH+4 to diazotrophically growing cells. NH+4, alone or together with tetracycline, was added to diazotrophically growing cultures and samples were taken for SDS–PAGE/Western blot with antibodies against glutamine synthetase, after 1 and 6.5 h. The same amount of protein was added to each lane. Lanes: 1, 5 and 9, no additions at 0, 1 and 6.5 h respectively; 2 and 6, 30 mM NH4Cl; 3 and 7, 30 mM NH4Cl+tetracycline (10 μg ml−1); 4 and 8, tetracycline (10 μg ml−1). Lanes 2–5 after 1 h and lanes 6–9 after 6.5 h.

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The effect of Mg2+ on the transferase activity of the enzyme from N+ cells indicated that regulation of glutamine synthetase is exerted by adenylylation, as in enterics. As shown in Fig. 3A, the enzyme purified from N cells migrated as one band on SDS–PAGE, whereas two bands are clearly seen with the enzyme from N+ cells. Furthermore, when the N+ enzyme was incubated with snake venom phosphodiesterase, which catalyzes hydrolysis of AMP from adenylylated glutamine synthetase subunits from other bacteria, the intensity of the upper band decreased with a corresponding increase in the lower (Fig. 3B). This change was also accompanied by a 2-fold increase in the transferase activity in the presence of Mg2+, as would be expected if the enzyme became deadenylylated. Incubation of glutamine synthetase from diazotrophic cells with snake venom phosphodiesterase had no effect on the transferase activity, with or without added Mg2+ (data not shown). Taken together, the results presented provide clear evidence for the operation of a control mechanism involving adenylylation of glutamine synthetase in A. diazotrophicus.

image

Figure 3. SDS–PAGE of glutamine synthetase from cells grown diazotrophically or in the presence of NH+4, and the effect of treatment with snake venom phosphodiesterase. A: Purified enzyme was subjected to SDS–PAGE and stained with Coomassie blue. Lane 1, enzyme from N cells and lane 2, enzyme from N+ cells. The arrows indicate the two bands obtained. B: Western blot and activity measurements of glutamine synthetase from N+ cells before and after treatment with snake venom phosphodiesterase. Lane 1 before treatment and lane 2 after 60 min treatment. The activity values are relative activity in the presence of 60 mM Mg2+. The arrows indicate the two bands obtained.

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In E. coli, glutamine synthetase is also regulated by feedback effectors representing the end products of biosynthetic pathways of nitrogenous compounds [13]. The effect of three of these effectors on the two forms of purified A. diazotrophicus glutamine synthetase was investigated. As shown in Table 2, in the transferase assay, glycine and CTP caused clear inhibition, whereas AMP had no effect, similar to the situation in R. rubrum[21]. In the biosynthetic assay, only CTP was inhibitory, but the two forms of glutamine synthetase were inhibited to the same degree. It is thus not likely that feedback regulation of glutamine synthetase plays a significant role in A. diazotrophicus.

Table 2.  Effect of glycine, AMP and CTP on glutamine synthetase activity
   Inhibitor:Gly (5 mM)AMP (5 mM)CTP (5 mM)
   Cells:N+NN+NN+N
  1. Enzyme purified from cells grown either in the presence of 30 mM NH4Cl or diazotrophically, was used. All assays were run in duplicate and the values shown are activity in % of the controls, which were for each condition the same assay without the inhibitor. Standard deviation was less than ±10% for all values.

Transferase assay7056891262457
Biosynthetic assay10113894843636

In conclusion we have shown that glutamine synthetase in A. diazotrophicus is regulated by covalent modification, but in contrast to other diazotrophs there is no upregulation of its synthesis under nitrogen fixing conditions. A detailed understanding of the transcriptional regulation of glutamine synthetase will have to include genes encoding regulatory proteins, e.g. ntrBC and glnB. This will be a prerequisite to establish the role of glutamine synthetase in the processes leading to export of fixed nitrogen from A. diazotrophicus to the host plant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This investigation was supported by grants to S.N. from Carl Tryggers Foundation and the Swedish Council for Forestry and Agricultural Research.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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