Enzymes of ammonia assimilation in <i>Rhizobium leguminosarum</i> bacteroids

Kurz, Wolfgang; Rokosh, D. A.; LaRue, Thomas A.

doi:10.1139/m75-149

Cited by 52 publications

(24 citation statements)

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“…Glutamate serves as the central nitrogen metabolite in the plant nodule cells for the synthesis of the other amino acids, nucleic acids, and other nitrogen-containing compounds such as ureides that are the principal nitrogen compounds transported from the nodules to the plant shoots and leaves of N 2 -fixing soybeans. Consistent with this hypothesis are the observations by several laboratories that the ammonium assimilation enzymes of bacteroids are repressed relative to the levels found in cultured cells (3)(4)(5)(6)(7).…”

supporting

confidence: 66%

Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids

Waters

Hughes

Purcell

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

119

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Symbiotic nitrogen fixation, the process whereby nitrogen-fixing bacteria enter into associations with plants, provides the major source of nitrogen for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobialeguminous plant symbioses, the current model of nitrogen transfer from the symbiotic form of the bacteria, called a bacteroid, to the plant is that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids outside of the bacteroid. We purified soybean nodule bacteroids by a procedure that removed contaminating plant proteins and found that alanine was the major nitrogencontaining compound excreted. Bacteroids incubated in the presence of 15 N 2 excreted alanine highly enriched in 15 N. The ammonium in these assays neither accumulated significantly nor was enriched in 15 N. The results demonstrate that a transport mechanism rather than diffusion functions at this critical step of nitrogen transfer from the bacteroids to the plant host. Alanine may serve only as a transport species, but this would permit physiological separation of the transport of fixed nitrogen from other nitrogen metabolic functions commonly mediated through glutamate.In 1956 (1), ammonium was shown to be the product of nitrogenase, the bacterial enzyme that reduces atmospheric dinitrogen. In free-living, nitrogen-fixing microorganisms, the nitrogenase-generated ammonium is assimilated into glutamate through the glutamine synthetase͞glutamate synthase pathway. Various transaminases utilize glutamate to generate all of the other amino acids, which are then used to synthesize proteins, nucleic acids and other nitrogen-containing molecules.In rhizobia-leguminous plant symbioses, the majority of the dinitrogen reduced by the microsymbionts, referred to as bacteroids, is transferred to the plant. Because the rhizobia are physically separated from the plant cytosol by a plant-derived symbiosome membrane, the ammonium must move across the bacteroid and symbiosome membranes. The ammonia diffusion hypothesis has been the prevalent model surmising the initial steps of leguminous nodule nitrogen transfer. In this model, the product of nitrogenase exists in two forms within the cell, ammonium [NH 4 ϩ ] and ammonia [NH 3 ], which are readily interconvertible. The ammonium ion cannot diffuse across membranes, but ammonia is believed to diffuse freely out of the bacteroid. Once outside the bacteroid but within the symbiosome space, the ammonia may either diffuse or be transported across the symbiosome membrane (2) where it is assimilated by the host plant into glutamate by the concerted action of glutamine synthetase and glutamate synthase. Glutamate serves as the central nitrogen metabolite in the plant nodule cells for the synthesis of the other amino acids, nucleic acids, and other nitrogen-containing compounds such as ureides that are the principal nitrogen compounds transported from the nodules to the plant shoots and leaves o...

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supporting

confidence: 66%

Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids

Waters

Hughes

Purcell

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

119

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“…The same specific activity of this enzyme (Fig. 7 B) during stages with high and low nitrogenase activities further support the idea (also found with Rhizobium leguminosarum bacteroids [26]) that this enzyme in bac teroids does not function similarly as in free living diazotrophic cells. Even a negative correlation be tween both enzymes, a falling glutamine synthetase with an increasing nitrogenase, was found in bac teroids of Rhizobium leguminosarum (Planque and Kijne, personal communication) during nodule de velopment.…”

Section: Discussionsupporting

confidence: 84%

“…8). In bacteroids from other slow growing rhizobia this was also found [11,25,27] but there was no activity in bacteroids of Rhizobium leguminosarum [26,27], A significant increase in the activity of this en zyme in the cytoplasm of lupin nodules was seen [28] after separation of low molecular weight com pounds such as glycine and glutamate by filtration through a Sephadex G 25 column [28]. We obtained no increase in the cytoplasm enzyme activity of our soybean varieties after the same procedure.…”

Section: Discussionsupporting

confidence: 68%

Differentiation of Rhizobium japonicum. II. Enzymatic Activities in Bacteroids and Plant Cytoplasm during the Development of Nodules of Glycine max

Stripf

Werner

1978

Zeitschrift Für Naturforschung C

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Phytotron grown plants of Glycine max var. Caloria infected with Rhizobium japonicum 61-A-101 under controlled conditions as 14 d old seedlings develop a sharp maximum of nitrogenase activity of 13 ± 3 nmol C2H4 · h-1 · mg nodule fresh weight-1 19 d after infection, followed by a long period of reduced activity (3 -5 nmol) between 30 and 45 days after infection. A higher maximum activity (18 nmol C2H4 · h-1 · mg nodule-1, lasting 7 days was found in Glycine max var. Mandarin, with a similar peroid of low activity (3 - 4 nmol) following, between 35 and 50 d after infection. Nitrogenase activity in the varieties infected with Rhizobium japonicum strain 3I1 b 85 is very similar. In both varieties, the leghaemoglobin continues to increase (3 fold) after the maximum nitrogenase activity is reached and starts to decline only after 35 to 40 d. Specific activities of the three enzymes aspartate aminotransferase (E.C.2.6.1.1.), glutamate dehydrogenase (E.C.1.4.1.2.) and alanine aminotransferase (E.C.2.6.1.2.) in the plant cell cytoplasm are changing very similarly to nitrogenase activity in the bacteroids, whereas the specific activities of the three enzymes in the bacteroids decrease only very slightly between 19 and 45 d. For these 3 enzymes the specific activity in the bacteroids during the phase of maximum nitrogenase activity is only 20 - 40% of the specific activity in the cytoplasm. A constant low activity (0.350 units) of glutamine synthetase (E.C.6.3.1.2.) is found in bacteroids from 19 to 45 d old nodules, whereas the specific activity in the plant cytoplasm increases from about 1.2 units at 19 d to more than 6 units at 45 d. The specific activity of GOGAT (E.C.1.4.13.) in bacteroids is 2 - 3 times higher than in the plant cell cytoplasm and increases slightly. Alanine dehydrogenase (E.C.1.4.1.1.) and 3-hydroxybutyrate dehydrogenase (E.C.1.1.1.30.) activities in bacteroids increase between 19 and 35 d after infection by a factor of 2 - 3. In 35 - 45 d old nodules, the specific activity of aianine-dh in bacteroids of the same Rhizobium japonicum strain (61-A-101) from plant var. Caloria is significantly smaller than in bacteroids from plant var. Mandarin, whereas for 3-hydroxybutyrate-dh the activity in bacteroids from var. Caloria is enhanced compared to bacteroids from var. Mandarin.

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“…A. CASTILLO and OTHERS Although it has been accepted that Rhizobium bacteroids do not assimilate ammonium during symbiosis (Kurz et al, 1975), there is abundant evidence indicating that bacteroids synthesize different nitrogencontaining metabolites during symbiosis (Salminen & Streeter, 1987 ;Streeter, 1987 ;Kouchi et al, 1991 ;Rastogi & Watson, 1991). Furthermore, significant GS and GOGAT activities have been detected in bacteroids isolated from nodules formed on different legume species, including bean plants inoculated with Rhizobium leguminosarum bv.…”

Section: Introductionmentioning

confidence: 99%

Role of GOGAT in carbon and nitrogen partitioning in Rhizobium etli The GenBank accession number for the sequence reported in this paper is AF107264.

et al. 2000

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The isolation and characterization of a Rhizobium etli glutamate auxotroph, TAD12, harbouring a single Tn5 insertion, is reported. This mutant produced no detectable glutamate synthase (GOGAT) activity. The cloning and physical characterization of a 72 kb fragment of R. etli DNA harbouring the structural genes gltB and gltD encoding the two GOGAT subunits GltB and GltD is also reported. In comparison with the wild-type strain (CFN42), the GOGAT mutant strain utilized less succinate and glutamate and grew less with this and other amino acids as nitrogen source. R. etli assimilates ammonium by the glutamine synthetase (GS)-GOGAT pathway and a GOGAT mutant prevents the cycling of glutamine by this pathway, something that impairs nitrogen and carbon metabolism and explains the decrease in the amino-nitrogen during exponential growth, with glutamate as nitrogen source. GOGAT activity also has a role in ammonium turnover and in the synthesis of amino acids and proteins, processes that are necessary to sustain cell viability in non-growing conditions. The assimilation of ammonium is important during symbiosis and glutamate constitutes 20-40 % of the total amino-nitrogen. In symbiosis, the blockage of ammonium assimilation by a GOGAT mutation significantly decreases the amino-nitrogen pool of the bacteroids and may explain why more N 2 is fixed in ammonium, excreted to the plant cell, transported to the leaves and stored in the seeds.

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Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroids

Cited by 52 publications

References 8 publications

Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids

Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids

Differentiation of Rhizobium japonicum. II. Enzymatic Activities in Bacteroids and Plant Cytoplasm during the Development of Nodules of Glycine max

Role of GOGAT in carbon and nitrogen partitioning in Rhizobium etli The GenBank accession number for the sequence reported in this paper is AF107264.

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Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroids

Cited by 52 publications

References 8 publications

Alanine, not ammonia, is excreted from N 2 -fixing soybean nodule bacteroids

Alanine, not ammonia, is excreted from N 2 -fixing soybean nodule bacteroids

Differentiation of Rhizobium japonicum. II. Enzymatic Activities in Bacteroids and Plant Cytoplasm during the Development of Nodules of Glycine max

Role of GOGAT in carbon and nitrogen partitioning in Rhizobium etli The GenBank accession number for the sequence reported in this paper is AF107264.

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Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids

Alanine, not ammonia, is excreted from N ₂ -fixing soybean nodule bacteroids