Ammonia Assimilation by Rhizobium Cultures and Bacteroids

Abstract

S U M M A R YThe enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.3. I . 2), glutamate synthase (Lglutamine : a-oxoglutarate amino transferase) and glutamate dehydrogenase (EC I . 4 . I .4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slowgrowing R. japonicum and R. lupini.Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M . sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system. I N T R O D U C T I O NThe assimilation of ammonia by bacteria proceeds via either glutamate dehydrogenase or the glutamine synthetase/glutamate synthase system, depending upon the organism and the ammonia concentration of the environment (Tempest, Meers & Brown, 1973; Brown, Macdonald-Brown & Meers, 1974). Dainty (1972) reported the presence of glutamate synthase in a strain of Clostridium pasteurianum which lacked glutamate dehydrogenase, while Nagatani, Shimizu & Valentine (1971) demonstrated the presence of glutamate synthase in a range of nitrogen-fixing bacteria and in bacteroids of Rhizobium japonicum prepared from Glycine max (soya bean) root nodules. Nagatani et al. (1971) claimed that the glutamine synthetase/glutamate synthase system was operative in ammonia assimilation

Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max

“…Rhizobium japonicum strain OSR-2 was cultured in 1-liter flasks containing 500 ml of medium containing the following components dissolved in 1 liter of water: K2HPO4, 0.23 g; MgSO4-7H2O, 0.10 g; sodium glutamate, 1.10 g; glycerol, 4 g; CaC12, 5 mg; H3B03, 145 ,ug; FeSO4-7H20, 125 Mg; CoSO4 7H2O, 70 Mg; CuSO4 7H2O, 5 Mug; MnCl2-4H20, 4 Preparation of Bacteroids. Soybean root nodules (7-9 g) from plant cultured by methods previously described (10) were mixed with 24 ml of 0.1 M K-phosphate (pH 7.6) buffer containing 0.2 M sucrose (sucrose-phosphate buffer) and 3 g of insoluble PVP and were macerated with a mortar and pestle at 4 C. The macerate was filtered through four layers of cheesecloth and then centrifuged at 40,000g for 15 min.…”

The Use of Activities of Carbon Catabolic Enzymes as a Probe for the Carbon Nutrition of Snakebean Nodule Bacteroids

“…Ucker & Signer (1978) Cells of NGR234 were grown in fumarate (10mM) -minimal salts medium to midexponential phase (OD&T, approx. 0.6), centrifuged, washed twice with salts (Brown & Dilworth, 1975) and resuspended in fumarate (10 mM) or sucrose (10 mM) or fumarate plus sucrose (both at 10 mM). Samples (50 ml) were taken at 2 h intervals for the preparation of cellfree extracts and the activities of glucose-6-phosphate dehydrogenase and the EntnerDoudoroff enzymes were determined (Fig.…”

Properties of Organic Acid Utilization Mutants of Rhizobium leguminosarum Strain 300

“…Batch cultures of bacteria were grown at 28 "C in the liquid minimal salts medium of Brown & Dilworth (1975) with carbon sources at 10 mM except for glycerol which was used at 20 mM. The nitrogen source was NHJ (10 mM), with phosphate at 0.3 mM and pH maintained at 7.2 with 40 mM-HEPES buffer.…”