The Enzymes in Ammonia Assimilation Pathways Have Been Examined in a Strictly Anaerobic Bacterium Bacteroides Fragilis

J. Gen. Appl. Microbiol., 30, 499-508 (1984) THE PATHWAY OF AMMONIA ASSIMILATION IN BACTEROIDES FRAGILIS ISAMU YAMAMOTO, ATSUKO ABE, HIROYUKI SAITO AND MAKOTO ISHIMOTO Department of Chemical Microbiology, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060 (Received January 21, 1985) The enzymes in ammonia assimilation pathways have been examined in a strictly anaerobic bacterium Bacteroides fragilis. The activities of NADPH- and NADH-linked glutamate dehydrogenase and glutamine synthetase were demonstrated in extracts of cells, but very low activity of NADPH-dependent glutamate synthase and no activity of alanine dehydrogenase were demonstrated. Both activities of the glutamate dehydrogenase were not distinguished electrophoretically, indicating the presence of a dual pyridine nucleotide-specific enzyme. At low concentrations of ammonia in batch cultures and at low dilution rates of continuous flow cultures, higher activities of glutamate dehydrogenase were found in the cells. The values of Km of NADPH-linked glutamate dehydrogenase were 0.8 mM, 0.15 mM, and 7 uM for ammonia, 2-oxoglutarate, and NADPH, respectively. Although glutamine synthetase activity was also higher in cultures with limited ammonia and at low dilution rates of continuous cultures, this enzyme may not be important for ammonia incorporation into amino acids, since cell growth was not affected by the addition to the culture of methionine sulfoximine, a glutamine synthetase inhibitor. This evidence suggests that ammonia assimilation is mainly carried out by the glutamate dehydrogenase in B. fragilis. Glutamate and glutamine are the important intermediates in biosynthesis of cell materials of organisms. Two pathways have been known to be mainly in- volved in ammonia incorporation into glutamate in many bacteria (1, 2). When ammonia is available in excess, glutamate dehydrogenase (GDH; L-glutamate: NADP+ oxidoreductase, EC 1.4.1.4) catalyzes the assimilation of ammonia, whereas at limited concentrations of ammonia a combined system of glutamine synthetase (GS; L-glutamate : ammonia lygase, ATP requiring, EC 6.3.1.2) and 1 Abbreviations used in this text are as follows: GDH , glutamate dehydrogenase; GS, glutamine synthetase; GOGAT, glutamate synthase; and MSX, L-methionine sulfoximine. 499 500 YAMAMOTO, ABE, SAITO and ISHIMOTO VOL. 30 glutamate synthase (GOGAT; L-glutamine : 2-oxoglutarate aminotransferase, EC 2.6.1.53) is active. This is generally supported by the findings that GS is re- pressed and GDH is induced when ammonia is present in excess and that the affinity of GS for ammonia i s higher than that of GDH. Bacteroides fragilis is a strictly anaerobic bacterium predominating in the human intestinal tract (3, 4) and in abscesses (S). Ammonia is required and not replaced by amino acids as the source of nitrogen for growth (6). Little is known about ammonia assimilation in this organism and the related species of Bacteroides. GLASSand HYLEMONreported properties of the purified GDH from B. thetaiotao- micron and the presence of the enzyme in other members of the Bsfragilis group (7). The affinity of the purified enzyme for ammonia is not so high (Km of 5.0 mM) that the enzyme seems to function only at high concentrations of ammonia. In a rumen bacterium B. amylophilus (8), activities of GS and NADPH-dependent GDH but not of GOGAT have been found under conditions of ammonia-limited continuous cultures. GS is inactivated by the addition of excess ammonia to this culture, while GDH is active in any conditions and shows relatively high affinity for ammonia (Km of 1 mM). We have investigated the assimilation pathways of ammonia by determining activities of the enzymes in extracts of B. fragilis grown at high and low concentrations of ammonia in batch and continuous cultures. High activities of NADPH- linked GDH were found at low concentrations of ammonia in batch and at low dilution rates in continuous cultures. Furthermore, Km value of this enzyme for ammonia was less than 1 mM. This is the first example that a higher level of GDH was observed in cells grown on limited ammonia than in cells grown at high concentrations of ammonium. The function of GDH for ammonia assimilation in B. fragilis will be discussed. MATERIALS AND METHODS Bacterial strain and cultivation. Bacteroides fragilis ATCC 23745 was used throughout the experiments. Two kinds of medium were employed for the cell growth. a) VL medium contained per liter : Trypticase peptone (BBL), 10 g; yeast extract (Difco), 5 g; beef extract (Difco), 2 g; salt solution I, 7.5 ml; salt solution II, 7.5 ml; hemin, 7 mg; Na2CO3, 2 g; L-cysteine-HCl H2O, 0.34 g; glucose, 10 g; and resazurin, 1 mg. Salt solution I was 3 % K2HP04 and salt solution II contained KH2P04, 3 g; (NH4)2504, 3 g; NaCI, 6 g; MgSO4 7H2O, 0.3 g; and CaCl2 2H2O, 0.3 g per 100 ml. Hemin was dissolved in 0.01 N NaOH and added to the medium. Before sterilization the medium was bubbled with CO, for more than 30 min and trans- ferred to test tubes which were then flashed with CO2 and sealed with butyl rubber stoppers. These medium-containing tubes were autoclaved at 115° for 15 min. b) Defined medium which was used for subcultures and experimental cultures 1984 Ammonia Assimilation in Bacteroides fragilis 501 contained the following (per liter) : K2HP04, 0.5 g; KH2P04, 0.4 g; NaCI, 0.9 g; CaC12, 20 mg; MgSO4.7H2O, 25 mg; MnC12.4H2O, 10 mg; CoC12.6H2O, 1 mg; FeSO4.7H2O, 10 mg; hemin, 2 mg; Na2CO3, 2 g; vitamin B12,20 ug; glucose, 5 g; L-cysteine-HC1• H2O, 0.53 g; and NH4CI at indicated concentrations. The medium was bubbled with CO2 before autoclaving. Glucose and cysteine were separately sterilized by autoclaving and filtration, respectively, and added to the medium. In growth studies, L-methionine sulfoximine (MSX) which was sterilized by filtration was added to cultures at the concentration of 0.5 mM. The cultures were anaerobically incubated in rubber-stoppered tubes or flasks at 37° under CO2 atmosphere which was attained by flashing CO2 for 2 to 5 min or bubbling CO2 during incubation. The defined medium containing 5 mM NH4CI was inoculated with an overnight culture in VL medium and incubated for about 15 hr. The obtained culture was used as inoculum for experimental cultures. The inoculum size was 5 % volume of media for each culture. Cells were harvested by a centrifuge after 14 or 18 hr incubation from batch cultures, washed three times with 50 mM Tris-HCI, pH 7.6, and stored at -20° until use. In continuous cultivation the culture volume was 2.81. The pH of the culture was maintained at pH 7.0 by a pH stat. Mixed gas (5 % CO2 in nitrogen) was bubbled into the culture and the medium in a reservoir. The concentration of ammonia was 1.5 mM in the medium. About 2 l of the effluent from the culture vessel was anaerobically collected in a flask in an ice-water bath and then cells were harvested and stored as above. Cell growth was followed by measuring the optical density of cultures at 650 nm in a 10 mm-cuvette with a spectrophotometer. When the optical density was over 0.5, cultures were diluted with 0.85 % NaCI to give a density below 0.5. Preparation of crude extracts. Cells suspended in 50 mM Tris-HC1 buffer, pH 7.6, were disrupted with a sonicator (Tomy model 150P) at 74 W, 20 kHz for 5 min, and centrifuged at 15,000><g for 15 min. The obtained supernatants were used as crude extracts. Assays of enzyme activities. The reductive amination activities of GDH were assayed at 30° by monitoring the absorbance of NADPH or NADH at 340 nm (~= 6.22 mM-1 cm-1) in the reaction mixture containing 100 mM Tris-HCI, pH 8.0, 40 mM NH4CI, 0.15 mM NADPH, and 5 mM 2-oxoglutarate. When NADH was used in place of NADPH, Tris-HC1, pH 7.4, was used as the buffer. The difference of oxidation rates of NADPH in the presence and absence of 2-oxoglutarate was obtained as the activity of the enzyme. The activity of GOGAT was also determined by measuring the rates of NADPH oxidation in the reaction mixture containing 0.1 M Tris-HCI, pH 8.0, 0.15 mM NADPH, 5.0 mM 2-oxoglutarate, and 4.0 mM L-glutamine which was added last after observation of NADPH oxidation in the absence of glutamine. The biosynthetic and transferase activities of GS were determined according 502 YAMAMOTO, ABE, SAITO and ISHIMOTO VOL. 30 to the method described by WooLFoLK et al. (9). For assay of the synthetic activity of GS, phosphate released from ATP was determined in the reaction mixture (0.5 ml) containing 0.1 M imidazole-HCI, pH 7.0, 50 mM NH4CI, 50 mM MgC12,0.1 M L-glutamate, and 7.6 mM ATP. After incubation at 30° for 20 min, 0.5 ml of 24 % trichloroacetic acid was added to stop the reaction. The mixture was allowed to stand for 5 min and then centrifuged to remove precipitates. The following reagents were added to 0.5 ml of the supernatant: 0.3 ml each of 15 H2S04, 0.4% amidol containing 8 % NaHSO3, and 3.3 % ammonium molybdate, and 1.6 ml of H2O (10). The absorbance was measured at 600 nm. The obtained values were corrected for nonenzymic and ATPase-catalyzed ATP hydrolysis. An aliquot of the deproteinized reaction mixture was also subjected to paper chromatography to detect glutamine. The solvent system was composed of n- propanol and 0.2 N NH4OH (3: 1) (11).

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