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 involved 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 repressed 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. thetaiotaomicron 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>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). One unit (U) of the enzyme activities was defined as the amount of enzymes that catalysed the oxidation of 1 µmol of NAD (P) H or the release of 1,imol of phosphate per min in each enzyme reaction. Protein was assayed by the method of LowRY et al. (12) with bovine serum albumin as a standard. Zymography on acrylamide gel. The activities of GDH were visualized on acrylamide gels (7.5%) after disc electrophoresis. Electrophoresis of crude extracts was performed according to the method of DAVIS(13). The gels were soaked in the staining solution containing 1 mM NADP+ or NAD+, 10 mM L-glutamate, 1 mM N-methylphenazinium methylsulfate, 0.2 % triphenyltetrazolium chloride, and 50 mM Tris-HCI. The pHs were 8.6 and 8.0 for NADP- and NAD-linked activities, respectively. Stained gels were washed with water and kept in a mixture of acetic acid (7 %) and methanol (25 %) in distilled water. Chemicals. Pyridine nucleotide coenzymes, L-methionine sulfoximine, and N-methylphenazinium methylsulfate were purchased from Sigma Chemical Co. ATP was a product of Boehringer-Mannheim. All other chemicals for assay of enzyme activities were reagent grade.

RESULTS Enzyme activities for ammonia assimilation Activities of GDH, GS, and GOGAT presumed to be involved in the process of ammonia assimilation were determined in the extracts from cells of B. fragilis grown in batch cultures with different concentrations of ammonia and in continuous cultures with limited ammonia. As shown in Table 1, activities of GDH, GS, and GOGAT were observed in the extracts of B. fragilis harvested at an early stationary phase of the growth. GS activity was found by glutamate-dependent ATP hydrolysis but not by the 7-glutamyltransferase method (9) in the presence of Mn2+ or Mgt+. After the biosynthetic reaction, glutamine was detected in the 1984 Ammonia Assi milation in Bacteroides fragilis 503

Table 1. Enzyme activities in extracts of cells grown in media containing different concentrations of ammonia and glutamate.

reaction mixture, indicating that GS was really present in this organism. Activities of these enzymes were not much decreased by exposing extracts to air for about 7 hr. This suggests that these enzymes are not especially sensitive to oxygen. The activity of GS was decreased by freezing. Alanine dehydrogenase activities were not detected by either amination or deamination assays at pH 9.0. In the batch cultures, the highest activity of NADPH-linked GDH was observed in the cells grown on 0.5 mM ammonia. In the 5 mM and 50 mM ammonia cultures, the specific activities of the enzyme were 50 and 15 % of that in the 0.5 mM ammonia cultures, respectively. GDH activities have been found to be low in many microorganisms when grown on limited ammonia. The regulation mech- anism of GDH in B. fragilis appears to be quite different from that in other organisms. In the continuous flow culture (1.5 mM ammonia in the medium), higher activities of NADPH-GDH were found at low dilution rates of the culture (Fig. 1). These findings suggest that the GDH may serve for assimilation of ammonia at low concentrations. The NADH-linked activity of GDH was also the highest in the 0.5 mM ammonia cultures as shown in Table 1. The activities of both NADPH-GDH and NADH-GDH were not altered significantly by addition of 20 mM L-glutamate to the growth media. The ratios of the activities (NADH/NADPH) were about 0.2 in the 0.5 mM and 5 mM ammonia cultures, but 1.5-fold higher in the 50 mM ammonia cultures. The crude extracts from cells grown at different concentrations of ammonia were electrophoresed on acrylamide gels and then GDHs were visualized on gels by activity staining. As shown in Fig. 2, values of the relative mobility (Rm) of NADP- and NAD-linked enzyme activities were almost the same (Rm of 0.17) in all extracts, indicating that an identical enzyme catalyzed both 504 YAMAMOTO, ABE, SAITO and ISHIMOTO VOL. 30

Fig. 1. Enzyme activities in extracts from cells grown in the ammonia-limited continuous culture. • : The activities of NADPH-linked glutamate dehydrogenase, • : glutamine synthetase, A : glutamate synthase.

Fig. 2. Activity staining of glutamate dehydrogenase on acrylamide gels. The extracts prepared from cells grown on 1 mM (98 pg protein, 1 and 2) or 50 mM ammonia (84 pg protein, 3 and 4) were subjected to electrophoresis. NADP- (1 and 3) and NAD-linked activities (2 and 4) were stained under the conditions described in MATERIALS AND METHODS.

NADP- and NAD-linked reactions in Bsfragilis as found in B. thetaiotaomicron (7), Selenomonas ruminantium (14), and Mycoplasma laidlawii (15). In addition, the second activity band which was specific for NAD with Rm of 0.25 was found in the extracts from the 50 mM ammonia cells. The higher ratio of NADH- and NADPH- linked activities found in these extracts may be due to the presence of NAD- specific GDH in addition to the dual pyridine nucleotide-specific GDH. The activity of GS was higher in the 0.5 mM than in the 50 mM ammonia cul- 1984 Ammonia Assimilation in Bacteroides fragilis 505 tures (Table 1), and also at low dilution rates rather than at high ones in the continuous culture (Fig. 1), as shown in many microorganisms. The specific activity of GS was not lowered immediately after the addition of high concentration of ammonia (50 mM) to the ammonia-limited culture (data not shown), suggesting that the GS of B, fragilis was not regulated by the modification of the enzyme protein like that which occurred in Enterobacteriaceae (2). GOGAT activity was also present but very low, only 0.1 % of that of NADPH- linked GDH in the extracts of B. fragilis (Table 1). The GOGAT might be as- sociated with GS to produce glutamate from 2-oxoglutarate and ammonia, but seems to be too weak to play a main role in ammonia assimilation in this organism.

Kinetic properties of glutamate dehydrogenase As the higher activity of GDH was found in cells grown under ammonia- limited conditions, some kinetic properties of the GDH were investigated with the extracts of cells grown on 1 mM ammonia. In NADPH-linked reactions, the optimal pH range was pH 7.8 to 8.4 and the values of Km were 0.8 mM, 0.15 mM, and 7 µM for ammonia, 2-oxoglutarate, and NADPH, respectively. In NADH- linked reactions, the pH optimum was 7.4 and the values of Km were 3.6 mM, 3.1 mM, and 0.4 mM for ammonia, 2-oxoglutarate, and NADH, respectively. 2- Oxoglutarate gave substrate inhibition at concentrations higher than 1 mM in the NADPH-linked reactions, but this effect was not observed in the reactions with NADH. The relatively low values of Km for ammonia and 2-oxoglutarate in the reactions with NADPH suggest that the GDH is able to function in ammonia assimilation even under ammonia-limited conditions.

Growth studies The growth of B. fragilis was examined in the defined medium with various amounts of ammonia. The growth levels and rates were dependent on the concentrations of ammonia in the media but not exactly proportional to the used concentrations (Fig. 3). The growth rates were 0.13-0.15 hr-1, 0.20-0.22 hr- i, and 0.26-0.32 hr-1 in the 1.0 mM, 5.0 mM, and 50 mM ammonia cultures, respectively. L-Glutamate (20 mM) exhibited neither effect on the cell growth nor on activities of the enzymes (Table 1). No growth of cells was observed in the medium containing glutamate instead of NH4CI as a sole source of nitrogen. The growth rates and levels were also not affected by the addition of 0.5 mM MSX, an irreversible inhibitor of GS (16), to the 1 mM ammonia cultures (Fig. 3). However, as shown in Table 2, the activity of GS was lowered in the presence of MSX to 1500 of that in cells grown in its absence. The activities of GDH and GOGAT showed only small changes. These results suggest that the GS is not the major enzyme for ammonia assimilation and the GDH which has relatively high affinity for ammonia may serve for glutamate production as an amino-N supply even under limited ammonia conditions in B. fragilis. 506 YAMAMOTO, ABE, SAITo and ISHIMOTo VoL. 30

Fig. 3. The growth of B. fragilis in the defined medium. Concentrations of ammonia in the medium were 50 mM (o), 5 mM (ii), and 1 mM The arrow indicates the time when 0.5 mM methionine sulfoximine was added to the 1 mM ammonia culture (.).

DISCUSSION Two findings have confrmed that GDH of B. fragilis functions primarily in ammonia assimilation even under ammonia-limited conditions. One of them is the observation that the higher activity of the enzyme was present in the cells grown at low concentrations of ammonia. Such a phenomenon had not been observed through the studies of ammonia assimilation in any other organisms. The other finding is that GDH of this organism showed a relatively high affinity for ammonia (Km of 0.8 mM). Though the participation of the combined pathway of GS/GOGAT in ammonia assimilation was not completely excluded, this pathway was presumably not important in B. fragilis because the GOGAT activity was very low and the addition of MSX did not cause any changes in cell growth, irrespective of depressing GS (Fig. 3 and Table 2). In some bacteria which contain the GS/ GOGAT system as the major route of ammonia incorporation, inhibitory effects of MSX have been reported on cell growth as well as on the activity of GS (17, 18). Since the discovery of GOGAT in many bacteria (19, 20), algae (21), and plants (22), the significant role of GS/GOGAT coupling system has been emphasized in ammonia assimilation in those organisms. One of the points of argument is the affinity of enzymes for ammonia. Considering the relatively low value of Km for ammonia, GDH may also be involved in the assimilation of ammonia at low concentrations in some microorganisms even though this may not be a major route. These organisms are Clostridium SB4 (Km for ammonia=0.32 mM, NAD-specific GDH (23)), Thiobacillus novel/us (0.5 mM, NAD-specific (24)), B. amylophilus 1984 Ammonia Assimilation in Bacteroides fragilis 507

Table 2. Effect of methionine sulfoximine on activities of the enzymes in cells grown on limited ammonia.

(1.0 mM, NADP-specific (8)), Escherichia coli (1.1 mM, NADP-specific (25)), Neurospora crassa (0.5 mM, NADP-specific (26)), and Chlorella pyrenoides (0.6 mM, NADP-specific (26)). B, fragilis also contained the dual pyridine nucleotide- specific GDH, the Km value of which was 0.8 mM for ammonia in the reaction with NADPH, Another GDH which was presumably specific for NAD appeared when B. fragilis was grown on 50 mM ammonia. A few electrophoretically distinct forms of GDH have been noted in some species of Bacteroides (27), although their properties are not known. The presence of both NADP-dependent GDH and NAD-dependent GDH and their regulation have been reported in several bacteria (28, 29). For instance, NADP-GDH is induced by ammonia and repressed by nitrogen starvation, while NAD-GDH is induced by glutamate in Pseudomonas aeruginosa (29) and Hydrogenomonas H16 (30). The NADH-linked activity of GDH was not much increased by the addition of glutamate to the cultures of B, fragilis. The physiological function of the second enzyme remains unclear. Most GSs are known to have not only biosynthetic but also -glutamyltransferase activities (2, 9). In B, fragilis the former activity was demonstrated by the glutamate-dependent ATP hydrolysis and by the formation of glutamine, but the latter activity could not be detected. The lack of transferase activity has been noted in spite of the presence of biosynthetic activity in extracts of Bacillus licheniformis (31), S. ruminantium (14), and Methanobacterium thermoautotrophicum (32). Only in B. licheniformis the cause of the low activity is presumed to be the inhibitory effect of glutamine. The oxidation of NADPH depending on glutamine and 2-oxoglutarate was measured as the GOGAT activity in B, fragilis. Ferredoxin-dependent GOGAT, in the reaction of which methyl viologen can substitute for ferredoxin as an electron donor, has been reported to take part in ammonia assimilation in algae (21) and higher plants (22). Recently, a similar activity of methyl viologen-linked GOGAT has been found in the strictly anaerobic bacteria S. ruminantium (14), M. thermoautotrophicum, and Methanosarcina barkeri (32), in which NAD(P)H-dependent GOGAT is deficient. We did not attempt to assay methyl viologen-dependent activity of GOGAT because of the presence of NADPH-dependent activity of 508 YAMAMOTO, ABE, SAITO and ISHIMOTO VoL. 30

GOGAT in B. fragilis.

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and by a grant from the Yakult Institute.

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