APPLIZD AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1977, p. 69-73
Copyright © 1977 American Society for Microbiology
Fermentation
Vol. 33, No. 1 Printed in U.S.A.
L-Aspartate by a Saccharolytic Strain of Bacteroides melaninogenicus of
JASON C. WONG, JOHN K. DYER,* AD JACK L. TRIBBLE Department of Oral Biology, College of Dentistry,* and Section of Microbiology, Immunology, and Plant Pathology, School of Life Sciences, University of Nebraska, Lincoln, Nebraska 68588 Received for publication 7 July 1976
Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, C02, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L_-[4C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L_[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form -aspartate, which was subsequently, but less actively, fermented.
Bacteroides melaninogenicus represents a heterogeneous group of microorganisms that can vary widely from strain to strain with regard to biochemical and serological characteristics (2, 9). These organisms utilize amino acids as sources of energy and, in addition, some strains ferment a variety of carbohydrates. Studies with four strains of B. melaninogenicus showed that they can ferment amino acids when present as peptides, but may be limited in fermenting free amino acids (12). Recently, the influence of single amino acids on growth of either saccharolytic or asaccharolytic strains of B. melaninogenicus was reported (6). Of all the amino acids tested, L-aspartate and L-asparagine induced maximal growth stimulation of both strains. Preliminary results indicated that the growth-stimulating capacity was dependent upon fermentation of these specific amino acids. The present study was initiated to determine both qualitatively and quantitatively the products of i-aspartate fermentation by the saccharolytic strain of B. melaninogenicus. Carbon and nitrogen balances of these products were calculated. These data suggest that L-aspartate is mainly reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate.
ture Collection, Rockville, Md., was used in these studies. The culture media was a Trypticase-yeast extract-hemin (TYH) medium of the following composition: Trypticase (BBL, Cockeysville, Md.), 3%; yeast extract (Difco, Detroit, Mich.), 0.3%; sodium thioglycolate, 0.05%; hemin (equine type III, Sigma Chemical Co., St. Louis, Mo.), 5 ug per ml; and NaCl, 0.5%. Resting cells for the fermentation studies were grown anaerobically at 37°C in TYH medium supplemented with 0.5% L-aspartate (Sigma, reagent grade). Cells were harvested in the log phase (18 to 20 h) by centrifugation at 12,000 x g at 5°C and suspended to a concentration of 35 + 10 mg (dry weight) per ml in 0.1 M potassium phosphate buffer (pH 7.0). Growth responses to individual amino acids in combination with L-aspartate were tested in T-YH medium with amino acids added at 0.5% concentration. The amino acids (Sigma, reagent grade) tested in combination with L-aspartate included L-cysteine, L-serine, L-alanine, L-proline, L-ornithine, L-leucine, glycine, L-threonine, and L-glutamate. The inocula for growth studies were grown anaerobically at 37°C for 18 to 20 h. Cell suspensions were standardized to an absorbance of 0.7 to 0.8 in 13-mm tubes at 600 nm in buffer (0.05 M potassium phosphate, pH 7.0, with 0.05% sodium thioglycolate) using a Bausch and Lomb Spectronic 20 colorimeter. For growth determinations (repeated four times), matched test tubes (13 by 100 mm) containing 6 ml of medium were closed with rubber serum stoppers immediately after autoclaving, cooled, and inoculated to an absorbance of 0.03 using a tuberculin syringe. The tubes were incubated at 37°C, and absorbance readings were taken as indicated. Fermentations. Fermentations of radioactive substrates were conducted in test tubes (20 by 150
MATERIALS AND METHODS Organism and cultural methods. B. melaninogenicus subsp. melaninogenicus ATCC 25261, a saccharolytic strain obtained from the American Type Cul69
70
WONG, DYER, AND TRIBBLE
mm) at 37°C. A glass vial containing 0.5 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The tubes were fitted with an entry port covered with a rubber serum stopper to allow addition of 2 M H2SO4 to stop the reaction and release 14CO2. The system was flushed with argon immediately after adding the components of the reaction mixture. Analytical methods. Reaction mixtures were quantitatively transferred to centrifuge tubes, and the cells were removed by centrifugation. Volatile acids were separated from the supernatant by steam distillation. The acids were neutralized with NaOH and concentrated under vacuum. The individual volatile acids from the concentrate were separated and identified using the chromatographic procedure of Seki (10) by use of a column (1.2 by 70 cm) of Amberlite CG-50, 100 to 200 mesh (Mallinckrodt Chemical Works, St. Louis, Mo.). Fractions (1 ml) were collected at a flow rate of 6 ml/h. The total radioactivity for each acid was determined on subsamples of the fractions. Ammonia was quantitated by steam distillation from 1 M NaOH, followed by titration with standard acid. The total anions were separated from the amino compounds with a small Dowex resin column (0.6 by 6 cm) as described by Mitruka and Costilow (7). Individual nonvolatile anionic compounds were separated by an ion-exchange chromatographic procedure similar to that of Busch et al. (1) using a column (1.1 by 15.5 cm) of Dowex 1-formate form (Bio-Rad Laboratories, Richmond, Calif., AG 1- x 10, 100 to 200 mesh). A 6 N formic acid elution gradient was used. Fractions (2 ml) were collected at a flow rate of 24 ml/h. The total radioactivity for each acid was determined. The identity of the organic acids was confirmed by thin-layer chromatography using cellulose plates (Brinkmann Instruments, Inc., Westbury, N.Y.) with a layer thickness of 0.10 mm. Diethyl ether-formic acid (90%)-water (7:2:1) was used as the solvent. Cations were eluted from the column with 1 M NH40H concentrated to dryness and made to volume in water. Amino acids were separated and identified by thin-layer chromatography (as above) with four solvent systems: n-propanol-concentrated NH40H (7:3); chloroform-methanol-17% NH40H (40:40:20); phenol-water (75:25); and n-butanolacetic acid-water (80:20:20, upper phase). For quantitation, duplicate samples of the total cations were applied 2 cm apart on the cellulose plates. For locating the individual amino acids, ninhydrin reagent was used as the indicator, with the space above the duplicate sample shielded by a glass plate during spraying. The thin-layer plates were then heated at 100°C for several minutes until blue-colored spots became visible. Zones of the nonsprayed sample corresponding to the visible acids were scraped off, and radioactivity was determined. The scintillation fluid Insta Gel (Packard Instrument Co., Inc., Downers Grove, Ill.) was used in all radioactive assays. Internal standards were used for quench corrections. A Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc.), model 2002, was used for counting the radioactivity. Radiochemicals. Uniformly labeled L-['4C]aspar-
APPL. ENVIRON. MICROBIOL. tic acid and uniformly labeled L-[14C]asparagine were obtained from New England Nuclear Corp., Boston, Mass.
RESULTS
Quantitative analysis of the radioactive components in the supernatant showed that resting-cell suspensions were able to ferment Laspartate as a single substrate (Table 1). The cells fermented 88.1% of the i_[14C]aspartate added (120 ,umol) in a 6-h period. The 14C-labeled products included CO2, formate, acetate, oxaloacetate, succinate, malate, fumarate, alanine, and glycine in the relative percentages 9.9, 1.8, 15, 2.7, 68, 1, 0.06, 0.5, and 0.7, respectively, based on the total counts per minute of the L_['4C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96.3% of the L-aspartate fermented was deaminated. In this experiment, the total radioactivity recovery was 99.6%. The high recoveries of carbon and nitrogen (99.6 and 98.2%, respectively) indicated that essentially all products were recovered. Resting-cell suspensions were also able to ferment L_['4C]asparagine as a single substrate (Table 2). Essentially all L-asparagine added (60 ,umol) was fermented in a 6-h period with high accumulation of aspartate (33.1% of the total counts per minute of the L-asparagine fermented). The other labeled products were similar to those found for L-aspartate fermentation and quantitatively proportional based on the amount of L-asparagine utilized. Analysis of the ammonia produced showed that 160.8 ,umol of ammonia was formed from 100 umol of substrate fermented. The total radioactivity recovery was 97%. The growth responses of the organism to the addition of individually selected amino acids, together with L-aspartate, are shown in Table 3. A moderate increase in growth rate and total cell yield was observed with the addition of icysteine, L-serine, L-threonine, or L-alanine to TYH medium, supplemented with L-aspartate, as compared with the growth rate in unsupplemented TYH medium with L-aspartate. Addition of L-proline, L-ornithine, L-leucine, glycine, and L-glutamate had no effect on growth. The results of L-['4C]aspartate fermentation in the presence of individually selected amino acids or glucose by resting cells are shown in Table 4. The products detected in all cases were qualitatively and quantitatively similar to those found in the L-aspartate fermentation. However, in the presence of glucose, less ammonia and CO2 were produced, whereas more alanine was detected. Recovery of added radioactivity was essentially complete.
L-ASPARTATE FERMENTATION
VOL. 33, 1977
71
TABLE 1. Carbon and nitrogen balances of L-aspartate fermentation a Amt per 100 ,umol of L-aspartate fermented
Radioactivity (cpm)
Determination
Substrate L-['4C]aspartate Added Fermented Products Co2 Formate Acetate Oxaloacetate Succinate Malate Fumarate Alanine Glycine NH3
2.1912 x 106 1.9319 x 106 1.9084 3.5319 2.9049 5.2824 1.3131 2.0220 1.2270 9.3000 1.2750
x x x x x x x x x
105 104 105 104 106 104 103 103 104
% of total cpm
Product ( ,umol)
Carbon
(j,uatoms)
100 88.1 9 9b
1.8 15.0 2.7 68.0 1.0 0.0635 0.5 0.7
400
39.5 7.3 30.1 2.7 68.0 1.0 0.06 0.7 1.3 96.3
39.5 7.3 60.2 10.8 272.0 4.0 0.2 2.0 2.6
Nitrogen
(,uatoms)
100
0.6 1.3 96.3d
98.2 Total in productsc 1.9248 x 106 398.6 99.6 a Reaction mixtures in tubes (20 by 150 mm) contained 400 ,umol of potassium phosphate buffer (pH 7.0), 120 Atmol of L-['4C]aspartate (specific activity, 18,260 cpm/4mol), 84 mg (dry weight) of cells, and water to 5.6 ml. To trap 14CO2, a glass vial containing 0.4 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The reaction was incubated under argon at 37°C for 6 h and was stopped by addition of 0.4 ml of 2 M H2SO4. b Values represent percentages of total counts per minute of the L-['4C]aspartate fermented. c Carbon recovery = 99.6%; nitrogen recovery = 98.2%. d Values for NH3 in the reaction mixture without substrate were subtracted.
TABLE 2. Products of L-asparagine fermentation a Substrate
Radioactivity (cpm)
Product
L-['4C]asparagine Added Fermented
CO2 Anionic compounds Aspartate
Glycine Alanine
1.1130 1.1048 7.6253 6.1335 3.6531 8.9600 4.6080
x x x x x x x
106 106 104 105 105 103 103
% of total cpm
100 99.3 6.9" 55.5 33.1 0.8 0.4
NH3C Total in products 96.7 1.0685 x 106 a Reaction mixtures in tubes (20 by 150 mm) contained 200 ,umol of potassium phosphate buffer (pH 7.0), 60 /Imol of L-[14C]asparagine (specific activity, 18,550 cpm/,umol), 33.4 mg (dry weight) of cells, and water to 2.8 ml. To trap '4CO2, a glass vial containing 0.4 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The reaction was incubated under argon at 37°C for 6 h and was stopped by addition of 0.2 ml of 2 M H2SO4. b Values represent percentages of total counts per minute of the L-['4C]asparagine fermented. of substrate fermented. c A total of 160.8 ,umol of NH3 was formed from 100
/.mol
DISCUSSION
The present study shows that resting cells of B. melaninogenicus are able to ferment L-aspartate as a single substrate with succinate as the major product (Table 1). This finding is significant because succinate has never been demonstrated as a major product of amino acid fermentation by B. melaninogenicus. Since de-
tectable amounts of oxaloacetate, malate, and fumarate were also present among the products, the major pathway of L-aspartate fermentation by B. melaninogenicus may be that Laspartate first undergoes oxidative deamination, forming oxaloacetate. Transamination may be unlikely, since all of the ammonium from L-aspartate was recovered in the form of ammonia, except for trace amounts found in
72
APPL. ENVIRON. MICROBIOL.
WONG, DYER, AND TRIBBLE
alanine and glycine. Subsequently, oxaloace- oxidized in the presence of fumarate (13). Macy a number of inet al. (5) found a type b cytochrome, possibly a termediate reactions involving enzymes of the type c cytochrome, and a very active fumarate tricarboxylic acid cycle via malate and fuma- reductase in the cells of a strain of Bacteroides rate to succinate. Also, L-aspartate may be di- fragilis grown with hemin present in a glucoserectly converted to fumarate, catalyzed by the based medium. However, this organism was unable to synthesize cytochromes and a funcenzyme aspartase, which was demonstrated in many facultative anaerobic bacteria (11). tional fumarate reductase when grown in the Metabolism of B. melaninogenicus appears absence of hemin. These authors suggested to require the presence of a functional mem- that hemin is required by B. fragilis to induce brane-bound electron transport system (8). This synthesis of a functional fumarate reductase respiratory system includes cytochrome c, a and that the hemin-dependent increase of the carbon monoxide-binding pigment, and possi- growth yield may be due to adenosine 5'-tribly flavoproteins. Rizza et al. (8) also reported phosphate production during reduction of fumathat the pigments could be reversibly reduced rate to succinate. by reduced nicotinamide adenine dinucleotide Based upon the evidence that a functional (NADH) or endogenous metabolism and could electron transport system with cytochromes is be oxidized anaerobically by fumarate or by present in various Bacteroides species, it is shaking in air. C. Reddy and M. P. Bryant tempting to speculate that a similar electron (Bacteriol. Proc., p. 40, 1967) observed that one transport system is also present in the strain of strain of B. melaninogenicus contained cyto- B. melaninogenicus investigated in this study chrome c and a second strain contained cyto- and that hemin is required for synthesizing the chromes b and o. Another heme-requiring an- cytochrome-containing enzyme system. If this aerobe, Bacteroides ruminicola, contains a postulation is correct, L-aspartate fermentation membrane-bound electron transport system by B. melaninogenicus may be associated with that is reduced in the presence of NADH and is the membrane-bound electron transport system by which adenosine 5'-triphosphate is syntheTABLE 3. Effect of single amino acid additions to sized via anaerobic electron transport phosphoTYH medium supplemented with 0.5% L-aspartate rylation during the reduction of fumarate to on the growth of B. melaninogenicus ATCC 25261 form succinate. Since CO2, acetate, formate, and trace Amt of growth atb Amino acid adof glycine and alanine were also found amounts deda 10 h 20 h 30 h 40 h 50 h as products of L-aspartate fermentation (Table 1), an oxidative pathway of L-aspartate fermenNone 0.07 0.13 0.20 0.32 0.48 L-Cysteine 0.07 0.14 0.25 0.40 0.58 tation must also be present. However, there is L-Serine 0.07 0.15 0.30 0.50 0.60 not enough evidence from the present study to L-Threonine 0.08 0.14 0.28 0.45 0.58 postulate the oxidative pathway because there L-Alanine 0.07 0.14 0.28 0.50 0.60 are too many probable pathways by which a Each amino acid was added in 0.5% concentra- these products could be formed. Based on the facts that NADH can only be partly furnished tion. b Optical density at 600 nm. by the oxidative deamination of L-aspartate
tate could be reduced through
TABLE 4. Effect of various compounds on L-[14C]aspartate fermentationa L-[4C]aspartate fermented
Additive
None L-Alanine L-Serine L-Threonine
cpm (x 10-5)
%b
9.1955 9.1290 9.2040 9.1854 9.2210
87.6 87.0 87.7 87.5 87.8
Products (% of total cpme)
Anionic
compoundO
c
12.6 12.1 12.2 11.3 9.1
Alanine
Glycine
Amt of NH3 formed Total
(,.mol)
86.6 0.5 0.4 100.1 51.8 87.3 0.8 0.5 100.7 54.92 85.9 0.4 0.7 99.3 53.45 86.2 0.5 0.5 98.5 52.98 '-(+)-Glucose 86.6 2.3 0.7 98.8 45.81 a Reaction mixtures in tubes (20 by 150 mm) contained 200 ,umol of potassium phosphate buffer (pH 7.0); 60 gmol of L_[44C]aspartate (specific activity, 17,495 cpm/,umol); 60 Amol of L-alanine, L-serine, or Lthreonine, or 0.1% D-(+)-glucose; 35.7 mg (dry weight) of cells; and water to 2.8 ml. To trap 14C02, a glass vial containing 0.4 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The reaction was incubated under argon at 37°C for 6 h and was stopped by addition of 0.2 ml of 2 M H2SO4. b Values represent percentages of total counts per minute of the L-[14C]aspartate added (1.0497 x 106 cpm). c All values are based on the amount of L_['4C]aspartate fermented.
L-ASPARTATE FERMENTATION
VOL. 33, 1977
73
and that NADH from endogenous metabolism may not be similar to that by E. coli and B. of the resting cells is not sufficient for the re- fragilis. ductive pathway of L-aspartate fermentation to ACKNOWLEDGMENTS be operable, NADH must be supplied by the This investigation was supported by Public Health Service Grant DEO-3672 from the National Institute of oxidative pathway of L-aspartate catabolism. Research. The accumulation of aspartate resulting from Dental We appreciate the assistance of Donna Dinges in the the exposure of L-['4C]asparagine to resting preparation of the manuscript. cells of B. melaninogenicus (Table 2) indicates LITERATURE CITED that L-asparagine is actively deaminated to 1. Busch, H., R. B. Huribert, and V. Potter. 1952. Anion aspartate, which is subsequently, but less acexchange chromatography of acids of the citric acid cycle. J. Biol. Chem. 196:717-727. tively, fermented. The formation of aspartate Biochemical and ammonia from L-asparagine may by cata- 2. Courant, P. R., and R. J. Gibbons. of1967. and immunological heterogeneity Bacteroides mellyzed by the enzyme L-asparaginase, which has aninogenicus. Arch. Oral Biol. 12:1605-1613. been demonstrated in Escherichia coli (4). 3. Davis, B. D. 1973. Biosynthesis, p. 57-88. In B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg, and L-Alanine, L-serine, and L-threonine were W. B. Wood (ed.), Microbiology, 2nd ed. Harper and shown to be growth stimulating when added to Row Publishers, Hagerstown, Md. TYH medium supplemented with L-aspartate 4. Jackson, R. C., and R. E. Handschumacher. 1970. (Table 3). It was decided to determine whether Escherichia coli L-asparaginase. Catalytic activity and subunit nature. Biochemistry 9:3585-3590. the enhancement effect of these amino acids 5. Macy, J., I. Probst, and G. Gottschalk. 1975. Evidence was due to fermentation by a coupled oxidationfor cytochrome involvement in fumarate reduction reduction reaction with L-aspartate fermentaand adenosine 5'-triphosphate synthesis by Bactetion. However, these stimulatory amino acids roides fragilis grown in the presence of hemin. J. Bacteriol. 123:436-442. did not show any effect on L[-4C]aspartate fer1976. Influmentation (Table 4). In addition, very little 6. Miles, D. O., J. K. Dyer, and J. C. Wong. ence of amino acids on the growth of Bacteroides ammonia other than that from L-aspartate fermelaninogenicus. J. Bacteriol. 127:899-903. mentation was produced in all three cases, 7. Mitruka, B. M., and R. N. Costilow. 1967. Arginine and ornithine catabolism by Clostridium botulinum. J. which indicates that these three amino acids Bacteriol. 93:295-301. apparently are not fermented by resting cells of 8. Rizza, V., P. R. Sinclair, D. C. White, and P. R. B. melaninogenicus. This is similar to the obCourant. 1968. Electron transport system of the proservation of Wahren and Gibbons (12) that toheme-requiring anaerobe Bacteroides melaninogenicus. J. Bacteriol. 96:665-671. most amino acids tested were not fermented R. J. Gibbons. by the resting-cell suspensions of their strains 9. Sawyer, S. J., J. B. Macdonald, and 1962. Biochemical characteristics of Bacteroides melaof B. melaninogenicus. ninogenicus. Arch. Oral Biol. 7:685-691. Glucose is fermented to form succinate, with 10. Seki, T. 1958. Chromatographic separation of lower phosphoenolpyruvate, pyruvate, oxaloacetate, fatty acid. J. Biochem. Tokyo 45:855-860. A. I., and N. Elifolk. 1955. Aspartase, p. 386malate, and fumarate as intermediates, by E. 11. Virtanen, 390. In S. P. Colowick, and N. 0. Kaplan (ed.), Methcoli under anaerobic conditions (3) and B. fraods in enzymology, vol. 2. Academic Press Inc., New gilis grown anaerobically in the presence of York. hemin (5). Results of L-[4Claspartate fermenta- 12. Wahren, A., and R. J. Gibbons. 1970. Amino acid fermentation by Bacteroides melaninogenicus. Antonie tion in the presence of glucose (Table 4) show van Leeuwenhoek J. Microbiol. Serol. 36:149-159. that glucose has no effect on L-aspartate fer- 13. White, D. C., M. P. Bryant, and D. R. Caldwell. 1962. mentation, which indicates that the pathway of Cytochrome-linked fermentation in Bacteroides ruminicola. J. Bacteriol. 84:822-828. glucose fermentation by B. melaninogenicus L-
Copyright © 1977 American Society for Microbiology
Fermentation
Vol. 33, No. 1 Printed in U.S.A.
L-Aspartate by a Saccharolytic Strain of Bacteroides melaninogenicus of
JASON C. WONG, JOHN K. DYER,* AD JACK L. TRIBBLE Department of Oral Biology, College of Dentistry,* and Section of Microbiology, Immunology, and Plant Pathology, School of Life Sciences, University of Nebraska, Lincoln, Nebraska 68588 Received for publication 7 July 1976
Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, C02, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L_-[4C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L_[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form -aspartate, which was subsequently, but less actively, fermented.
Bacteroides melaninogenicus represents a heterogeneous group of microorganisms that can vary widely from strain to strain with regard to biochemical and serological characteristics (2, 9). These organisms utilize amino acids as sources of energy and, in addition, some strains ferment a variety of carbohydrates. Studies with four strains of B. melaninogenicus showed that they can ferment amino acids when present as peptides, but may be limited in fermenting free amino acids (12). Recently, the influence of single amino acids on growth of either saccharolytic or asaccharolytic strains of B. melaninogenicus was reported (6). Of all the amino acids tested, L-aspartate and L-asparagine induced maximal growth stimulation of both strains. Preliminary results indicated that the growth-stimulating capacity was dependent upon fermentation of these specific amino acids. The present study was initiated to determine both qualitatively and quantitatively the products of i-aspartate fermentation by the saccharolytic strain of B. melaninogenicus. Carbon and nitrogen balances of these products were calculated. These data suggest that L-aspartate is mainly reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate.
ture Collection, Rockville, Md., was used in these studies. The culture media was a Trypticase-yeast extract-hemin (TYH) medium of the following composition: Trypticase (BBL, Cockeysville, Md.), 3%; yeast extract (Difco, Detroit, Mich.), 0.3%; sodium thioglycolate, 0.05%; hemin (equine type III, Sigma Chemical Co., St. Louis, Mo.), 5 ug per ml; and NaCl, 0.5%. Resting cells for the fermentation studies were grown anaerobically at 37°C in TYH medium supplemented with 0.5% L-aspartate (Sigma, reagent grade). Cells were harvested in the log phase (18 to 20 h) by centrifugation at 12,000 x g at 5°C and suspended to a concentration of 35 + 10 mg (dry weight) per ml in 0.1 M potassium phosphate buffer (pH 7.0). Growth responses to individual amino acids in combination with L-aspartate were tested in T-YH medium with amino acids added at 0.5% concentration. The amino acids (Sigma, reagent grade) tested in combination with L-aspartate included L-cysteine, L-serine, L-alanine, L-proline, L-ornithine, L-leucine, glycine, L-threonine, and L-glutamate. The inocula for growth studies were grown anaerobically at 37°C for 18 to 20 h. Cell suspensions were standardized to an absorbance of 0.7 to 0.8 in 13-mm tubes at 600 nm in buffer (0.05 M potassium phosphate, pH 7.0, with 0.05% sodium thioglycolate) using a Bausch and Lomb Spectronic 20 colorimeter. For growth determinations (repeated four times), matched test tubes (13 by 100 mm) containing 6 ml of medium were closed with rubber serum stoppers immediately after autoclaving, cooled, and inoculated to an absorbance of 0.03 using a tuberculin syringe. The tubes were incubated at 37°C, and absorbance readings were taken as indicated. Fermentations. Fermentations of radioactive substrates were conducted in test tubes (20 by 150
MATERIALS AND METHODS Organism and cultural methods. B. melaninogenicus subsp. melaninogenicus ATCC 25261, a saccharolytic strain obtained from the American Type Cul69
70
WONG, DYER, AND TRIBBLE
mm) at 37°C. A glass vial containing 0.5 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The tubes were fitted with an entry port covered with a rubber serum stopper to allow addition of 2 M H2SO4 to stop the reaction and release 14CO2. The system was flushed with argon immediately after adding the components of the reaction mixture. Analytical methods. Reaction mixtures were quantitatively transferred to centrifuge tubes, and the cells were removed by centrifugation. Volatile acids were separated from the supernatant by steam distillation. The acids were neutralized with NaOH and concentrated under vacuum. The individual volatile acids from the concentrate were separated and identified using the chromatographic procedure of Seki (10) by use of a column (1.2 by 70 cm) of Amberlite CG-50, 100 to 200 mesh (Mallinckrodt Chemical Works, St. Louis, Mo.). Fractions (1 ml) were collected at a flow rate of 6 ml/h. The total radioactivity for each acid was determined on subsamples of the fractions. Ammonia was quantitated by steam distillation from 1 M NaOH, followed by titration with standard acid. The total anions were separated from the amino compounds with a small Dowex resin column (0.6 by 6 cm) as described by Mitruka and Costilow (7). Individual nonvolatile anionic compounds were separated by an ion-exchange chromatographic procedure similar to that of Busch et al. (1) using a column (1.1 by 15.5 cm) of Dowex 1-formate form (Bio-Rad Laboratories, Richmond, Calif., AG 1- x 10, 100 to 200 mesh). A 6 N formic acid elution gradient was used. Fractions (2 ml) were collected at a flow rate of 24 ml/h. The total radioactivity for each acid was determined. The identity of the organic acids was confirmed by thin-layer chromatography using cellulose plates (Brinkmann Instruments, Inc., Westbury, N.Y.) with a layer thickness of 0.10 mm. Diethyl ether-formic acid (90%)-water (7:2:1) was used as the solvent. Cations were eluted from the column with 1 M NH40H concentrated to dryness and made to volume in water. Amino acids were separated and identified by thin-layer chromatography (as above) with four solvent systems: n-propanol-concentrated NH40H (7:3); chloroform-methanol-17% NH40H (40:40:20); phenol-water (75:25); and n-butanolacetic acid-water (80:20:20, upper phase). For quantitation, duplicate samples of the total cations were applied 2 cm apart on the cellulose plates. For locating the individual amino acids, ninhydrin reagent was used as the indicator, with the space above the duplicate sample shielded by a glass plate during spraying. The thin-layer plates were then heated at 100°C for several minutes until blue-colored spots became visible. Zones of the nonsprayed sample corresponding to the visible acids were scraped off, and radioactivity was determined. The scintillation fluid Insta Gel (Packard Instrument Co., Inc., Downers Grove, Ill.) was used in all radioactive assays. Internal standards were used for quench corrections. A Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Inc.), model 2002, was used for counting the radioactivity. Radiochemicals. Uniformly labeled L-['4C]aspar-
APPL. ENVIRON. MICROBIOL. tic acid and uniformly labeled L-[14C]asparagine were obtained from New England Nuclear Corp., Boston, Mass.
RESULTS
Quantitative analysis of the radioactive components in the supernatant showed that resting-cell suspensions were able to ferment Laspartate as a single substrate (Table 1). The cells fermented 88.1% of the i_[14C]aspartate added (120 ,umol) in a 6-h period. The 14C-labeled products included CO2, formate, acetate, oxaloacetate, succinate, malate, fumarate, alanine, and glycine in the relative percentages 9.9, 1.8, 15, 2.7, 68, 1, 0.06, 0.5, and 0.7, respectively, based on the total counts per minute of the L_['4C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96.3% of the L-aspartate fermented was deaminated. In this experiment, the total radioactivity recovery was 99.6%. The high recoveries of carbon and nitrogen (99.6 and 98.2%, respectively) indicated that essentially all products were recovered. Resting-cell suspensions were also able to ferment L_['4C]asparagine as a single substrate (Table 2). Essentially all L-asparagine added (60 ,umol) was fermented in a 6-h period with high accumulation of aspartate (33.1% of the total counts per minute of the L-asparagine fermented). The other labeled products were similar to those found for L-aspartate fermentation and quantitatively proportional based on the amount of L-asparagine utilized. Analysis of the ammonia produced showed that 160.8 ,umol of ammonia was formed from 100 umol of substrate fermented. The total radioactivity recovery was 97%. The growth responses of the organism to the addition of individually selected amino acids, together with L-aspartate, are shown in Table 3. A moderate increase in growth rate and total cell yield was observed with the addition of icysteine, L-serine, L-threonine, or L-alanine to TYH medium, supplemented with L-aspartate, as compared with the growth rate in unsupplemented TYH medium with L-aspartate. Addition of L-proline, L-ornithine, L-leucine, glycine, and L-glutamate had no effect on growth. The results of L-['4C]aspartate fermentation in the presence of individually selected amino acids or glucose by resting cells are shown in Table 4. The products detected in all cases were qualitatively and quantitatively similar to those found in the L-aspartate fermentation. However, in the presence of glucose, less ammonia and CO2 were produced, whereas more alanine was detected. Recovery of added radioactivity was essentially complete.
L-ASPARTATE FERMENTATION
VOL. 33, 1977
71
TABLE 1. Carbon and nitrogen balances of L-aspartate fermentation a Amt per 100 ,umol of L-aspartate fermented
Radioactivity (cpm)
Determination
Substrate L-['4C]aspartate Added Fermented Products Co2 Formate Acetate Oxaloacetate Succinate Malate Fumarate Alanine Glycine NH3
2.1912 x 106 1.9319 x 106 1.9084 3.5319 2.9049 5.2824 1.3131 2.0220 1.2270 9.3000 1.2750
x x x x x x x x x
105 104 105 104 106 104 103 103 104
% of total cpm
Product ( ,umol)
Carbon
(j,uatoms)
100 88.1 9 9b
1.8 15.0 2.7 68.0 1.0 0.0635 0.5 0.7
400
39.5 7.3 30.1 2.7 68.0 1.0 0.06 0.7 1.3 96.3
39.5 7.3 60.2 10.8 272.0 4.0 0.2 2.0 2.6
Nitrogen
(,uatoms)
100
0.6 1.3 96.3d
98.2 Total in productsc 1.9248 x 106 398.6 99.6 a Reaction mixtures in tubes (20 by 150 mm) contained 400 ,umol of potassium phosphate buffer (pH 7.0), 120 Atmol of L-['4C]aspartate (specific activity, 18,260 cpm/4mol), 84 mg (dry weight) of cells, and water to 5.6 ml. To trap 14CO2, a glass vial containing 0.4 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The reaction was incubated under argon at 37°C for 6 h and was stopped by addition of 0.4 ml of 2 M H2SO4. b Values represent percentages of total counts per minute of the L-['4C]aspartate fermented. c Carbon recovery = 99.6%; nitrogen recovery = 98.2%. d Values for NH3 in the reaction mixture without substrate were subtracted.
TABLE 2. Products of L-asparagine fermentation a Substrate
Radioactivity (cpm)
Product
L-['4C]asparagine Added Fermented
CO2 Anionic compounds Aspartate
Glycine Alanine
1.1130 1.1048 7.6253 6.1335 3.6531 8.9600 4.6080
x x x x x x x
106 106 104 105 105 103 103
% of total cpm
100 99.3 6.9" 55.5 33.1 0.8 0.4
NH3C Total in products 96.7 1.0685 x 106 a Reaction mixtures in tubes (20 by 150 mm) contained 200 ,umol of potassium phosphate buffer (pH 7.0), 60 /Imol of L-[14C]asparagine (specific activity, 18,550 cpm/,umol), 33.4 mg (dry weight) of cells, and water to 2.8 ml. To trap '4CO2, a glass vial containing 0.4 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The reaction was incubated under argon at 37°C for 6 h and was stopped by addition of 0.2 ml of 2 M H2SO4. b Values represent percentages of total counts per minute of the L-['4C]asparagine fermented. of substrate fermented. c A total of 160.8 ,umol of NH3 was formed from 100
/.mol
DISCUSSION
The present study shows that resting cells of B. melaninogenicus are able to ferment L-aspartate as a single substrate with succinate as the major product (Table 1). This finding is significant because succinate has never been demonstrated as a major product of amino acid fermentation by B. melaninogenicus. Since de-
tectable amounts of oxaloacetate, malate, and fumarate were also present among the products, the major pathway of L-aspartate fermentation by B. melaninogenicus may be that Laspartate first undergoes oxidative deamination, forming oxaloacetate. Transamination may be unlikely, since all of the ammonium from L-aspartate was recovered in the form of ammonia, except for trace amounts found in
72
APPL. ENVIRON. MICROBIOL.
WONG, DYER, AND TRIBBLE
alanine and glycine. Subsequently, oxaloace- oxidized in the presence of fumarate (13). Macy a number of inet al. (5) found a type b cytochrome, possibly a termediate reactions involving enzymes of the type c cytochrome, and a very active fumarate tricarboxylic acid cycle via malate and fuma- reductase in the cells of a strain of Bacteroides rate to succinate. Also, L-aspartate may be di- fragilis grown with hemin present in a glucoserectly converted to fumarate, catalyzed by the based medium. However, this organism was unable to synthesize cytochromes and a funcenzyme aspartase, which was demonstrated in many facultative anaerobic bacteria (11). tional fumarate reductase when grown in the Metabolism of B. melaninogenicus appears absence of hemin. These authors suggested to require the presence of a functional mem- that hemin is required by B. fragilis to induce brane-bound electron transport system (8). This synthesis of a functional fumarate reductase respiratory system includes cytochrome c, a and that the hemin-dependent increase of the carbon monoxide-binding pigment, and possi- growth yield may be due to adenosine 5'-tribly flavoproteins. Rizza et al. (8) also reported phosphate production during reduction of fumathat the pigments could be reversibly reduced rate to succinate. by reduced nicotinamide adenine dinucleotide Based upon the evidence that a functional (NADH) or endogenous metabolism and could electron transport system with cytochromes is be oxidized anaerobically by fumarate or by present in various Bacteroides species, it is shaking in air. C. Reddy and M. P. Bryant tempting to speculate that a similar electron (Bacteriol. Proc., p. 40, 1967) observed that one transport system is also present in the strain of strain of B. melaninogenicus contained cyto- B. melaninogenicus investigated in this study chrome c and a second strain contained cyto- and that hemin is required for synthesizing the chromes b and o. Another heme-requiring an- cytochrome-containing enzyme system. If this aerobe, Bacteroides ruminicola, contains a postulation is correct, L-aspartate fermentation membrane-bound electron transport system by B. melaninogenicus may be associated with that is reduced in the presence of NADH and is the membrane-bound electron transport system by which adenosine 5'-triphosphate is syntheTABLE 3. Effect of single amino acid additions to sized via anaerobic electron transport phosphoTYH medium supplemented with 0.5% L-aspartate rylation during the reduction of fumarate to on the growth of B. melaninogenicus ATCC 25261 form succinate. Since CO2, acetate, formate, and trace Amt of growth atb Amino acid adof glycine and alanine were also found amounts deda 10 h 20 h 30 h 40 h 50 h as products of L-aspartate fermentation (Table 1), an oxidative pathway of L-aspartate fermenNone 0.07 0.13 0.20 0.32 0.48 L-Cysteine 0.07 0.14 0.25 0.40 0.58 tation must also be present. However, there is L-Serine 0.07 0.15 0.30 0.50 0.60 not enough evidence from the present study to L-Threonine 0.08 0.14 0.28 0.45 0.58 postulate the oxidative pathway because there L-Alanine 0.07 0.14 0.28 0.50 0.60 are too many probable pathways by which a Each amino acid was added in 0.5% concentra- these products could be formed. Based on the facts that NADH can only be partly furnished tion. b Optical density at 600 nm. by the oxidative deamination of L-aspartate
tate could be reduced through
TABLE 4. Effect of various compounds on L-[14C]aspartate fermentationa L-[4C]aspartate fermented
Additive
None L-Alanine L-Serine L-Threonine
cpm (x 10-5)
%b
9.1955 9.1290 9.2040 9.1854 9.2210
87.6 87.0 87.7 87.5 87.8
Products (% of total cpme)
Anionic
compoundO
c
12.6 12.1 12.2 11.3 9.1
Alanine
Glycine
Amt of NH3 formed Total
(,.mol)
86.6 0.5 0.4 100.1 51.8 87.3 0.8 0.5 100.7 54.92 85.9 0.4 0.7 99.3 53.45 86.2 0.5 0.5 98.5 52.98 '-(+)-Glucose 86.6 2.3 0.7 98.8 45.81 a Reaction mixtures in tubes (20 by 150 mm) contained 200 ,umol of potassium phosphate buffer (pH 7.0); 60 gmol of L_[44C]aspartate (specific activity, 17,495 cpm/,umol); 60 Amol of L-alanine, L-serine, or Lthreonine, or 0.1% D-(+)-glucose; 35.7 mg (dry weight) of cells; and water to 2.8 ml. To trap 14C02, a glass vial containing 0.4 ml of 1 M hydroxide of hyamine in methanol was suspended above the reaction mixture. The reaction was incubated under argon at 37°C for 6 h and was stopped by addition of 0.2 ml of 2 M H2SO4. b Values represent percentages of total counts per minute of the L-[14C]aspartate added (1.0497 x 106 cpm). c All values are based on the amount of L_['4C]aspartate fermented.
L-ASPARTATE FERMENTATION
VOL. 33, 1977
73
and that NADH from endogenous metabolism may not be similar to that by E. coli and B. of the resting cells is not sufficient for the re- fragilis. ductive pathway of L-aspartate fermentation to ACKNOWLEDGMENTS be operable, NADH must be supplied by the This investigation was supported by Public Health Service Grant DEO-3672 from the National Institute of oxidative pathway of L-aspartate catabolism. Research. The accumulation of aspartate resulting from Dental We appreciate the assistance of Donna Dinges in the the exposure of L-['4C]asparagine to resting preparation of the manuscript. cells of B. melaninogenicus (Table 2) indicates LITERATURE CITED that L-asparagine is actively deaminated to 1. Busch, H., R. B. Huribert, and V. Potter. 1952. Anion aspartate, which is subsequently, but less acexchange chromatography of acids of the citric acid cycle. J. Biol. Chem. 196:717-727. tively, fermented. The formation of aspartate Biochemical and ammonia from L-asparagine may by cata- 2. Courant, P. R., and R. J. Gibbons. of1967. and immunological heterogeneity Bacteroides mellyzed by the enzyme L-asparaginase, which has aninogenicus. Arch. Oral Biol. 12:1605-1613. been demonstrated in Escherichia coli (4). 3. Davis, B. D. 1973. Biosynthesis, p. 57-88. In B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg, and L-Alanine, L-serine, and L-threonine were W. B. Wood (ed.), Microbiology, 2nd ed. Harper and shown to be growth stimulating when added to Row Publishers, Hagerstown, Md. TYH medium supplemented with L-aspartate 4. Jackson, R. C., and R. E. Handschumacher. 1970. (Table 3). It was decided to determine whether Escherichia coli L-asparaginase. Catalytic activity and subunit nature. Biochemistry 9:3585-3590. the enhancement effect of these amino acids 5. Macy, J., I. Probst, and G. Gottschalk. 1975. Evidence was due to fermentation by a coupled oxidationfor cytochrome involvement in fumarate reduction reduction reaction with L-aspartate fermentaand adenosine 5'-triphosphate synthesis by Bactetion. However, these stimulatory amino acids roides fragilis grown in the presence of hemin. J. Bacteriol. 123:436-442. did not show any effect on L[-4C]aspartate fer1976. Influmentation (Table 4). In addition, very little 6. Miles, D. O., J. K. Dyer, and J. C. Wong. ence of amino acids on the growth of Bacteroides ammonia other than that from L-aspartate fermelaninogenicus. J. Bacteriol. 127:899-903. mentation was produced in all three cases, 7. Mitruka, B. M., and R. N. Costilow. 1967. Arginine and ornithine catabolism by Clostridium botulinum. J. which indicates that these three amino acids Bacteriol. 93:295-301. apparently are not fermented by resting cells of 8. Rizza, V., P. R. Sinclair, D. C. White, and P. R. B. melaninogenicus. This is similar to the obCourant. 1968. Electron transport system of the proservation of Wahren and Gibbons (12) that toheme-requiring anaerobe Bacteroides melaninogenicus. J. Bacteriol. 96:665-671. most amino acids tested were not fermented R. J. Gibbons. by the resting-cell suspensions of their strains 9. Sawyer, S. J., J. B. Macdonald, and 1962. Biochemical characteristics of Bacteroides melaof B. melaninogenicus. ninogenicus. Arch. Oral Biol. 7:685-691. Glucose is fermented to form succinate, with 10. Seki, T. 1958. Chromatographic separation of lower phosphoenolpyruvate, pyruvate, oxaloacetate, fatty acid. J. Biochem. Tokyo 45:855-860. A. I., and N. Elifolk. 1955. Aspartase, p. 386malate, and fumarate as intermediates, by E. 11. Virtanen, 390. In S. P. Colowick, and N. 0. Kaplan (ed.), Methcoli under anaerobic conditions (3) and B. fraods in enzymology, vol. 2. Academic Press Inc., New gilis grown anaerobically in the presence of York. hemin (5). Results of L-[4Claspartate fermenta- 12. Wahren, A., and R. J. Gibbons. 1970. Amino acid fermentation by Bacteroides melaninogenicus. Antonie tion in the presence of glucose (Table 4) show van Leeuwenhoek J. Microbiol. Serol. 36:149-159. that glucose has no effect on L-aspartate fer- 13. White, D. C., M. P. Bryant, and D. R. Caldwell. 1962. mentation, which indicates that the pathway of Cytochrome-linked fermentation in Bacteroides ruminicola. J. Bacteriol. 84:822-828. glucose fermentation by B. melaninogenicus L-
