xi.“ . _. . 31 m x .? Wu; '0 . A va- . I. c .0}... . n . .~ - f. .0. uvflh‘. c. , Q ‘h \‘ IWI' CO. . . .‘ . ANA”... . A . . 9 V _ -1 xA ... .. .‘.. . . . curb“... L .A:.,... A . n. . v . ‘ (.LnfnnCDVATwnuU . A A. . .. . .33.:WW ‘11.- . . .3... H I .0. . fiwfitmfi. ... ”wfiufimhdxl 0 A 1 2.. , Ivy.“ 0 'D.I.b . . 2vnblll‘ . ,Mo ta'ltvoV. Ufi..§hflnw-bfi 1...... $.14 9L... -ollll. . ".13; I. Itintllhvu .r.. nvgllnlol..ylvc.l|.|!|l‘1,u I.»\.-nr1 I; ‘3 .. 11.3.1. . . La¢z com 0mm com om. ooa F d 1‘ cor owm P 1 1 J! 1 d!- d .i O¢o. : coo. one .. 87 : om". BONHGHOSQU 58 Figure 4. Dithionite-reduced versus air oxidized difference spectrum of the 104,000 x g supernatant frac- tion (20 mg of protein per ml) of B. fragilis ( ); air oxidized versus air oxidized (~H———). 59 00b 9 ohsmfim AmmMszozngzpoZMJm>¢z one cow cum com om¢ 00+ GP b d I. d- .- n p I q q q d .ovo. coo. 0N". om“. BONUQHOSQU 60 Figure 5. Dithionite-reduced versus air oxidized difference spectrum of the 104,000 x g pellet fraction (3.8 mg of protein per m1) of B. fragilis ( ); air oxidized versus air oxidized (—-n—-). 61 m ohswqm Ammuhmzozczurhozw4m>¢z 02. 0mm com 0mm com omv 00V 1.1. , I Ll DON. \\ com. C .. 8.. 0m? Dom. BJNUQHOSQU 62 of a protoheme which is known to be the prosthetic group of the b-type cytochromes in general (12). In studies by Macy et al. (15), a small absorption maxima was detected at 554 nm in cell extracts of B. fragilis when a liquid nitrogen difference spectrum was obtained. They suggested that a c-type cytochrome may be present in B. fragilis (15). In this study, there was no indication of a c-type cytochrome from the difference spectra at room temperature (Figures 3, 4 and 5). However, a pyridine hemochromogen was made of the residue remaining after the acid-acetone extraction of cell extracts (Figure 6). The dithionite-reduced versus air-oxidized difference spectrum of this residue had absorption maxima at 552, 520, and 414 nm, which are characteristic of a c-type cytochrome. Apparently the c-type cytochrome was completely masked by the b-type cytochrome in the different cell fractions. DISCUSSION Many chemoorganotrophic bacteria are thought to dispose of excess electrons by reducing protons to molecu- lar hydrogen by the enzyme hydrogenase (8,9). Hydrogenase activity has been demonstrated in whole cells and cell extracts of B. fragilis (Section 1). Further results showed that hydrogenase activity was higher in cells in the late stationary phase of growth as compared to those in the early stationary phase of growth. Potassium phosphate buffer (pH 7.0-7.6), used by several previous 63 Figure 6. Dithionite-reduced versus air oxidized difference spectrum of a pyridine hemochrome from the residue of the acid-acetone extract of B. fragilis. The residue of 94 mg of HCl-acetone extracted cell- protein was suspended in 7 ml of pyridine-KOH ( air oxidized versus air oxidized 6----). ); 64 o msswflu ammmpmzozczvrpozmqu>¢z r 00% 0mm com T.................. 1‘ 02. 0mm F 1 owe 8e r P d d uh- 4 J 1 .. DVD. $ coo. ‘J , ON". C : owl. Sw aaNuaaosgu 6S investigators for demonstrating hydrogenase activity in several microorganisms (10,12,14,17), appeared to be better than Tris HCl or HEPES buffer for the hydrogenase activity. It is certainly possible that the impurities such as iron in the P04 buffer may have contributed to the increase in hydrogenase activity with P04 buffer as compared to the activity with Tris HCl or HEPBS buffer. The slight variance in the pH values of the different buffers may have also been a factor in determining the rate of the hydrogenase activity. Macy and her colleagues (15) demonstrated HZ-fumarate coupling activity in B. fragilis but only in the presence of Clostridium pasteurianum extract and methyl viologen (MV). Clostridium pasteurianum extract apparently served as a source of hydrogenase and other essential cofactors and MV served as a required electron mediator (15). In the present investigation, 0. pasteurianum extract was not required for activity. In a few experiments, it was used to replace BV, FMN, or FAD, which are required for HZ-fumarate coupling activity (Section 1). Hydrogenase activity was readily demonstrated in the strain of B. fragilis used in this study, while Macy et al. (15) could not detect hydrogenase activity in their strain. The HZ-fumarate system in the study by Macy et a1. (15) may therefore be considered artificial since they had to use 0. pasteurianum extract and MV. In such an artificial system, MV may shunt the electrons around the natural carriers in the electron transport chain and the results 66 may not truly represent the events that occur in a natural electron flow. The results of the present investigation indicated that a majority (42%) of the fumarate reductase activity in B. fragilis was located in the particulate fraction (104,000 x g pellet) of the cell. The 104,000 x g super- natant (soluble fraction) contained approximately 21% of the total fumarate reductase activity. Similar results were obtained by Jacobs and Wolin for the location of fumarate reductase in Vibrio succinogenes (12). They observed 3 times more fumarate reductase activity in the 144,000 x g pellet than the 144,000 x g supernatant frac- tion (12). The majority of the fumarate reductase activity in Streptococcus faecalis and Desulfovibrio gigas were generally associated with large particles also (1,2,7). It is probable that multiple hydrogenases may be present in B. fragilis. From data previously shown (Section 1), the majority of the hydrogenase activity, 38%, was located in the soluble fraction of the cell. Only 2.9% of the hydrogenase activity was associated with the 104,000 x g pellet fraction. .Yet, Hz-fumarate coupling activity was demonstrated in the 104,000 x g pellet frac- tion. It may be possible that one hydrogenase in B. fragilis is soluble and a second hydrogenase is located in the particulate fraction and is involved exclusively in the HZ-fumarate electron transport system. 0f the various carboxylic acids examined, H2 consump- tion was observed only with fumarate and malate as 67 electron acceptors in the present study. H2 oxidation with malate suggested that malate was initially metabolized to fumarate by the fumarase in cell extracts and fumarate then accepted the electrons from H2. Fumarase activity has been detected in B. fragilis by Macy et al. (15). Similarly, C. A. Reddy (M.S. thesis, University of Illinois, 1967) showed that the addition of malate, fumarate, or oxaloacetate resulted in the oxidation of endogenously reduced cytochrome b in whole cells of B. fragilis. It was interesting that Hz was not oxidized in the presence of oxaloacetate in the strain used in this study in view of the fact that Reddy (M.S. thesis) observed that oxaloacetate oxidized the reduced b-type cytochrome in other strains of B. fragilis. Malate dehydrogenase, the enzyme that reduces oxaloacetate to malate, may be subject to catabolite repression from the fumarate added to the growth medium. In this study, fumarate was shown to be reduced to succinate in the presence of H2. The fumarate added initially was quantitatively recovered as fumarate and succinate at the end of the reaction. There was not a perfect stoichiometry between the quantity of H2 consumed and the quantity of succinate formed in the reaction. This discrepancy is probably due to the lack of sensi- tivity of the gas chromatograph to low levels of the acids. The results of the present study in conjunction with those in Section 1 suggested that NAD+ may be a component 63 in the Hz-fumarate electron transport system in B. fragilis. The results presented in the preceding section indicated that NAD+ was reduced by H2 in the presence of B. fragilis extract and a catalytic quantity of ferredoxin from C. pasteurianum, or BV. Contrary to findings con- cerning the Hz-fumarate complex in B. fragilis, Jacobs and Wolin (12) and Barton et al. (1,2) reported that pyridine nucleotides were not involved in the hydrogen- fumarate electron transport systems in V. succinogenes and D. gigas, respectively. Cytochromes have been shown to be involved in the reduction of fumarate by H2 or reduced pyridine nucleo- tides in many organisms (4,5,6,10,12,13,16,18,19); cytochrome-free S. faecalis, which reduces fumarate with NADH, is an exception, however (7). The involvement of the b-type cytochrome has been reported for fumarate reduction in D. gigas (10), V. succinogenes (13), Escherichia coli (16), Selenomonas ruminantium (6), Anaerovibrio Zipolytica (6), Propionibacterium freuden- reichii (5), and Propionibacterium pentosaceum (5). Cytochrome b has been demonstrated in several Bacteroides species including B. ruminicola (19), B. oralis, B. succinogenes (C. A. Reddy, M.S. thesis), and B. fragilis (C. A. Reddy, M.S. thesis; 15). A c—type cytochrome detected in Bacteroides melaninogenicus may be linked to fumarate reduction by NADH (18). Cytochromes of the b and c types were demonstrated in the strain of B. fragilis used in the present study. At the present time, the role 69 of the c-type cytochrome, if any, in fumarate reduction is not known. . The results in this study in conjunction with those in Section 1 suggested that fumarate is reduced to succinate in B. fragilis via an anaerobic electron trans- port system. The apparently oxygen labile low potential electron carrier in B. fragilis may be a protein with a flavin (FAD or FMN) prosthetic group. This hypothesis is supported by the observations that FAD or FMN accepted electrons from H2 and transferred electrons from H2 to fumarate in B. fragilis (Section 1). The oxidation- reduction potential of the low potential carrier may be similar to that of benzyl viologen (~350 millivolts), since benzyl viologen but not methyl viologen (-440 milli- volts) could serve as an electron mediator in the Hz- ' fumarate electron transport system. These observations were again consistent with the idea that the low poten- tial electron carrier may be a flavodoxin-like protein since the oxidation-reduction potential of flavodoxin from other anaerobic organisms has been similar to that of BV (20). The anaerobic electron transport system involved in the transfer of electrons from H2 to fumarate in B. fragilis may consist of a low potential flavodoxin- like protein, NAD+, flavoprotein, quinone, and cytochrome b (Section 1, Figure l). LITERATURE CITED Barton, L. L., J. LeGall, and H. D. Peck, Jr. 1970. Phosphorylation coupled to oxidation of hydrogen with fumarate in extracts of the sulfate reducing bacterium Desquovibrio gigas. Biochem. Biophys. Res. Commun. 41:1036-1042. Barton, L. L., J. LeGall, and H. D. Peck, Jr. 1972. Oxidative phosphorylation in the obligate anaerobe, Desquovibrio gigas. p. 33-51. Horizons of Bioenergetics. Chance, B. 1954. Spectrophotometry of intracellular respiratory pigments. Science 120:767-776. DerVartanian, D. V., and J. LeGall. 1974. A mono- molecular electron transfer chain: Structure and function of cytochrome C3. Biochim. Biophys. Acta 346:79-99. DeVries, W., W. M. C. van Wijck-Kapteyn, and A. H. Stouthamer. 1973. Generation of ATP during cytochrome- linked anaerobic electron transport in propionic acid bacteria. J. Gen. Microbiol. 76:31-41. DeVries, W., W. M. C. van Wijck-Kapteyn, and S. K. H. Oosterhuis. 1974. The presence and function of cytochromes in Selenomonas ruminantium, Anaerovibrio Zipolytica, and VeiZZoneZZa aanZescens. J. Gen. Microbiol. 81:69-78. Faust, P. J., and P. J. Vandemark. 1970. Phosphoryla- tion coupled to NADH oxidation with fumarate in Streptococcus faecaZis 10Cl. Arch. Biochem. Biophys. 137:392-398. Gest, H. 1954. Oxidation and evolution of molecular hydrogen by microorganisms. Bacteriol. Rev. 18:43-73. Gray, C. T. and H. Gest. 1965. Biological formation of molecular hydrogen. Science 148:186-192. 70 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 71 Hatchikian, E. C., and J. LeGall. 1972. Evidence for the presence of a b-type cytochrome in the sulfate reducing bacterium Desulfonibrio gigan, and its role in the reduction of fumarate by molecular hydrogen. Biochim. Biophys. Acta 267:479-484. Holdeman, L. V., and W. E. C. Moore (eds.). 1973. Anaerobe Laboratory Manual, 2nd ed. VPI Anaerobe Laboratory, Blacksburg, Virginia. Jacobs, N. J., and M. J. Wolin. 1963. Electron transport system of Vibrio succinogenes. I. Enzymes and cytochromes of the electron transport system. Biochim. Biophys. Acta 69:18-28. Jacobs, N. J., and M. J. Wolin. 1963. Electron transport system of Vibrio succinogenes. II. Inhi- bition of electron transport by 2-hepty1-4-hydroxy- quinoline-N-oxide. Biochim. Biophys. Acta 69:29-39. Joyner, A. E., W. T. Winter, and D. M. Godbout. 1977. Studies on some characteristics of hydrogen production in cell-free extracts of rumen anaerobic bacteria. Can. J. Microbiol. 23:346-353. Macy, J., I. Probst, and G. Gottschalk. 1975. Evidence for cytochrome involvement in fumarate reduction and adenosine S'triphosphate synthesis by Bacteroides fragilis grown in the presence of hemin. J. Bacteriol. 123:436-442. Macy, J., H. Kulla, and G. Gottschalk. 1976. Hz-dependent anaerobic growth of Escherichia coli on L-malatezsuccinate formation. J. Bacteriol. 125: 423-428. Peck, H. D., Jr., and H. Gest. 1967. Hydrogenase of Clostridium butylicum. J. Bacteriol. 73:569-580. Rizza, V., P. R. Sinclair, D. C. White, and P. R. Courant. 1968. Electron transport system of the protoheme-requiring anaerobe Bacteroides meZaninogenicus. J. Bacteriol. 96:665-671. White, D. C., M. P. Bryant, and D. Caldwell. 1962. Cytochrome linked fermentation in Bacteroides ruminicoZa. J. Bacteriol. 84:822-828. Youch, D. C., and R. C. Valentine. 1972. Ferredoxins and flavodoxins of bacteria. Annu. Rev. Microbiol. 26:139-162. SECTION 3 (ARTICLE 3) ATTEMPTS TO DEMONSTRATB ADENOSINE 5'-TRIPHOSPHATE PRODUCTION COUPLED TO FUMARATE REDUCTION BY H2 IN BACTEROIDE’S FRAGILIS By Martha A. Harris and C. Adinarayana Reddy 72 73 INTRODUCTION Previous investigators have demonstrated phosphoryla- tion of adenosine S'-diphosphate (ADP) to adenosine 5'-triphosphate (ATP) in several organisms that reduce fumarate to succinate with either H2 or reduced nicotina- mide adenine dinucleotide (NADH) (1,2,11,21, C. A. Reddy and H. D. Peck, Jr. Abst. Annu. Meet. Amer. Soc. Micro- biol., 1973, 194, C. A. Reddy, 1973, Rumen Function Conference, Chicago, unpublished). Many and her colleagues (21) have presented indirect evidence to suggest that additional ATP is produced when fumarate is reduced to succinate in Bacteroides fragilis. Recently, Harris and Reddy (15) have demonstrated a Hz-fumarate electron trans- port system in B. fragilis. Therefore, the primary objective of this investigation was to attempt to demon- strate more direct evidence for ATP production during fumarate reduction by H2 in cell extracts of B. fragilis. MATERIALS AND METHODS Except as indicated below, methods for growth of bacteria, preparation of extracts, and enzymatic and chemical analysis were as described previously (15). In all experiments designed to demonstrate ATP forma- tion, the cells were grown in the basal medium (15) supplemented with 0.005% ferrous sulfate, 0.001% sodium molybdate, cobalt chloride, manganese chloride, and sodium selenate, and 0.5% of the B vitamin solution of 74 Varel and Bryant (31), which also contained 0.01% flavin mono- and adenine dinucleotides (FMN) and (FAD). Molar growth yields were calculated from dry weight determinations of cells grown on media with varying glucose and fumarate concentrations. Specific components of each medium are given in the results section. Cells were grown in 400 m1 of each medium in a 500 ml round bottom flask. The inoculum for each medium was prepared as previously described (15) and duplicate flasks of each medium were inoculated. The cells were harvested batch- wise in a Sorvall RC-S refrigerated centrifuge and washed twice with distilled H20. The washed cells were dried to constant weight at 100°C. Unfermented glucose in the various media was determined as described by Dubois et a1. (10) and Johnson et al. (19) as modified by Montgomery et al. (24). Gas chromatographic analysis of organic acids and gas Volatile and non-volatile acids were extracted and analyzed, from cells grown in Hungate anaerobic tubes containing 10 m1 of medium for 22 h, in a Dohrmann gas chromatograph as described by Holdeman and Moore (17). A Hewlett Packard 5700 A gas chromatograph was used to detect H2 production. The gas in the head space of the tube, 0.2 cc, was used for injection. The oven and detector temperatures were 50°C and 100°C, respectively. The flow rate of the carrier gas (argon) was 60 cc/min. 75 The quantitation and identification of acids and gases were based on the comparison of peak heights and retention times with that of standard concentrations of acids and gases. Each sample was injected at least twice in order to determine a mean peak height value. Adenosine triphosphatase (ATPase) assay The procedures for assaying ATPase activity were similar to those described by Pullman et al. (25). The assay mixture contained 25 umoles of N-Z-hydroxyethyl- piperazine-N-Z-ethanesu1fonic acid (HEPES) buffer, pH 7.6, 1 umole of magnesium chloride, 2 umoles of ethylene diaminetetraacetic acid (EDTA), 7 umoles of phosphoenol- pyruvate (PEP), 7 umoles of ATP, 14 ug of pyruvate kinase, and 5 mg of B. fragilis extract in a volume of 2.8 m1. All reactions were done at 37°C for 20 min and stopped by adding 1 m1 of 10% TCA. After centrifugation, the inor- ganic phosphate present was determined by the procedures of Fiske and Subbarow (12) as modified by Clark (6). ATP determination by the hexokinase assay The hexokinase assay (28) was one method employed to determine if phosphorylation of ADP to ATP is coupled to electron transfer from H2 to fumarate in cell extracts of B. fragilis. Hexokinase catalyzes the production of D-glucose-6-phosphate and ADP from D-glucose and ATP (28). The consumption of H2 was measured by standard mano- metric techniques as described previously (30). Inorganic phosphate, ADP, and Z-deoxy-D-glucose were present in one 76 sidearm. An analog of glucose, Z-deoxy-D-glucose, was used in the assay since B. fragilis metabolizes glucose but not 2-deoxy-D-glucose. Fumarate and the uncoupler, pentachlorophenol (PCP) (suSpended in ethanol), were present in the second sidearm, where indicated. Bacteroides fra- gilis and CZostridium pasteurianum extracts, HEPES buffer, FAD or FMN, magnesium chloride (MgClz), sodium fluoride (NaF), mercapto-ethanol, hexokinase, and bovine serum albumin (BSA). The BSA used in the assay was treated with norite by the method of Chen (5), for the purpose of removing fatty acid impurities. The center well contained 0.2 m1 of freshly prepared 2-% KOH absorbed onto a fluted filter paper. The reaction was started by tipping the components from the sidearm into the main vessel. The concentration of inorganic phosphate and that remaining after the completion of the reaction were determined in each cup (6,12). A decrease in the inor- ganic phosphate concentration after completion of the reaction in the experimental cup as compared to that in the control cup containing the uncoupler PCP or in the presence of a N2 instead of a H2 gas phase was used as an index of the Pi esterified. The concentration of inor- ganic phosphate in the sample was calculated by comparing the absorbance values to those of a standard containing 0.1—0.6 umoles of inorganic phosphate treated the same way as the sample (6). 77 The formation of Z-deoxy-D-glucose-6-phosphate, a product of the hexokinase assay, was measured spectro- photometrically by taking a known volume of the completed reaction from the manometric assay and observing an increase in absorbance at 340 nm in the presence of NADP+ and g1ucose-6-phosphate dehydrogenase (8). ATP determination by the modified luciferifi¥Iuciferase assay The modified luciferin-luciferase assay (7,23,26,27,28) was also used to determine if ATP is produced in fumarate reduction by H2 in cell extracts of B. fragilis. HZ consumption was measured manometrically as described previously (15). The components of the reaction mixture for the luciferase assay were the same as those for the hexokinase assay except Z-deoxy-D-glucose and hexokinase were deleted and 2.5 umoles of ADP and 10 umoles of inorganic phosphate were added. The perchloric acid method described by Cole et a1. (7) and Roberton and Wolfe (26) was used to extract ATP from the unknown samples and the ATP standards. A Z-ml sample was mixed with 0.5 ml of cold 30% (v/v) perchloric acid (7). After standing for 3 h at 4°C, the suspension was neutralized with 4 N KOH and centrifuged in a clinical centrifuge (26). The supernatant was used immediately to determine the presence of ATP or stored at -20°C (26). Firefly lantern extract in arsenate-magnesium buffer (Sigma Chemical Co., St. Louis, MO) was suspended in 5 ml of H20 and activated by procedures of Kimmich et a1. (20). 78 The suspension was mixed with 80 mg of calcium phosphate (tribasic) and kept at room temperature for 10 min. Following centrifugation at 400 xlg for 2 min, the super- natant was treated with Ca3(P04)2 again and allowed to stand at room temperature for an additional 10 min. The suspension was then centrifuged at 18,000 x g for 10 min and the supernatant was used as the activated firefly lantern extract. The ATP present in the ATP standards and unknown samples was assayed in a Searle Delta 300 liquid scintil- lation counter operated at ambient temperature. Standard scintillation vials contained 2.7 m1 of 5 mM sodium arsenate, 4 mM MgSO4, and 20 mM glycyl-glycine, pH 8.0, 0.15 ml of the sample, and 0.15 ml of the activated fire- fly lantern extract to the scintillation vials. Approxi- mately 10 sec after adding and mixing the firefly lantern extract, the sample was counted for 4 consecutive periods of 30 sec each. Chemicals and enzymes The firefly lantern extract, BV, FMN, NAD+, NADH, ATP, ADP, 2-deoxy-D-glucose, and 2-deoxy-D-glucose-6- phosphate were purchased from the Sigma Chemical Co. (St. Louis, MO). Glucose-6-phosphate dehydrogenase, hexokinase, and pyruvate kinase were purchased from the Sigma Chemical Co. (St. Louis, MO). Methylene blue (MB) was purchased from the Allied Chemical Co. (New York, NY). All other chemicals were of reagent grade or higher quality. 79 RESULTS Molar growthyyields as affected by various fumarate concentratibns From the data presented previously (15, Section 2), the Hz-fumarate coupling activity was demonstrated in cell extracts of B. fragilis. The calculated free energy change for the oxidation of H2 with fumarate is more than sufficient to account for the formation of at least one ATP. If indeed fumarate is involved in energy generation in B. fragilis, then this should be reflected by an increase in cell yield of cells grown with fumarate present in the medium. Experiments were designed to determine if additional energy for growth is available when fumarate is reduced to succinate. Table 1 shows the effect of various fumarate concentrations on the growth of B. fragilis. The molar growth yields in Med. #1 and #2, which contained substrate quantities of glucose, were essentially the same regardless of the additional fumarate in Med. #1. Media #3, #4, and #5 had 0.05% glucose and various quantities of fumarate present. There was an increase of 25 g of cells per mole of glucose in Med. #3 as compared to Med. #5. These results suggested that fumarate reduction to succinate may be a source of additional ATP when the glucose concentration is limiting in the medium. 80 Table 1. Molar growth yields as affected by fumarate concentration in the medium Mediuma % Glucose % Fumarate wt (g) Ymb 1 0.400 0.20 .380 44.19 2 0.400 0.00 .399 46.39 3 0.050 0.20 .111 123.30 4 0.050 0.05 .105 116.10 5 0.050 0.00 .089 98.80 aMed. #1 is the basal medium described previously (15). Media 2-5 are the same as Med. #1 except for the varying glucose and fumarate concentrations. b Ym = grams dry weight/mole of glucose. 81 The effect of added fumarate on acids and gas production From the results shown in Table 1, the molar growth yields from cells grown with substrate quantities of glucose (Med. #1 and #2) were essentially the same regardless of the additional fumarate in Med. #1. Experiments were then designed to determine the effect, if any, of added fumarate on acid end products produced by cells on a medium that contained fumarate (Med. #1) and a medium that contained no fumarate (Med. #2). The results (Table 2) showed that in Med. #1, approximately equal quantities of acetate and succinate were produced while in Med. #2, there was a four-fold increase in succinate as compared to acetate production. The lower quantities of H2 detected in Med. #1 as compared to that in Med. #2 may be due to the use of H2 in Med. #1 for the reduction of additional fumarate to succinate. ATPase activity ATPase activity was examined in cell extracts of B. fragilis since it is usually considered to be an expression of the same enzyme that is involved in the 2+-stimulated production of ATP. As shown in Table 3, A Mg ATPase activity was demonstrated. The addition of NaF and the deletions of PEP and pyruvate kinase caused a 30% and 45% decrease in the ATPase activity, respectively. EDTA had no apparent effect on the enzymatic activity. Cell membrane, ATP, and Mg++ were essential components 82 Table 2. The effects of exogenous fumarate on hydrogen, acetate, and succinate production in B. fragilis _—.—- a b Acetate Succinate Medium H2 umoles umolesC umolesC Med. #1 2.90 127 155 Med. #2 6.34 25 102 aMed. #1 is the same as the basal medium described previously (15). Med. #2 is the same as Med. #1 except that fumarate has been deleted. sz production was measured by gas chromatographic techniques described in the Materials and Methods section. The numbers represent the total amount of H2 produced per tube. CAcetate and succinate production was measured by gas chromatographic techniques described in the Materials and Methods section. The numbers represent the amount per tube. 83 Table 3. Requirements for ATPase activity8 Reaction mixture Activityb Complete 1.52 plus NaF 1.06 minus EDTA 1.67 minus pyruvate kinase 0.84 minus PEP 1.06 minus membrane 0.23 minus ATP 0.68 minus Mg++ 0.61 minus Mg++, plus Na+ 0.76 minus Mg++, plus K+ 0.84 aThe composition of the complete reaction mixture is given in the Materials and Methods section. The membrane protein concentration was 5 mg. bActivity expressed as umoles of Pi liberated per 20 min per mg of membrane protein. 84 to demonstrate ATPase activity. No appreciable ATPase activity was observed on substitution of Na+ and K+ for ++ Mg Attempts to demonstrate phosphorylation coupledito fumarate reduction by HE_ Experiments were designed to determine directly if phosphorylation of ADP to ATP occurs during fumarate reduction to succinate by H2 in cell extracts of B. fragilis. Results obtained with the 2-deoxy-D-glucose hexokinase trap are presented in Table 4. In the complete assay and likewise in the complete assay without ADP, P04, and ADP and hexokinase, hydrogen consumption was readily detected in the presence of fumarate. Minimal levels of hydrogen were consumed when FAD, C. pasteurianum extract, or B. fragiZis extract was deleted from the com- plete assay. Deletion of fumarate or substitution of N2 for H2 resulted in no hydrogen consumption. There was, however, no detection of esterification of orthophosphate or 2-deoxy-D-g1ucose-6-phosphate, a product of the 2-deoxy-D-g1ucose hexokinase trap, in the complete reac- tion. As shown in Table 5, attempts to demonstrate phosphorylation of ADP to ATP coupled to fumarate reduc- tion with the particulate or soluble fraction were unsuccessful. Attempts to demonstrate ATP production during fuma- rate reduction with NADH as the electron donor were done using the 2-deoxy-D-glucose-hexokinase trap. The calcu— lated free energy change for fumarate reduction by NADH 85 Table 4. Attempt to demonstrate phosphorylation coupled to fumarate reduction by H2 using the hexokinase assay Pi 2-deoxy-D- H esteri- glucose 6- a oxidized fied phosphate Reaction mixture (umoles) (umoles)b (umoles)C Complete 16.50 0.0 0.0 minus fumarate 0.0 minus ADP 17.60 minus P04 14.30 minus ADP, hexo- 16.80 kinase minus FAD 1.86 minus C. pasteuri- 0.98 anum extract minus B. fragilis 1.30 extract Complete--N2 gas 0.0 aThe complete reaction contained 50 umoles of HEPES buffer (pH 7.6), 40 pmoles of MgClz, 50 umoles of NaF, 20 umoles of P04 (pH 7.0), l umole of FAD, 1 umole of ADP, 100 umoles of 2-deoxy-D-g1ucose, 3O umoles of sodium fumarate (pH 6.5), 5 mg of BSA, 0.1 mg of hexokinase, 3 mg of C. pasteurianum extract, and 5 mg of B. fragiZis extract in a total volume of 2.8 ml. The reaction was done at 37°C for 20 min with a H2 gas phase, unless other- wise specified. bCalculated from the difference in esterification between each reaction and the one with the uncoupler pentachlorophenol (6 x 10'4 M) present. CDetermined spectrophotometrically. 86 Table 5. Attempt to demonstrate phosphorylation coupled to fumarate reduction by Hz with various cell fractionsa Hz oxidized Pi esterified Cell fraction (umoles) (umoles) A. 12,100 x g 12.50 0.0 Supernatant B. 104,000 x g 2.98 0.0 Supernatant C. 104,000 x g 19.98 0.0 Pellet D. B + C 16.3 0.0 aReaction conditions were the same as those given in Table 4, except that 10 mg of the various cell frac- tions were present. The reaction was done for 10 min. 87 is more than sufficient to account for the formation of at least one ATP. In these experiments C. pasteurianum extract and FAD or FMN, which substituted for the oxygen labile electron carrier in B. fragilis, were deleted. The primary objective of this experiment was to avoid the site of the oxygen labile, low potential electron carrier that is apparently involved in the HZ-fumarate electron transport system. Bacteroides fragilis can reduce fumarate by NADH directly without any additional electron mediators (Section 2). Direct evidence for ATP produc- tion during fumarate reduction by NADH, however, was not detected. The luciferin-luciferase assay was a second inde- pendent method used to attempt to demonstrate direct phosphorylation of ADP to ATP. There was no detection of ATP in the complete assay with cell extracts of B. fragiZis (Table 6). DISCUSSION Indirect evidence for phosphorylation coupled to fumarate reduction has been obtained in several organisms from molar growth yield studies (16,18,21,22). Anomalous growth yields have been reported in the facultative anaerobe Proteus rettgeri (21), in the propionic acid bacteria Propionibacterium freudenreichii and Propioni- bacterium pentaosaceum (9), in the rumen anaerobes Selenomonas ruminantium and Anaerovibrio Zipolytica (16,18), and the human Bacteroides species B. fragiZis (22). 88 Table 6. Attempt to demonstrate phosphorylation coupled to fumarate reduction by H2 using the luciferin- luciferase assay a H2 oxidized ATP formed Reaction mixture (umoles) (umoles) Complete 8.13 0.0 minus fumarate 0.33 minus ADP 9.20 minus P04 8.43 minus FMN 2.13 minus C. pasteurianum 1.14 extract minus B. fragilis 0.27 extract Complete--N2 gas 0.0 aThe reaction conditions were the same as those given in Table 4, except that 2-deoxy-D—glucose and hexokinase were deleted and 10 umoles of P04 and 2.5 umoles of ADP were added. The samples were extracted with perchloric acid (7) and assayed in a Searle Delta 300 scintillation counter. 89_ Macy and her colleagues (22) calculated that 4.5 ATPs per mole of glucose were produced by B. fragilis on a hemin medium as compared to 1.7 ATPs per mole of glucose on a hemin deficient medium. The major acid end products produced by the cells on the hemin medium were acetate, propionate, and succinate as compared to acetate, lactate, and fumarate on a hemin-deficient medium (22). Macy et al. (22) interpreted these results to suggest that pro- pionate and succinate production may be involved in additional energy production in B. fragilis. In the present study, more direct evidence for the involvement of fumarate in additional energy production was presented. From a molar growth yield value of 44.19 g/mole of glu- cose (Table l) and a YATP of 10.5 (3), 4.2 ATPs/mole of glucose were made by B. fragilis. Higher molar growth yields obtained on a hemin medium supplemented with fumarate indicated that additional energy is probably produced when fumarate is reduced to succinate in B. fragilis. In the present investigation, the addition of fumarate to modifications of the basal medium was found to affect the molar growth yields. When substrate quan- tities of glucose (Med. #1 and #2, Table l) were present, the molar growth yields were approximately the same regardless of the fumarate concentration. However, dif— ferent quantities of the acids and hydrogen gas were detected in the media. If additional energy were produced from fumarate reduction in Med. #1, it may not have been 90 reflected in an increase in cell yield, but rather in the increased quantities of acetate and succinate produced by the cells on Med. #1 as compared to Med. #2. The lower quantity of H2 detected in Med. #1 as compared to Med. #2 may have been due to the use of H2 in the reduction of the exogenously added fumarate to succinate. When cells were grown on media with limiting glucose and high fuma- rate concentrations (Med. #3, Table 1), a 20% increase was observed in the cell yield when compared to growth of the cells on media with limiting glucose and lower fuma- rate concentrations (Med. #4 and #5, Table 1). Again, these data implied that additional ATP is generated from fumarate reduction. These observations are consistent with the calculated free energy change (-20.4 Kcal/mole) for H2 oxidation with fumarate, which should be sufficient to allow for the formation of at least one ATP in B. fragilis. ATP production from the transfer of electrons from H2 to fumarate may occur at the flavoprotein and/or cytochrome b site(s). Divalent cation stimulated ATPases, particularly Mg++ or Ca++, have been shown to be generally associated with phosphorylation of ADP to ATP (1,2,4,13,14). ATPases stimulated by monovalent cations such as Na+ or K+ have been usually associated with active transport (4,13,14). Mg++ or Ca++ stimulated ATPase activity associated with phosphorylation during fumarate reduction has been demon- strated in Desquovibrio gigas (1,2,13), Escherichia coli (4,14), and Vibrio succinogenes (C. A. Reddy and H. D. 91 Peck, Jr., Abst. Annu. Meet. Amer. Soc. Microbiol., 1973, 194). In the present investigation, ATPase activity was detected in cell extracts of B. fragilis. The monovalent cations Na+ or K+ did not stimulate the ATPase activity, which suggested that the ATPase is not involved in active transport in B. fragilis. Direct evidence for electron transport phosphoryla- tion coupled to fumarate reduction has been demonstrated in several organisms that reduce fumarate to succinate with either H or NADH (1,2,11, C. A. Reddy and H. D. 2 Peck, Jr., Abst. Annu. Meet. Amer. Soc. Microbiol., 1973, 194; C. A. Reddy, Rumen Function Conference, Chicago, 1973, unpublished). Barton et al. (1,2) have demonstrated phosphorylation of ADP to ATP when fumarate is reduced to succinate by H2 in cell extracts of D. gigas. Similar results have been obtained by C. A. Reddy and H. D. Peck, Jr. (Abst. Annu. Meet. Amer. Soc. Microbiol., 1973, 194) in V. succinogenes. Direct evidence for ATP formation coupled to fumarate reduction by NADH in cell extracts of Streptococcus faecalis and Bacteroides ruminicoZa has been presented by Faust and Vandemark (11) and C. A. Reddy (1973, Rumen Function Conference, Chicago, unpub- lished), respectively. Efforts to demonstrate direct evidence for ATP pro- duction during fumarate reduction in cell extracts of B. fragiZis have been unsuccessful to date in the present investigation. Barton et al. (1,2) and C. A. Reddy and H. D. Peck, Jr. (Abst. Annu. Meet. Amer. Soc. Microbiol., 92 1973, 194) employed isotOpic and chemical procedures of the glucose-hexokinase trap to demonstrate esterification of orthophosphate during fumarate reduction in D. gigas and V. succinogenes. Using chemical procedures of the 2-deoxy-D-glucose hexokinase trap in the present study, phosphorylation during fumarate was not detected in B. fragilis. Attempts to show ATP formation coupled to fumarate reduction by the luciferin-luciferase assay were also unsuccessful. The luciferin-luciferase assay was chosen as a second independent means to attempt to demonstrate ATP production from fumarate reduction because of its extreme sensitivity (7,23,26,27,28). The assay can detect nanomole or picomole quantities of ATP (7,23,26, 27,28). Direct evidence for phosphorylation of ADP to ATP during fumarate reduction in B. fragiZis was not obtained, probably due to the lack of coupling factors or some essential coupling proteins which may be extremely sensi- tive to oxygen and therefore destroyed during the frac- tionation procedures. The lack of an essential trace element may also have been a factor in being unable to demonstrate ATP formation during fumarate reduction. Turner and Stadman (29) have shown the requirement of selenium for a protein that is involved in ATP production in the glycine reductase system of CZostridium sticklandii. In this study, however, trace quantities of several com- pounds including selenium and molybdenum were added to 93 basal medium in all experiments designed to demonstrate phosphorylation in B. fragilis. In the present investigation, the assays employed would have only detected phosphorylated compounds as an index of energy production from fumarate reduction in cell extracts of B. fragilis. It may be possible that the high energy compound is a non-phosphorylated com- pound such as an acyl thioester and therefore it would not have been demonstrated in the assay procedures used. It is also possible that pyrophosphate (PPi) may actually have been produced instead of ATP from fumarate reduction and therefore it would not have been detected in the assays. Additional energy production from fumarate reduction in B. fragilis may be utilized for a favorable conformational change in the membrane and not for the synthesis of a high energy compound. Additional studies, of course, are needed to investigate these possibilities. LITERATURE CITED Barton, L. L., J. LeGall, and H. D. Peck, Jr. 1970. 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