Phosphorylating Enzymes Involved in Glucose Fermentation of ...

JOURNAL OF BACTERIOLOGY, Oct. 1995, p. 5806–5811 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 177, No. 20

Phosphorylating Enzymes Involved in Glucose Fermentation of Actinomyces naeslundii NOBUHIRO TAKAHASHI,1* SOTIRIOS KALFAS,2

AND

TADASHI YAMADA1

Department of Oral Biochemistry, Tohoku University School of Dentistry, Sendai 980, Japan,1 and Department of Oral Microbiology, Umeå University, S-901 85 Umeå, Sweden2 Received 24 May 1995/Accepted 17 August 1995

Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucosegrown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phosphoglycerate kinase, pyruvate kinase, and acetate kinase coupled GDP as well as ADP, but Pi compounds were not their phosphoryl acceptor. Cell extracts showed GDP-dependent activity of phosphoenolpyruvate carboxykinase, which assimilates bicarbonate and phosphoenolpyruvate into oxaloacetate, a precursor of succinate. Considerable amounts of GTP, polyphosphate, and PPi were found in glucose-fermenting cells, indicating that these compounds may serve as phosphoryl donors or acceptors in Actinomyces cells. PPi could be generated from UTP and glucose 1-phosphate through catalysis of UDP-glucose synthase, which provides UDP-glucose, a precursor of glycogen. Actinomyces naeslundii (formerly A. naeslundii and Actinomyces viscosus [18]) is one of the predominant bacteria in the oral microflora (10, 12). These bacteria are among the pioneer microbial species on tooth surfaces (28) and have been implicated in oral diseases, such as dental caries (3, 11, 45) and gingivitis (25, 31, 32). Actinomyces cells metabolize carbohydrates to organic acids and can also accumulate intracellular polysaccharides. These properties are considered to be related to the cariogenic potential of these bacteria (22). Although carbohydrates are a main energy source, only limited information is available on their metabolism by A. naeslundii. Since the studies of Buchanan and Pine (5), it was generally accepted that Actinomyces cells degrade carbohydrates through the Embden-Meyerhof-Parnas pathway and can also assimilate bicarbonate into oxaloacetate, a precursor for succinate formation in this bacterial genus (4, 47, 48). In a recent study on the initial sorbitol catabolism by some selected oral isolates of A. naeslundii (20), we found that these bacteria had a hexokinase activity that catalyzed the phosphorylation of fructose to fructose 6-phosphate in the presence of GTP. The enzyme activity appeared essential for sorbitol metabolism by these strains since they lacked a phosphotransferase system for sorbitol (20). These findings prompted us to further study the metabolism of glucose by Actinomyces cells, especially the activities of enzymes involved in energy turnover.

inoculation), washed twice with 40 mM potassium phosphate buffer (pH 7.0) containing 5 mM MgCl2, and then stored as cell pellets at 2808C until use. To prepare permeabilized cells, thawed cell pellets were suspended in 50 mM potassium phosphate buffer (pH 7.0) at a concentration of 10 mg (dry weight)/ml and mixed vigorously with 0.03 volume of toluene for 1 min at 48C. The cells were then washed twice with potassium phosphate buffer and suspended in the same buffer. For cell extracts, the cell pellets were thawed and suspended in 40 mM potassium phosphate buffer (pH 7.0) containing 5 mM MgCl2 and 1 mM dithiothreitol. The cells were disrupted by sonic oscillation for 10 min at 48C (2 A, 200 W; Insonator, Kubota, Japan), and the cell extracts obtained after centrifugation (15 min, 48C, 10,000 3 g) were dialyzed for 2 h at 48C against the same buffer. The protein content of the extracts was estimated with a dye-binding method (protein assay; Bio-Rad Laboratories, Richmond, Calif.) using bovine serum albumin as the standard. Assays of enzymatic activities. Glycolytic enzymes except acetate kinase (EC 2.7.2.1 and EC 2.7.2.12) were assayed photometrically (340 nm) at 358C by reactions coupled to NADP reduction or NADH oxidation. When measured as NADH oxidation, the activities were corrected for contaminating NADH oxidase activity of the sample. The reaction mixture for glucokinase (EC 2.7.1.1 and EC 2.7.1.63) contained 5 mM MgCl2, 1 mM glucose, 1 mM NADP, 0.1 U of glucose6-phosphate dehydrogenase (yeast) per ml, and cell extract in 50 mM Tris-HCl buffer (pH 7.2). The reaction was started by the addition of 0.5 mM phosphoryl donors, ATP, GTP, ITP, CTP, UTP, phosphoenolpyruvate (PEP), acetyl phosphate, PPi, tripolyphosphate (T5633; Sigma, St. Louis, Mo.), and polyphosphate (PPn) (PP15 [mean number of polymerized Pis 5 15] [S6003; Sigma] and PP65 [mean number of polymerized Pis 5 65] [S6253; Sigma]). The reaction mixture for phosphofructokinase (EC 2.7.1.11 and EC 2.7.1.90) contained 5 mM MgCl2, 1 mM fructose 6-phosphate, 0.15 mM NADH, 0.1 U of aldolase (rabbit muscle) per ml, 5 U of triose phosphate isomerase (rabbit muscle) per ml, 0.3 U of glycerol-3-phosphate dehydrogenase (rabbit muscle) per ml, and cell extract in 50 mM Tris-HCl buffer (pH 7.2). The reaction was started by the addition of 0.5 mM phosphoryl donors. Phosphoglycerate kinase (EC 2.7.2.3) was measured from the formation of 1,3-diphosphoglycerate (55). The reaction mixture contained 20 mM 3-phosphoglycerate, 5 mM MgCl2, 83 mM EDTA, 3 mM cysteine, 0.15 mM NADH, 4 U of glyceraldehyde-phosphate dehydrogenase (rabbit muscle) per ml, and cell extract in 100 mM Tris-HCl buffer (pH 7.2). The reaction was started by the addition of phosphoryl donors. Acetate kinase was assayed colorimetrically as the formation of acetyl phosphate from acetate and phosphoryl donors (33). The reaction mixture for pyruvate kinase (EC 2.7.1.40) contained 5 mM MgCl2, 75 mM KCl, 0.15 mM NADH, 3 U of lactate dehydrogenase (rabbit muscle) per ml, cell extract, and 1 mM phosphoryl acceptor (ADP, GDP, IDP, CDP, UDP, Pi, tripolyphosphate, PP15, or PP65) in 50 mM Tris-HCl buffer (pH 7.2). The reaction was started by the addition of 1 mM PEP. To some mixtures, 0.5 mM AMP, acetyl coenzyme A, glucose 6-phosphate, fructose 1,6bisphosphate, or ribose 5-phosphate was added to serve as an activator of pyruvate kinase. The reaction mixture for the assay of pyruvate:Pi dikinase (EC 2.7.9.1) was as for pyruvate kinase except that the phosphoryl acceptor was 1 mM AMP and the phosphoryl donor was 1 mM PPi. The reaction mixture for PEP carboxykinase (EC 4.1.1.32 and EC 4.1.1.49) contained 2 mM PEP, 50 mM

MATERIALS AND METHODS Permeabilized cells and cell extracts. A. naeslundii genospecies 1 ATCC 12104 and WVU 398A, A. naeslundii genospecies 2 WVU 627 and W1053, and A. viscosus ATCC 15987 were used. Strain ATCC 15987 was isolated from the mouth of a hamster, while the other strains are isolates from humans (18). Bacteria were grown in a phyton-peptone-based culture medium supplemented with 0.2% glucose under anaerobic conditions (10% H2 and 10% CO2 in N2) as described previously (48). The purity of the culture was regularly checked by microscopic examination of Gram-stained smears and by culturing on blood agar plates. Cells were harvested at the exponential phase of growth (15 to 18 h after

* Corresponding author. Mailing address: Department of Oral Biochemistry, Tohoku University School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Phone: 81-22-273-9344. Fax: 81-22-263-9867. 5806

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PHOSPHORYLATING ENZYMES OF ACTINOMYCES NAESLUNDII

NaHCO3, 0.15 mM NADH, 6 U of malate dehydrogenase (pig heart) per ml, and cell extract in 100 mM Tris-HCl buffer (pH 7.2). The reaction was started by the addition of 0.5 mM phosphoryl acceptors. The composition of the mixture for estimation of PEP carboxylase (EC 4.1.1.31) was as for PEP carboxykinase except that the phosphoryl donors were omitted. The reaction was started by the addition of NaHCO3. To some mixtures, 0.5 mM ATP, GTP, ITP, CTP, or UTP was added as an activator for PEP carboxylase. The activity of PEP carboxyphosphotransferase (EC 4.1.1.38) was determined by the method of Wood et al. (54). The reaction mixture for glucosephosphate isomerase (EC 5.3.1.9) contained 5 mM MgCl2, 1 mM NADP, 2 mM fructose 6-phosphate, 0.1 U of glucose-6phosphate dehydrogenase (yeast) per ml, and cell extract in 100 mM Tris-HCl buffer (pH 7.6). The reaction mixture for fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) contained 5 mM MgCl2, 0.1 mM NADH, 5 mM fructose 1,6-bisphosphate, 5 U of triosephosphate isomerase per ml, 1.6 U of glycerol-3-phosphate dehydrogenase per ml, and cell extract in 100 mM Tris-HCl buffer (pH 7.6). The reaction mixture for triosephosphate isomerase (EC 5.3.1.1) contained 0.15 mM NADH, 0.5 mM glyceraldehyde 3-phosphate, 1.7 U of glycerol-3-phosphate dehydrogenase per ml, and cell extract in 150 mM triethanolamine-HCl (TEA) buffer (pH 7.6). The reaction mixture for phosphoglyceromutase (EC 2.7.5.3) contained 5 mM MgCl2, 0.1 mM NADH, 2 mM ADP, 2 mM 3-phosphoglycerate, 2 U of enolase (rabbit muscle) per ml, 1.2 U of pyruvate kinase (rabbit muscle) per ml, 2.8 U of lactate dehydrogenase per ml, and cell extract in 100 mM Tris-HCl buffer (pH 7.6). The reaction mixture for enolase (EC 4.2.1.11) contained 5 mM MgCl2, 0.1 mM NADH, 2 mM ADP, 2 mM 2-phosphoglycerate, 1.2 U of pyruvate kinase per ml, 2.8 U of lactate dehydrogenase per ml, and cell extract in 100 mM Tris-HCl buffer (pH 7.6). Glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12) was assayed by the method of Duggleby and Dennis (9). The following enzymatic activities, related to nucleotide and PPi metabolism, were measured in the cell extracts from strains WVU 627 and ATCC 12104. Nucleoside-diphosphate (NDP) kinase (EC 2.7.4.6), nucleoside-triphosphate (NTP) pyrophosphatase (EC 3.6.1.8 and EC 3.6.1.19) and NTP:Pi phosphotransferase were estimated by measuring the formation of NTP from NDP and ATP, from nucleoside monophosphate (NMP) and PPi, and from NDP and PPi, respectively. To a mixture of 5 mM MgCl2, cell extract, and 75 mM TEA buffer (pH 7.0), the following compounds were added: 0.5 mM NDP (GDP, IDP, CDP, or UDP) and 0.5 mM ATP for NDP kinase activity; 0.5 mM NMP (GMP, IMP, CMP, or UMP) and 0.5 mM PPi for NTP pyrophosphatase activity; and 0.5 mM NDP (GDP, IDP, CDP, or UDP) and 0.5 mM PPi for NTP:Pi phosphotransferase. The mixture was incubated at 358C for 10 min and then mixed with perchloric acid (final concentration of 0.5 N). After 10 min at 48C, the mixture was neutralized with 5 M K2CO3 and centrifuged to remove denatured protein and precipitated salts. The supernatant was assayed for the concentration of NTP in the presence of 5 mM MgCl2, 0.5 mM glucose, 0.5 mM NADP, 0.02 mM adenosine (59)pentaphosphate (59)adenosine and 300 mM TEA buffer (pH 7.0). The reaction was started by sequential addition of 0.1 U of glucose-6-phosphate dehydrogenase per ml, 0.3 U of hexokinase (yeast) per ml, and 0.8 U of NDP kinase (bovine liver) per ml, and the formation of NADPH was monitored by a fluorescence spectrophotometer (model 650-40; Hitachi, Tokyo, Japan) with an excitation wavelength of 350 nm and an emission wavelength of 450 nm. The amount of NADPH formed after the addition of NDP kinase corresponded to the amount of NTP in the sample. ATP pyrophosphatase and ATP:Pi phosphotransferase activities were estimated from the formation of NADPH measured at 340 nm and 358C, after addition of 0.5 mM PPi to the reaction mixtures. For ATP pyrophosphatase, the mixture contained 5 mM MgCl2, 0.5 mM glucose, 0.5 mM NADP, 0.5 mM AMP, cell extract, 0.3 U of hexokinase per ml, and 0.1 U of glucose-6-phosphate dehydrogenase per ml in 100 mM TEA buffer (pH 7.0). For ATP:Pi phosphotransferase, the mixture was the same except that AMP was replaced by ADP. Activity of NMP kinase (EC 2.7.4.3 and EC 2.7.4.4) was determined by following the formation of ADP and NDP from ATP and NMP. The mixture contained 5 mM MgCl2, 0.5 mM ATP, 0.15 mM NADH, 1 mM PEP, 2 U of pyruvate kinase per ml, 3 U of lactate dehydrogenase per ml, and cell extract in 100 mM TEA buffer (pH 7.0). The reaction was started by the addition of 0.5 mM NMP (AMP, GMP, IMP, CMP, or UMP), and the decrease in NADH was monitored. NDP-glucose synthase (EC 2.7.7.9, EC 2.7.7.27, EC 2.7.7.33, and EC 2.7.7.34) was estimated from the formation of glucose 1-phosphate in the presence of NDP-glucose and PPi. The reaction mixture contained 0.5 mM NDP-glucose (ADP-glucose, GDP-glucose, CDP-glucose, or UDP-glucose), 0.5 mM NADP, 1 U of phosphoglucomutase (yeast) per ml, 0.1 U of glucose-6phosphate dehydrogenase per ml, and cell extract in 100 mM TEA buffer (pH 7.0). The reaction was started by the addition of 1 mM PPi, and the reduction of NADP was monitored. Inorganic pyrophosphatase (EC 3.6.1.1) was measured by the method of Josse (19). Measurement of intracellular glucose, glucose 6-phosphate, PPn, PPi, Pi, ATP, ADP, GTP, and GDP. The following experiments were performed under anaerobic conditions (10% H2 in N2) as described previously (48) unless otherwise stated. Cells of strains WVU 627 and ATCC 12104 were harvested at the exponential phase of growth and washed twice with 40 mM potassium phosphate buffer containing 5 mM MgCl2. The cells were suspended at a final concentration of 4 mg (dry weight)/ml in 4 mM potassium phosphate buffer (pH 7.0) containing 150 mM KCl, 5 mM MgCl2, and 4 mM NaHCO3. Three milliliters of the cell suspension was incubated at 358C for 4 min, and then 20 mM glucose was added.

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The acids produced from glucose were titrated at pH 7.0 with 50 mM KOH by a pH-stat (model AT-118; Kyoto Electronics, Kyoto, Japan). After 10 min, 1.0 ml of the suspension was filtered through a nitrocellulose membrane (0.22-mm pore size; Millipore, Bedford, Mass.). The cells retained on the membrane were washed rapidly (,10 s) by aspirating 2.0 ml of 150 mM KCl containing 5 mM MgCl2. The membrane with the cells was soaked into 1.0 ml of 0.6 N perchloric acid and vortexed vigorously at 48C to obtain cell extract. Following addition of 0.15 ml of TEA (1.0 M) and neutralization with 5 M K2CO3, the mixture was centrifuged (5,000 3 g, 48C, 10 min) to remove cell debris and precipitated salts. The clear filtrate was assayed for various intracellular metabolites except PPn in air. All the intracellular metabolites except PPi and Pi were measured by the NADPH or NADH coupled indicator system with a fluorescence spectrophotometer as described previously (46). Glucose was measured by the sequential addition of 0.1 U of glucose-6-phosphate dehydrogenase per ml and 0.3 U of hexokinase per ml to 1.0 ml of 100 mM TEA buffer (pH 7.0) containing 5 mM MgCl2, 2 mM ATP, 0.5 mM NADP, and the filtrate. The increase in NADPH after the addition of hexokinase corresponded to the amount of glucose. Glucose 6-phosphate was estimated as the increase in NADPH after the addition of glucose-6-phosphate dehydrogenase to 100 mM TEA buffer (pH 7.0) containing 5 mM MgCl2, 0.5 mM NADP, and the filtrate. Before the assay of ATP, ADP, GTP, and GDP, the filtrate was incubated for 30 min at 358C with 1 mM NADP, 0.2 U of glucose-6-phosphate dehydrogenase per ml, and 2 mM MgCl2 in 100 mM TEA buffer (pH 7.0) to remove glucose 6-phosphate, the concentration of which was much higher than those of nucleotides. The mixture was acidified by perchloric acid (final concentration of 0.3 N) to destroy formed NADPH and then neutralized with K2CO3. After precipitate was removed, the concentrations of ATP, GTP, and GDP in the solution were determined enzymatically by the methods of Keppler and Kaiser (21) except that the reactions for GMP assay was omitted and the increase in NADPH was monitored fluorophotometrically instead of photometrically. ADP was estimated from the amount of ATP converted from ADP after the addition of 1 mM PEP, 5 mM MgCl2 and 2 U of pyruvate kinase per ml in 100 mM TEA buffer (pH 7.0). The mixture was incubated at 358C for 30 min and then boiled for 2 min to inactivate the pyruvate kinase. The amount of ATP formed was estimated as described above. PPi was measured colorimetrically as PPi-molybdate complex (16). Pi was measured by the method of Fiske and SubbaRow (13). Intracellular PPn was isolated from the cells by following the procedure (steps 1 and 3 to 5) reported by Clark et al. (7). The amount of long-chain PPn isolated was estimated by measuring the metachromatic reaction with toluidine blue at 630 nm (14). Purchased PP65 preparation was used as the standard. The isolated PPn was also confirmed by measuring the shift of fluorescence of 49,6-diamidino2-phenylindole (DAPI) (50). Chemicals. Tripolyphosphate, PP15, PP65, NDP-glucose, fructose 2,6-bisphosphate, AP5A, and DAPI were obtained from Sigma. PPi, GTP, ITP, CTP, UTP, GDP, IDP, UDP, and glucose were obtained from Wako Chemical Industries, Tokyo, Japan. AMP, GMP, IMP, CMP, and UMP were obtained from Yamasa Co., Ltd., Choshi, Japan. Other reagents and enzyme preparations were purchased from Boehringer Mannheim GmbH, Mannheim, Germany.

RESULTS Enzymatic activities involved in the glycolytic pathway. Permeabilized Actinomyces cells could phosphorylate glucose in the presence of GTP or PPn, but not with PEP or ATP as the phosphoryl donor. The activities of GTP- and PP65-dependent glucose phosphorylation in strain WVU 627 were 3.37 and 2.81 mU/mg of dry cells, respectively. Those in strain ATCC 12104 were 3.50 and 1.24 mU/mg of dry cells, respectively. The cell extracts had glucokinase activity, and the enzyme utilized only GTP and PPn (Table 1). ITP supported only a slight activity in strain ATCC 12104. Phosphofructokinase utilized only PPi as a phosphoryl donor (Table 1). The activity was not affected by the addition of fructose 2,6-bisphosphate, AMP, cyclic AMP, or ammonium ion. Strains WVU 398A, W1053 and ATCC 15987 also showed activities of GTP- and PPn-dependent glucokinase and PPi-dependent phosphofructokinase (data not shown). Despite extensive examination of the cell extracts, no ATP-dependent activities of glucokinase and phosphofructokinase were found in any strain. The strains had activities of phosphoglycerate kinase, pyruvate kinase, PEP carboxykinase, and acetate kinase (Table 1), which couple substrate-level phosphorylation. Phosphoglycerate kinase utilized ATP, GTP, and ITP as the main phosphoryl donors. Both pyruvate and acetate kinases utilized all the nucleotides tested, although pyruvate kinase required AMP as an activator except for ADP-dependent activity. Fructose 1,6-

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TABLE 1. Activities of phosphorylating enzymes found in cell extracts of A. naeslundii Enzyme

Phosphoryl donor or acceptora

Sp act (mU/mg of protein)b in:

Sp act (mU/mg of protein)a in: Enzyme

WVU 627

ATCC 12104

Glucokinase

GTP ITP PP15 PP65

51.2 6 17.3 ND 81.8 6 0.3 74.5 6 17.3

167 6 100 7.8 6 1.1 268 6 126 224 6 132

Phosphofructokinase

PPi

958 6 391

717 6 115

Phosphoglycerate kinase

ATP

1,930 6 1,170 1,410 6 471

GTP CTP ITP UTP

812 6 471 2.5 6 0.8 34.8 6 11.8 1.5 6 0.4

661 6 364 ND 63.7 6 26.1 ND

ADP ADPc GDPc CDPc IDPc UDPc

67.1 6 11.0 102 6 3.9 119 6 30.3 99.3 6 22.6 82.6 6 12.7 56.6 6 12.5

235 6 216 351 6 178 254 6 36.9 237 6 15.7 208 6 7.3 162 6 28.8

PEP carboxykinase

ADP GDP IDP UDP

0.6 6 0.8 88.8 6 89.5 55.9 6 56.8 2.2 6 0.5

1.5 6 2.1 95.7 6 46.4 38.8 6 29.8 ND

Acetate kinase

ATP GTP CTP ITP UTP

1,270 6 248 976 6 965 811 6 144 751 6 148 705 6 157

1,840 6 864 1,590 6 728 1,170 6 538 976 6 392 875 6 347

Pyruvate kinase

TABLE 2. Activities of other enzymes involved in the EmbdenMeyerhof-Parnas and succinate pathways

Phosphoglucose isomerase Fructose-1,6-bisphosphate aldolase Triosephosphate isomerase Glyceraldehyde-phosphate dehydrogenase Phosphoglyceromutase Enolase PEP carboxylase With ATP as activator With GTP as activator

Other phosphoryl donors or acceptors were not utilized. Means 6 standard deviation obtained from three or more independent experiments. ND, not detected. c AMP was used as an activator.

bisphosphate, glucose 6-phosphate, ribose 5-phosphate or acetyl coenzyme A had no effect on the activity of pyruvate kinase. PEP carboxykinase utilized GDP and IDP mainly and showed only weak activity with ADP. Pyruvate:Pi dikinase was not found. Strains WVU 398A, W1053, and ATCC 15987 showed similar results (data not shown). All strains had the other glycolytic enzymes of the EmbdenMeyerhof-Parnas pathway as well as PEP carboxylase. The results from strains WVU 627 and ATCC 12104 are shown in Table 2. PEP carboxyphosphotransferase was not found in any strain. Enzymes related to nucleotide and PPi metabolism. The cells had NDP kinase and NMP kinase activities (Table 3). NDP kinase utilized all the nucleotides tested, whereas NMP kinase utilized mainly AMP and GMP. The cells had a high activity of UDP-glucose synthase with a low activity of ADP-glucose synthase (Table 3). A small activity of NTP:pyrophosphatase was found in the presence of AMP in the extracts from the strain WVU 627. NDP:Pi dikinase was not found. The cells had inorganic pyrophosphatase activity. Intracellular levels of glucose, glucose 6-phosphate, PPn, PPi, Pi, ATP, ADP, GTP, and GDP during glucose metabolism. Glucose, glucose 6-phosphate, PPn, PPi, Pi, ATP, ADP, GTP, and GDP were found in the cell extracts of both strains, after the cells had fermented glucose for 10 min (Table 4). The levels of ATP and GTP were higher than those of ADP and GDP, respectively.

ATCC 12104

4,200 6 1,600 1,910 6 1,100 8,940 6 2,820 970 6 120

100 6 12.0 1,960 6 761 6.57 6 3.38 16.5 6 8.93 18.6 6 12.9

2,420 6 1,780 5,480 6 2,290 3.02 6 2.55 15.7 6 8.02 42.8 6 23.2

a Means 6 standard deviation obtained from three or more independent experiments.

The rates of acid production were 97.7 and 35.2 nmol/min/mg of dry cells for strains WVU 627 and ATCC 12104, respectively. DISCUSSION The assays of glycolytic enzymes in the Actinomyces cell extracts (Tables 1 and 2) confirmed that Actinomyces cells have the Embden-Meyerhof-Parnas pathway for glucose catabolism

TABLE 3. Activities of enzymes involved in nucleoside phosphate metabolism and of inorganic pyrophosphatase Sp act (mU/mg of protein)a in: Enzyme (reaction catalyzed) WVU 627

ATCC 12104

134 6 34.6 32.0 6 10.3 42.2 6 20.9 60.2 6 25.7

159 6 57.0 56.4 6 16.9 104 6 41.6 86.6 6 27.7

NMP kinase (NMP 1 ATP N NDP 1 ADP) with: AMP GMP CMP IMP or UMP

1,430 6 331 29.8 6 6.15 ND ND

864 6 170 73.0 6 16.2 22.2 6 8.97 ND

NTP pyrophosphatase (NTP N NMP 1 PPi) with: AMP GMP, CMP, IMP, or UMP

4.71 6 3.26 ND

ND ND

ND

ND

1.11 6 0.10 123 6 107 ND

2.61 6 1.85 546 6 514 ND

23.3 6 7.28

24.5 6 6.78

a b

WVU 627

1,970 6 914 323 6 110 6,460 6 2,330 1,480 6 458

NDP kinase (NDP 1 ATP N NTP 1 ADP) with: GDP CDP IDP UDP

NTP:Pi phosphotransferase (NDP 1 PPi N NTP 1 Pi) with ADP, GDP, CDP, IDP, or UDP NDP-glucose synthase (NTP 1 glucose 1-phosphate N NDPglucose 1 PPi) with: ADP-glucose UDP-glucose GDP-glucose or CDP-glucose Inorganic pyrophosphatase (PPi 3 Pi 1 Pi)

a Mean 6 standard deviation obtained from three or more independent experiments. ND, not detected.


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TABLE 4. Concentrations of intracellular metabolites accumulated in the cells during glucose fermentation Metabolite or ratio

Glucose Glucose 6-phosphate PPn PPi Pi ATP ADP GTP GDP ATP/ADP GTP/GDP a b

Concn (nmol/mg [dry wt] of cells)a or ratio in: WVU 627

ATCC 12104

2.68 19.3 2.76b 1.96 29.0 3.62 1.52 0.55 0.24

4.54 9.73 4.26b 2.78 25.4 3.11 1.51 0.39 0.10

2.38 2.76

2.06 3.90

Mean obtained from two independent experiments. Value is in milligram per gram (dry weight) of cells.

(5), like most other saccharolytic bacteria. However, glucokinase and phosphofructokinase, catalyzing initial steps of glucose phosphorylation, did not utilize ATP as a phosphoryl donor. In most bacteria, the first step of glucose catabolism is the phosphorylation of glucose to glucose 6-phosphate by a glucokinase (hexokinase) with ATP as a phosphoryl donor. Glucose may also be phosphorylated by a phosphotransferase system with PEP as a phosphoryl donor. On the basis of the detection of a low glucose–PEP-phosphotransferase system activity in cells of A. viscosus grown in a continuous culture under glucose limitation, Hamilton and Ellwood predicted the existence of a nonphosphotransferase transport system for glucose in this bacterium (15). The strains used in this study phosphorylated glucose by a GTP- and PPn-dependent glucokinase. ATP- and PPn-dependent glucokinase activity has been found in some microorganisms, including Corynebacterium, Micrococcus, Mycobacterium, Nocardia, Propionibacterium, and Streptomyces spp. (23, 24, 35). To our knowledge, a GTP-dependent glucokinase has not previously been reported in any living cells (20). Glucose 6-phosphate was converted by a phosphoglucose isomerase into fructose 6-phosphate, which was further phosphorylated to fructose 1,6-bisphosphate by a phosphofructokinase. This phosphorylation was catalyzed by a PPi-dependent phosphofructokinase instead of the more usually found ATPdependent phosphofructokinase. Two types of PPi-dependent phosphofructokinase activities have previously been reported in other cells. The first type has been detected mainly in higher plants (42) and Euglena gracilis (30) together with ATP-dependent phosphofructokinase activity and is regulated by fructose 2,6-bisphosphate (52). The second type is found in Amycolatopsis (1), Bacteroides (26, 40), Entamoeba (39), Mycoplasma (36), Propionibacterium (2, 34), Trichomonas (29), and Spirochaeta (17) species. It is insensitive to fructose 2,6-bisphosphate and is not accompanied by ATP-dependent phosphofructokinase. Our results indicate that the Actinomyces enzyme may belong to the second type. Fructose 1,6-bisphosphate was degraded to PEP and finally turned to the end products lactate, formate, acetate, and succinate (Fig. 1). These pathways include four energy-yielding steps due to substrate-level phosphorylation, and the reactions are catalyzed by phosphoglycerate kinase, pyruvate kinase, PEP carboxykinase, and acetate kinase. These kinases phosphorylated GDP as well as ADP (Table 1), suggesting that GTP can be supplied for glucokinase by these reactions. The

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AMP-activating pyruvate kinase (Table 1) has been observed in several bacteria, including Escherichia coli (27), Rhodopseudomonas sphaeroides (43), and Alcaligenes eutrophus (53). However, it is not clear whether AMP is absolutely necessary for the phosphorylation of GDP, CDP, and IDP in Actinomyces cells, since there is a possibility that ADP is formed from AMP in the presence of GDP, CDP, or IDP in crude cell extracts. Besides conversion into pyruvate, PEP can also be converted into oxaloacetate by PEP carboxykinase or by PEP carboxylase in the presence of bicarbonate (Fig. 1). Further degradation of oxaloacetate leads to succinate formation, a metabolic characteristic of the genus Actinomyces. The Actinomyces PEP carboxykinase preferred GDP and IDP to ADP as phosphoryl acceptors for the reaction. This nucleotide specificity is typical for enzymes from animal sources. PEP carboxykinase in most bacteria, yeasts, and plants is usually coupled with phosphorylation of ADP to ATP (51). The Actinomyces PEP carboxykinase may be one of the very few bacterial enzymes requiring GDP and IDP, like the enzymes of Alcaligenes eutrophus (51) and Arthrobacter globiformis (44). The finding of GTP-dependent glucokinase, together with the existence of enzymes that supply GTP, leads to the idea that guanosine nucleotides function as main phosphoryl donors and acceptors in Actinomyces cells. However, the content of total adenosine nucleotide in Actinomyces cells was 7 to 10 times higher than those of guanosine nucleotide (Table 4), indicating the predominance of adenosine nucleotide as an energy currency. During glycolysis, the GTP/GDP ratio was as high as the ATP/ADP ratio, a finding indicating that GDP functions as a phosphoryl acceptor as well as ADP does in vivo. To operate the glycolysis smoothly, PPi has to be supplied continuously for the phosphofructokinase activity. Actinomyces strains in this study had UDP-glucose synthase that created PPi from glucose 1-phosphate and UTP with the formation of UDP-glucose, a precursor of glycogen synthesis (Table 3). Glucose 1-phosphate can be formed from glucose 6-phosphate, and UTP can be created through phosphorylation of UDP by glycolytic kinases (Table 1) or NDP kinase (Table 3). Dominance of UDP-glucose synthase activity as observed in Actinomyces cells is usually found in eukaryotes (8), while in prokaryotic cells, mainly ADP-glucose-dependent activity has been reported (37). In Propionibacterium cells, PEP carboxytransphosphorylase catalyzes the reaction PEP 1 Pi 1 HCO32 3 oxaloacetate 1 PPi and supplies PPi to PPi-dependent phosphofructokinase (38). A similar activity was not found in any of the Actinomyces strains tested. Neither could activities of pyruvate:Pi dikinase or PPi-dependent acetate kinase be detected, while the activity of ATP:pyrophosphatase was low. In growing bacteria, a large amount of PPi can be formed as a by-product of nucleic acid and protein syntheses. E. coli has inorganic pyrophosphatase activity to decrease the intracellular level of PPi since intracellular accumulation of PPi is toxic for the cells (6). In Actinomyces cells, besides inorganic pyrophosphatase (Table 3), coupling of PPi as a phosphoryl donor to glycolysis through phosphofructokinase is another way to remove PPi. This could salvage energy of PPi and give some advantage to Actinomyces cells for energy saving, possibly explaining the higher glucose-related growth yield of Actinomyces cells compared with that of Streptococcus cells (41). The findings of PPn formation and PPn-dependent glucokinase activity (Table 4) indicate that A. naeslundii can utilize PPn as an intracellular energy and phosphate reservoir and possibly as a metal ion chelator, as previously reported for other microorganisms (8, 23, 24). In the oral cavity, nutrients are supplied discontinuously and phosphate is provided mostly through saliva and tooth demineralization. When nutrients are

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J. BACTERIOL.

FIG. 1. Proposal of metabolic pathways for glucose in A. naeslundii. The numbers in circles indicate enzymes as follows: 1, glucokinase; 2, phosphofructokinase; 3, phosphoglycerate kinase; 4, pyruvate kinase; 5, PEP carboxykinase; 6, PEP carboxylase; 7, acetate kinase; and 8, UDP-glucose synthase. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; G1P, glucose 1-phosphate; FBP, fructose 1,6-bisphosphate; G3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3 DPG, 1,3-diphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate.

supplied in excess, Actinomyces strains may salvage inorganic phosphates from the environment and conserve the surplus of energy in PPn. A similar activity has been observed in a dental caries-associated Streptococcus strain (49). Since inorganic phosphate compounds such as PPn and PPi are considered more primitive as phosphoryl donors than are nucleotides (23), PPn- and PPi-dependent kinases such as those found in Actinomyces cells may preserve the primitive structure of phosphorylating enzymes. Moreover, Actinomyces cells had activities of GTP-dependent PEP carboxykinase and UDPglucose synthase, which are usually found in eukaryotes. Comparative studies of the phosphorylating enzymes among prokaryotes and eukaryotes may give valuable information on the evolution of phosphorylating enzymes and the phylogenetic position of the genus Actinomyces. ACKNOWLEDGMENTS S. Kalfas was an invited researcher of the Japan Society for the Promotion of Science. This study was supported in part by grant-in-aid

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