CO2. After utilization of CO2 was discovered the

Transcription

1 THE METABOLISM OF LABELED GLUCOSE BY THE PROPIONIC ACID BACTERIA1 HARLAND G. WOOD, RUNE STJERNHOLM, AND F. W. LEAVER Department of Biochemistry, Western Reserve University School of Medicine, and Department of Biochemistry, University of Pennsylvania School of Veterinary Medicine The propionic acid fermentation was one of the first bacterial fermentations from which phosphoglyceric acid was isolated (Stone and Werkman, 1937) and shortly thereafter it was demonstrated that phosphoglycerate, hexosediphosphate and glycerol phosphate are fermented to the usual end products (Wood et al., 1938). It therefore has been considered that glucose is metabolized by these bacteria through the reactions of the Embden- Meyerhof-Parnas scheme (EMP2). It also has been suggested that they possess a second pathway of glucose metabolism which is not inhibited by fluoride (Werkman et al., 1937; Wiggert and Werkman, 1939). However, Volk (1954) has presented evidence that the fluoride probably inhibited phosphatases and not enolase as had been assumed and that added phosphate esters are fermented only after they have been hydrolyzed to the free acid, in which form they penetrate the cell. When labeled glucose became available it seemed possible that the occurrence of pathways other than the traditional EMP pathway might be detected by determination of the distribution of C14 in the products of fermentation. Virtanen (1925) and Virtanen and Karstr6m (1931) have proposed that the propionic acid fermentation involves a cleavage to 2- and 4-carbon compounds and that succinate is formed from the latter. This mechanism originally was postulated because the yield of CO2 was low and this cleavage would account for the production of Buccinate without the accompanyiug formation of 1 This work was supported by grants from the Atomic Energy Commission under Contract No. AT-(30-1)-1320, from the Department of Health, Welfare and Education, Grant No. 3818, and by the Elisabeth Prentiss Fund, Western Reserve University. The C14 was obtained on allocation from the Atomic Energy Commission. 2 EMP will be used as an abbreviation for Embden-Meyerhof-Parnas, C, for three carbon compound, C-3 for carbon three of a compound. Received for publication March 7, 1955 CO2. After utilization of CO2 was discovered the proposal received little consideration since the necessity for the postulate was removed. However, the mechanism has never been disproved and in fact transketolase action on fructose phosphate represents a type of 2- and 4-carbon cleavage (Racker et al., 1954). In addition to the EMP type of cleavage there are at least two other cleavages which are now known to occur in bacteria. One occurs in the Leuconostoc mesenteroides fermentation (Gunsalus and Gibbs, 1952; Bernstein et al., 1955) and is represented in equation 1. C-C-C-C-C-C C02 + CHs.CH20H + COOH.CHOH.CH, (1) The second type of cleavage occurs with Pseudomonas sacchrophila (Entner and Doudoroff, 1952) and with Pseudomonas lindneri (Gibbs and DeMoss, 1954) and is illustrated in equation 2. C-C-C '-C-C C COOH.CO-CH, + OHC*CHOH.CH20PO,H2 (2) In these cleavages the metabolism of carbons 4, 5 and 6 would be expected to be similar to that of the EMP scheme but the metabolism of carbons 1, 2, and 3 would be different. C-1 becomes a methyl group of pyruvate in the EMP cleavage whereas in the cleavage with the pseudomonad it is C-3 that becomes the methyl. With L. mesenteroides the methyl of ethanol is from C-2. In addition to these cleavages there may be metabolism in bacteria via sedoheptulose and the transketolase and transaldolase reactions (Horecker, 1953; Racker, 1954). In preliminary studies with the propionic acid bacteria it was found, using labeled glucose, that the distribution of C14 in the products did not 510

2 1955] GLUCOSE METABOLISM BY PROPIONIC ACID BACTERIA 511 permit a clear indication of the type of cleavage that was occurring (Leaver and Wood, 1953). Therefore, in order to provide a better basis for the interpretation of the results, the pattern of C14 was determined in the products from labeled 3-carbon compounds. It was found (Leaver et al., 1955) with lactate-3-c14, lactate-2-c'4, pyruvate- 2_C14, and glycerol-2-c14 as substrates that the C14 activity from each Ca-labeled compound was almost equally randomized in the 2- and 3- carbons of the propionate, and that the activity of propionate was similar to that of the succinate. A small amount of activity appeared in the carboxyl carbons and in the CO2. However, the C14 in the acetate was not completely randomized; the substrates labeled at C-3 gave rise to acetate with the highest activity in the methyl group and those labeled at C-2 had highest activity in the carboxyl group. Accordingly, if three carbon intermediates labeled in the 2- or 3-positions were formed in the cleavage of labeled glucose the C14 would be at least partially randomized in all products, but the position with highest activity in the acetate would serve as an indicator of the position in the C3 intermediate which carried the greatest activity. Also, as judged from the results with carboxyl labeled lactate and pyruvate, if a carboxyl-labeled C3 compound were an intermediate, the propionate and succinate would be expected to be labeled almost exclusively in the carboxyl groups, and there would be very little activity in the acetate. Two different species of the genus Propionibacterium were used in this investigation: Propionibacterium arabinosum strain 34W and Propionibacterium shermanii strain 52W. The two species were selected as representing two extremes in the type of fermentation of glucose that is produced by propionic acid bacteria. Wood and Werkman (1936) found that the ratio of propionate to acetate was 7.82 to 14.7 with 34W and with 52W it was The ratio of C02 to acetate was 3.59 to 6.29 with 34W and 1.02 to 1.06 with 52W. It was suggested that this wide difference in ratios with the two species resulted from a fundamental difference in the mechanism of fermentation. The results obtained in the present study are not fully explained by any one of the cleavages discussed above. It is proposed that the observed results can be explained by the simultaneous occurrence of the EMP cleavage and of a second as yet undetermined cleavage. METHODS AND RESULTS The glucose-3,4ic14 was obtained from liver glycogen of rats which had received NaHC140O (Wood et al., 1945). The glycogen was hydrolyzed with 1.0 N sulfuric acid at 100 C for 3.0 hr and the sulfate was removed with barium. The glucose was chromatogrammed on filter paper. The glucose band was eluted and was diluted with glucose-c12. On degradation with Lactobacillu casei (Wood et al., 1945) 97 per cent of the C14 was in the 3- and 4-carbons. The glucose-1-c14, glucose-6-c14 and glucose- 2_C14 were obtained from the National Bureau of Standards, Washington, D. C. All the fermentations were carried out anaerobically with washed cell suspensions. For each experiment the bacteria were carried through three transfers with a 4 per cent inoculum on the following medium: 0.5 M K-phosphate buffer (ph 6.8), 5.0 g glycerol, 5.0 g yeast extract, 1.0 mg calcium pantothenate, 1.0 mg thiamin hydrochloride, and 0.2 mg biotin per liter. Growth was for 4 or 5 days at 30 C. The cells from the third transfer were washed twice with distilled water before use in the experiment. All experiments were at 30 C and were gassed continuously with N2 or He and the C02 was collected in 3 N alkali. The fermentation was stopped by addition of acid and the cells were removed from the reaction mixture by centrifugation and washed twice. The combined solutions were extracted with ether for 2 days.3 The waterether mixture in the extraction flask was steam distilled and the volatile acids in the ether-water distillate were titrated. The volatile acids were then separated on a celite column (Swim and Krampitz, 1954). The non-volatile acids in the residue of distillation were also titrated and separated on a celite column. After removal of the chloroform butanol, the fractions containing the succinate were oxidized with acid-kmno4 to destroy the lactate, and the succinic acid was then extracted from the solution by continuous ether extraction. The succinic acid was sublimed, 8 The recovery of volatile acids was not always quantitative because there was loss of volatile acids along with the ether during the extraction. This occurred when the condenser was not efficient, particularly during the summer when the tap water was warm. Water was added together with the ether in the receiving flask so that a larger part of the acids were partitioned into the water phase during the extraction.

3 512 WOOD, STJERNHOLM AND LEAVER [VOL. 70 the melting point determined, and the succinic Micrococcus lactilyticwu and the propionate was acid was weighed or titrated and diluted with separated on a celite column and degraded by the carrier. azide reaction. The propionic and acetic acids were degraded In some fermentations the residual glucose and by the azide reaction (Phares, 1951). The "non-reducing material" (Wood and Werkman, succinate was converted to propionate with 1934, 1940) were determined. The latter was TABLE 1 Fermentation of glucose-i -C4 and gluco8e-6-c14 by Propionibacterium Specific Activity in Per Cent of the Labeled Position of the Glucose Products/100 MI Medium Substrate Substrte and No. Culture Clture 5_CsKiXi Propionate Succinate Acetate co co UP CHs CH. COOH CH: COOH CH. COOH Id 8 mm meq mm mm mm Glucose-6-C"4 1 34W W W * Glucose-i-C Wt W$ W ' W * Part of sample lost. t No vitamins were added to the growth medium. t NaF was added to this fermentation. The 2- and 3-positions of the propionate were not separated, the value given is the average of the two positions. 9 hr. CO2 was 1.15 mm per 100 ml and its activity was No. 1: 7 hr 35 ml of glucose, m; phosphate buffer (ph 7.2), 0.15 M and cells 2.5 per cent in 125- ml Warburg flask. Gas phase N2. Alkali in center well. No. 1: 17 hr same conditions as 7-hr fermentation no. 1 except 25 ml of medium in Warburg flask. No. 2: 30 ml of glucose, m; phosphate buffer (ph 7.2), 0.15 m; and 5 per cent cells in 125-ml Warburg flask. Gas phase N2. Alkali in center well. No. 3: Same conditions as no. 1. No. 4: Same conditions as no. 2. No. 5: 60 ml of glucose, M; NaHCO,, M; phosphate buffer (ph 5.9), m; NaF, M; cells 5 per cent. All solutions except bicarbonate and bacteria were added to the 125-ml Warburg flask and it was made anaerobic with N2 when the bicarbonate and cells were added. CO2 was collected in alkali at end of fermentation. No. 6: Same conditions as no. 1. No. 7: Same conditions as no. 8, table 2, except 100 ml of reaction mixture were used. CO was collected at 9 and 29 hr, other products at 29 hr only.

4 1955] GLUCOSE METABOLISM BY PROPIONIC ACID BACTERIA TABLE 2 Fermentation of glucose-,4-c1' and glucose-2-c14 by Propionibacterium Specific Activity in Per Cent of the Labeled Position * Products per 100 of Glucose 9 MI Medium 513 Substrate and No. Culture Propionate Succinate Acetate l C02 &. 0 CHa CH2 COOH CHs COOH CHs COOH 0 mm m_q mm mm Glucose-2-Cl' 8 34W W t 19.4$ t 20.3t W W Glucose-3,4-CU 12 34W Wt t 5.95t W t 10.6t W t 4.88t W * The labeled position in the glucose-3,4-01' has one-half the total specific activity of the glucose. NaF was added to this fermentation. t 2- and 3-carbons were not separated. Value is the average. 9 hr CO2 was 1.00 mm per 100 ml and its activity was No. 8: 300-ml round bottom flask. Shaken and gassed with N2 continuously. CO2 collected in alkali and weighed. 120-ml reaction mixture containing 0.05 M glucose, 0.15 M phosphate buffer (ph 7.2), 2.5 per cent cells. 75 ml removed at 6 hr. Remainder fermented 16 hr more. No. 9: Same conditions as no. 8 except 180 ml of reaction mixture gassed with He, 100 ml removed at 6 hr. No. 10: Same conditions as no. 8. No. 11: Same conditions as no. 8 except gassed with He. No. 12: 30 ml of glucose, M, phosphate buffer (ph 7.2), 0.15 M and 5 per cent cells in 125-ml Warburg flask. Gas phase N,. Alkali in center well. No. 13: Same conditions as no. 5 of table 1. No. 14: Same conditions as no. 9. No. 15: Same conditions as no. 9. No. 16: Same conditions as no. 8 except 100 ml of reaction mixture were used. C02 was collected at 9 and 29 hr, other products at 29 hr only.

5 514 WOOD, STJERNHOLM AND LEAVER TABLE 3 Distribution of C14 in the fractions from the fermentation Per Cent Recovery of Added C4 Substrate and No. Culture Hours Residue CeUs EttO Propionate Acetate COs Succinate Total extract Glucose-i-C1 4 34W Glucose-2-C W Glucose-3,4-C W determined by the increase in reducing sugar after hydrolysis with 3.0 N HCl for 2.5 hr at 100 C. The method of Somogyi was used for determining the sugar. In other fermentations the total sugar was determined by the anthrone method (Koehler, 1952) and the decrease in total sugar is reported in the tables. For this determination a sample of the original cell suspension was hydrolyzed with 3.0 N HCl and the carbohydrate in the cells was determined; this together with the added glucose was considered the original total carbohydrate. At the conclusion of the fermentation an aliquot part of the mixture including the cells was hydrolyzed and the total carbohydrate was again determined and the decrease was calculated. All samples were treated with barium hydroxide and zinc sulfate prior to determination of the sugar. The original cells were found to contain about 20 mg of carbohydrate per g of wet weight of cells in the case of 34W and 50 mg per g of 52W. The degradation of each compound was checked for accuracy by comparing the sum of the activities of the individual fractions with the total activity of the compound as determined by total oxidation to CO2 with persulfate' (Osburn and Werkman, 1932) or with chromic acid (Van Slyke and Folch, 1940). The agreement was 4The persulfate oxidation was found to be unsatisfactory in the case of propionate. Apparently the 2-, and 3-positions of the propionate were not completely oxidized to C02 and for this reason the sum of the fractions by degradation did not equal the total activity of the compound as determined on the C02 of the total oxidation. [VOL. 70. usually within +3 per cent. All activities were determined on C02 gas by the method of Bernstein and Ballentine (1950). In a few fermentations the C14 in the cells and in the residue of ether extraction was determined after oxidation to C02 with chromic acid. The total C14 recovery was then calculated. The results are presented in tables 1, 2 and 3. DISCUSSION The values for the distribution of C14 in the products were obtained by determining the specific activity per,um of carbon in the given fraction and dividing it by the specific activity of the labeled position of the glucose and then expressing the results in per cent. This value is equivalent to the per cent of the carbon which has been derived from the labeled position in the glucose. It is clear from a survey of the tables that in no case was the carbon of any position derived exclusively from a labeled position of glucose. The highest values were the carboxyl groups of propionate and succinate with 3,4- labeled glucose (table 2). The situation is thus completely different from that with Leuconostoc mesenteroides, where almost 100 per cent of each position of a product is formed from a certain carbon of the glucose (Gunsalus and Gibbs, 1952; Bernstein et al., 1955). In the present experiments the C14 was converted to all positions of the products no matter which position of the glucose was labeled. From glucose-i-c14 and glucose-6-c4 (table 1) the activity in all of the carbons of the propionate, succinate, acetate and C02 was 15.7 or less with

6 1955] GLUCOSE METABOLISM BY PROPIONIC ACID BACTERIA 515 the exception of the CO2 of fermentations nos. 4 and 7. Since the average activity of the carbon of the original glucose would be 16.7 per cent (100/6) it is likely that a considerable part of the products was derived from carbon sources other than the added glucose. It is also possible that the C14 of the labeled position was converted differentially to some product other than the four compounds which were studied. There was a relatively rapid conversion of glucose to the "non-reducing material." In fermentation 1, for example, in 7 hr 5.22 of the 5.30 mm of glucose were fermented and 1.83 mm of "non-reducing material" were produced. Nothing is known about the structure of the "non-reducing material," and it is possible that there is preferential conversion of some positions of the glucose to this compound. There was substantial conversion of the glucose to cellular material. This is apparent from table 3 in which the distribution of C14 is shown in the fractions obtained from the fermentations. Fermentations 2, 4, 5 and 9 contained a 5.0 per cent wet weight suspension of cells, the others 2.5 per cent. In fermentation 4 there was 28.2 per cent of the C14 in the residual cells. The amount of C14 in the cells was much less at the shorter period of time than at the longer period. The C14 in the residue of ether extraction is probably in the form of unfermented glucose and "non-reducing material." The effects, if any, of endogenous metabolism and incorporation of C14 into cellular and "non-reducing material" on the distribution of isotope in the products are unknown, but in most instances when the fermentations were analyzed at two different times of incubation the distribution patterns were the same in the products. This was true even though much of the "non-reducing material" was formed early in the fermentation and then in some cases was partially fermented to propionate and other products. It is evident, however, that these unknown factors add to the uncertainty of interpretation of the data. The yields of volatile acids, succinate, CO2 are shown in tables 1 and 2 in order to provide some information on the amount and variation of products during the fermentation. The yield of succinate5 was not as high as found by Wood 5The accuracy of the degradation with a small amount of succinate was checked as follows. 10 M of succinate of known activity were added and Werkman (1940) with cell suspensions of Propionibacterium pentosaceum. In part this may be related to the fact that CO2 was continually removed from the medium in the present experiments (by gassing with nitrogen or helium) whereas in the earlier experiments NaHCO3 and CO2 were present in the medium. The presence of CO2 may favor succinate formation via CO2 fixation. The amount of propionate and acetate is not given in the tables because the chromatographic methods were not conducted in such a way as to be completely quantitative. In general, however, there appeared to be no difference in the ratio of propionate to acetate or of CO2 to acetate with 34W and 52W. The ratio of propionate to acetate varied from 2.2 to 6.0 for 34W and from 2.3 to 6.2 with 52W and of CO2 to acetate from 2.0 to 4.1 for 34W and 1.7 to 3.6 for 52W. The differences in ratios previously observed with the two species were obtained with proliferating cells (Wood and Werkman, 1936), and it is possible that similar differences do not occur with resting cells. There was, nevertheless, a difference in the isotope data from the two species. The isotope distribution pattern is complex and a complete explanation will not be attempted; however, the data form an important basis for future work and show many features which must be explained if the fermentation is to be comprehended fully. It is proposed that the observed distribution is a consequence of a simultaneous occurrence of the EMP type cleavage and a second unknown cleavage or cleavages. The divergence of the distribution of tracer in the to the chloroform butanol fractions obtained just prior to the succinate from a celite column of the non-volatile acids from a fermentation. The succinate was then carried through the steps of oxidation with KMnO4 ether extraction, rechromatographing, treatment with IR 100, titration and dilution with carrier succinate (71.3 times). It was degraded and the following values were obtained after correction for carrier dilution. COOH = 2.28, CH2 = 127.0, and CO2 of total oxidation = 64.3 (cpm./p&m of C) or per MM of succinate. The sum of the carboxyl and methyl groups = The total oxidation of the known succinate gave cpm per Mm of succinate. Thus the sample carried through the procedure gave 95 per cent of the original value. A similar procedure was used in fermentations 9, 11, 14 and 15 of table 2.

7 516 WOOD, STJERNHOLM AND LEAVER [VOL. 70 products from that predicted from the EMP pathway apparently are not accounted for by peculiarities in metabolism at the Cs level. This view is based on data from the study of fermentation of labeled C8 compounds (Leaver et al., 1955). The features which are explained by the EMP pathway will be considered first and then those features which are not explained will be considered in terms of our present knowledge of other pathways. The two species of pyruvate expected from the EMP cleavage are shown in equation 3. C* C0-C*-C*C CO C*H,sCOO*C@OOH (3) and COIE. CO * C*OOH From 1- and 6-labeled glucose, 3-labeled pyruvate would be formed; from 2-labeled glucose, 2-labeled pyruvate; and from 3,4-labeled glucose, 1-labeled pyruvate. The observed randomization of C14 into the 2,3-positions of the propionate and succinate with glucose-i-c14, glucose-6-c'4 and glucose-2-c"4 would be expected since this also occurred with lactate-3-c14, lactate-2-c14 and pyruvate-2-c14 (Leaver et al., 1955). The randomization may occur by formation of propionate from succinate or some other symmetrical compound; but, even if randomization of the C14 did not occur during the formation of the propionate, the C14 would be rapidly randomized by secondary reactions of the propionate (Wood et al., in preparation). The conversion of the 014 of glucose-l-c14, glucose-6-c14 and of glucose-2-c14 to the carboxyl groups of propionate and succinate also would be expected by the EMP pathway since the C14 from lactate-3-c14 and lactate-2-c14 was converted to these positions. This conversion may occur via a symmetrical Cs, by formation of succinate and propionate via citric acid, or via formation of a C4 compound by condensation of two C2 units (Leaver et al., 1955). In the present experiments there was no evidence of the formation of a symmetrical Cs, since, in contrast to the lactate fermentations the 3- and 2-positions of the propionate contained equal labeling. If a symmetrical Cs were involved the labeling in the 3 should be less than in the 2. The labeling in the acetate was in conformity with the EMP pathway in the case of the 1-, 6- and 2-labeled glucoses assuming that acetate arises via decarboxylation of pyruvate. Table 1 shows that acetate had the highest activity in the methyl position when formed from 1- and 6-labeled glucose. This is similar to the results found with lactate-3-c14. However, the isotope concentration in the acetate from glucose-6-c14 was not the same as that from glucose-l-c14. The methyl from C-1 was 9.13 to 15.1 as compared to 3.72 to 7.20 from C-6. This observation cannot be accounted for by the EMP pathway and will be discussed further in relation to other pathways. From glucose-2-c14, acetate was formed with the highest activity in the carboxyl group and just as with lactate 2_C14 and pyruvate- 2-C14 the activity in the carboxyl of acetate was higher than that of the 2,3-carbons of the propionate and succinate. Some of the major results not in accord with the EMP pathway are those obtained using glucose-3,4-c14. In this case by the EMP pathway C-1-labeled C, intermediates would be formed and results comparable to carboxyllabeled pyruvate and lactate would be expected. With pyruvate-i-c1' and lactate-l-cl1 practically all the label occurred in the carboxyls of propionate and succinate and in the C02. There was very little activity in the acetate and the 2,3- carbons of the propionate and succinate. In contrast with glucose-3,4-c14 there was considerable activity in the acetate and in the 2,3- carbons of the propionate and succinate (table 2). It is to be noted that there was a difference in the labeling of the acetate with the two microbial species. With 34W the methyl group had higher activity than the carboxyl group; with 52W the highest labeling was in the carboxyl group, and furthermore the activity was higher than it was in the 2,3-carbons of the propionate and succinate. Another portion of the data which does not appear to fit predictions for the EMP pathway is the difference in the yield of C02 from C-1 of glucose as compared to C-6. The yield from C-1 was 12.2 to 23.6 as compared to 1.67 to 5.39 from C-6 (table 1)..The question arises whether these variations from predictions of the EMP pathway may have an explanation other than the occurrence of a second type of cleavage of the glucose. In this regard it is important to note that the expectation that C-1 and C-6 will behave alike in the EMP cleavage involves the assumption that dihydroxy-

8 1955] GLUCOSE METABOLISM BY PROPIONIC ACID BACTERIA acetone phosphate and glyceraldehyde phosphate are metabolized identically. It is not inconceivable, for example, that the triosephosphate isomerase is not sufficiently active in the propionic acid bacteria to bring glyceraldehyde phosphate and dihydroxyacetone phosphate to complete isotopic equivalence. In this case if the metabolism of these compounds differed, then C-1- and C-6-labeled glucose would yield different results. This problem is of considerable importance in relation to studies of alternate pathways (Wood, 1955). A possible mechanism for the formation of CO2 from C-1 and for the conversion of C-3 of glucose to the 2,3-carbons of propionate and succinate via the EMP pathway is by removal of phosphate from dihydroxyacetone phosphate to yield symmetrical dihydroxyacetone. If the dihydroxyacetone then was converted to pyruvate the C-1 would be randomized into the carboxyl of pyruvate as illustrated in equation 4 and by decarboxylation would yield C1"02 from C-1. The C-3 likewise would be randomized into the methyl group and thus 2,3-labeled propionate and succinate would be formed from glucose- 3,4-C14. C*C*-C CO C*-C H20sPOC*. COO. CH20H H20,POC*.COO.C*H20H -* + OHC@ CHOH- COPOsH3 HOH2C*.COO.C*SH20H C*Has*COOCCOOH (4) and CIED COOC*OOH It is apparent, since dihydroxyacetone phosphate is formed from carbons 1, 2, and 3 in the EMP pathway, that in such circumstances C-1 would be a better precursor of CO2 than C-6. Also, if dihydroxyacetone phosphate were a better precursor of acetate than glyceraldehyde phosphate, then C-1 could be a better precursor of labeled acetate than C-6. Nevertheless these explanations do not appear to be completely adequate for the following reasons. The dihydroxyacetone arising from glucose-3,4-c14 would yield pyruvate-1,3-c14 and this would yield acetate with the highest labeling in the methyl group. With culture 52W 517 the highest labeling was in the carboxyl group of acetate, contrary to the above hypothesis. In addition, the C-1 of glucose would be expected to be a better source of the carboxyl group of pyruvate than C-6. If this occurred it would be expected that C-1 would be converted to carboxyl labeled propionate and succinate (via pyruvate) more extensively than would C-6. This did not occur. Perhaps the most important indication of the occurrence of a second pathway is the fact that the CO2 from glucose-l-c14 was higher in activity than any of the carboxyl groups of the end products. Since in the EMP pathway the carboxyl group of pyruvate gives rise to the CO, and to the carboxyl groups of propionate and succinate, each of these should have similar activities. This was usually the case with the sugars other than glucose-i-c14, but with glucose-i-c14 the CO2 had a higher activity than any of the carboxyl groups and it seems likely that there was direct formation of CO2 from C-1 by a pathway other than the EMP pathway. The results will next be considered in terms of the presently known alternate pathways. In the L. mesenteroides type of cleavage (equation 1) C-1 should give rise to CO2 and C-2 to the methyl of acetate. The acetate from glucose-2-c14 was predominantly labeled in the carboxyl position so the major cleavage does not appear to have occurred via this mechanism. Likewise the P. saccharophila type cleavage (equation 2) does not appear to be the major pathway, since in this case it would be expected that glucose- 3,4-C14 would yield acetate with the highest labeling in the methyl group. With 34W this was the case, but with 52W the labeling in the carboxyl group was highest. With both species C-1 was a good source of the methyl of acetate and the 2- and 3-carbons of propionate and succinate, which is not in accord with equation 2. The question of whether the metabolism may be via sedoheptulose and transketolase and transaldolase reactions is more difficult to evaluate since the mecha of these reactions are not clearly defined at present. Detailed consideration does not appear to be profitable unless evidence is obtained that sedoheptulose is an intermediate in the fermentation.6 6 One example of how a transformation might occur via the transketolase and transaldolase reactions is via ribose synthesis from glucose (Horecker, 1955). Pentoses are fermented by some

9 518 WOOD, STJERNHOLM AND LEAVER VOL. 70 Although a combination of the EMP pathway and an unknown alternate pathway appears at present to offer the best explanation of the present results, it is by no means certain that the EMP pathway actually occurs in these bacteria. Phosphoglyceric acid may be formed in this fermentation via an alternate pathway and the phosphate esters may be fermented after action of phosphatase but not by the EMP pathway. The inhibition of glucose fermentation by NaF is not necessarily at the enolase reaction (Volk, 1954). Fluoride does have other effects on the fermentation. For example, it inhibits net CO2 fixation and formation of the "non-reducing material" (Wood and Werkman, 1940). In the present study when NaF was added (experiment Nos. 5 and 13) there was no significant change in the isotope distribution in the propionate and succinate. Whether or not the cells were sufficiently permeable to NaF to permit the enolase reaction to be inhibited is not known. Further proof of the occurrence of the EMP pathway is needed. The fact that 52W and 34W behave differently adds to the difficulty of explaining the fermentation. The difference in the labeling of the acetate from glucose-3,4-c14 is particularly significant. It appears that the acetate either arises from different precursors or the precursors were formed differently by the two species. There are additional differences between the species. This is evident in the CO2 derived from glucose-3,4-c 4. The activity was usually lower with 52W than with 34W and the activity decreased with time with culture 52W in fermentation 15. The fact that the fermentation with 52W changes with time is also evident from the increase in activity of the acetate and C02 from glucose-2-c14. With culture 34W there also were some changes with time of incubation but the changes were usually small. Of particular interest is the fact that with glucose-2-c"4 the propionate formed by 34W had of the propionic acid bacteria and Rapaport and Barker (1954) have investigated the fermentation of arabinose-l-c14 with Propionibacterium pentosaceum and have found the isotope in all positions of the products. A combination of mechanism proposed by Horecker of ribose synthesis followed by a pentose fermentation via a C2 and Cs split together with C2 combination to succinate might explain many of the present results. a higher activity than the Euccinate.7 In experiment 8 at 6 hr, the 2- and 3-carbons of the propionate had an activity of 19.2 and 20.2 whereas the 2- and 3-carbons of succinate had an activity of The difference was not as great at 22 hr. Similar differences were obtained at 21 hr in experiment 9 but unfortunately the 6-hr succinate was lost in this experiment. The substantial difference between the propionate and succinate labeling makes it appear unlikely that succinate is the sole precursor of propionate' There also was considerable difference in the activity of the carboxyl groups of the succinate and propionate in experiments 14 and 15 with glucose-3,4-c'4. The question of succinatepropionate-interconversion is discussed by Wood et al. (in preparation). They found that the 2- and 3-carbons of the "intracellular" propionate and succinate come to rapid isotopic equivalence. Because of this rapid interconversion of propionate and succinate it is apparent that succinate and propionate usually will have similar labeling in the 2- and 3-carbons. Moreover, since the carbon of both the succinate carboxyls probably in large measure arises from the C-1 of a Cs intermediate, and since the carboxyl of the propionate also arises from C-1 of the Cs intermediate, it is likely that the carboxyls of the succinate and propionate will be alike. However, the fact that the labeling of the succinate and propionate does differ in some fermentations is very significant and points to independent mechanis of formation. SUMARY Different types of labeled glucose (1-, 2-, 6-, and 3,4-labeled) have been fermented by cell suspensions of two species of the genus Propionibacterium (P. arabinosum strain 34W) and the distribution of the C14 in the products has been determined. The following observations have been made: 7 Previously (Leaver and Wood, 1953; Wood et al., 1954) reported that the 2,3-carbons of suacinate from glucose-3,4-c4 had a higher activity than the 2,3-carbons of propionate. We have been unable to confirm these results and believe an error was made in the previous degradation. In these degradations the succinate carboxyls were obtained by the Schmidt reaction and the activity in the 2,3-carbons was calculated by difference on the basis of the total activity of the succinate.

10 1956] GLUCOSE METABOLISM BY PROPIONIC ACID BACTERIA (1) No matter which carbon of the glucose is labeled there is considerable activity in all positions of the products of fermentation. (2) The activity of the CO2 is highest from glucose-3,4-cl but there is considerable activity in the CO2 from glucose-l-c14. There is much less activity in the CO2 from glucose-2-c14 and glucose-6-ci4. (3) The acetate from both glucose-l-c14 and glucose-6-c14 has the highest activity in methyl group. However, the acetate from the glucose- 6-C14 was less active than that from the glucose- J-C14. (4) The acetate from glucose-2-c04 had the highest activity in the carboxyl group and the activity increased with the time of fermentation. (5) The acetate from glucose-3,4-c14 contained considerable activity. With culture 34W the methyl group had the highest activity but with 52W the carboxyl was highest. With culture 52W the activity increased with time. (6) The labeling of the 2- and 3-positions of the propionate was approximately equal with all types of labeled glucose, and usually the activity was the same as that of the 2- and 3-positions of the succinate. With glucose-2-c04 and culture 34W the 2- and 3-positions of the propionate were more active than the 2- and 3-positions of the succinate. It thus appears unlikely that succinate is the sole precursor of propionate in this fermentation. A difference in the labeling of the carboxyl groups of succinate and propionate was observed with glucose-3,4-c"4. It is tentatively proposed that a major part of the data can be explained on the basis of an Embden-Meyerhof type of cleavage of the glucose with a simultaneous second type of cleavage which is not yet identified. The second pathway appears necessary to account for the high yield of CO2 from C-1 of glucose and the conversion of the 3- and 4-carbons of glucose to acetate and the 2- and 3-carbons of propionate and succinate. On the basis of differences in isotope distribution in the products it appears likely that P. arabinosum and P. shermanii may have in part different mechanisms of glucose metabolism. REFERENCES BERNSTEIN, I. A., LENTZ, K., MALM, M., SCHAM- BYE, P., AND WOOD, H. G Degradation of glucose-c14 with Leuconostoc mesenteroides, 519 alternate pathways and tracer patterns. J. Biol. Chem., 215, BERNSTEIN, W., AND BALLENTINE, R Gas phase counting of low energy emitters. Rev. Sci. Instr., 21, ENTNER, N., AND DounoRo'FF, M Glucose and gluconic acid oxidation of Pseudomonas saccharophila. J. Biol. Chem., 196, GIBBS, M., AND DEMoss, R. D Anaerobic dissimilation of C14-labeled glucose and fructose by Pseudomonas lindneri. J. Biol. Chem., 207, GUNsALUS, I. C., AND GIBBS. M The heterolactic fermentation. II. Position of C14 in the products of glucose dissimilation by Leuconostoc mesenteroides. J. Biol. Chem., 194, HORECKER, B. L A new pathway for oxidation of carbohydrate. Brewers Dig., 28, HOREcKER, B. L Carbohydrate metabolism. Ann. Rev. Biochem., Vol. 24, KOEHLER, L. H Differentiation of carbohydrates by anthrone reaction rate and color intensity. Anal. Chem., 24, LEAVER, F. W., AND WOOD, H. G Evidence from fermentation of labeled substrates which is inconsistent with present concepts of the propionic acid fermentation. J. Cellular Comp. Physiol., 41, LEAVER, F. W., WOOD, H. G., AND STJERNHOLM, R The fermentation of three carbon substrates by Clostridium propionicum and by Propionibacterium. J. Bacteriol., 70, OSBURN, 0. L., AND WERKMAN, C. H Determination of carbon in fermented liquors. Ind. Eng. Chem., Anal. Ed., 4, PHARES, E. F Degradation of labeled propionic and acetic acids. Arch. Biochem. and Biophys., 33, RACKER, E Alternate pathways of glucose and fructose metabolism. Advances in Enzymol. 15, , Interscience, New York. RACKER, E., DE LA HABA, G., AND LEDER, I. G Transketolase-catalyzed utilization of fructose-6-phosphate and its significance in a glucose-6-phosphate oxidation cycle. Arch. Biochem. and Biophys., 48, RAPAPORT, D. A., AND BARKER, H. A Fermentation of arabinose-1-c14 by propionic acid bacteria. Arch. Biochem. and Biophys., 49, STONE, R. W., AND WERKMAN, C. H The occurrence of phosphoglyceric acid in the

11 520 WOOD, STJERNHOLM AND LEAVER [VOL. 70 bacterial dissimilation of glucose. Biochem. J. (London), 31, SWIM, H. E., AND KRAMPITZ, L Acetic acid oxidation by E8cherichia coli: quantitative significance of the tricarboxylic acid cycle. J. Bacteriol., 67, VAN SLYKE, D. D., AND FOLCH, J Manometric carbon determination. J. Biol. Chem., 136, VIRTANEN, A. I t)ber die propionsauregarung, II. Soc. sci. Fennica, Commentationes. Phys.-Math. 2, No. 20, VIRTANEN, A. I., AND KARSTR6M, A tjber die propionsauregarung, III. Acta Chem. Fennica Series B, 7, VOLK, W. A The effect of fluoride on the permeability and phosphatase activity of Propionibacterium pentosaceum. J. Biol. Chem., 208, WERRAN, C. H., STONE, R. W., AND WooD, H. G The dissimilation of phosphate esters by the propionic acid bacteria. Enzymologia, 4, WIGGERT, W. P., AND WERKMAN, C. H Fluoride sensitivity of Propionibacterium pento8aceum as a function of growth conditions. Biochem. J. (London), 83, WooD, H. G., AND WERKMAN, C. H The propionic acid bacteria. The mechanism of glucose dissimilation. J. Biol. Chem., 105, WOOD, H. G., AND WERKMAN, C. H Mechanism of glucose dissimilation by the propionic acid bacteria. Biochem. J. (London), 30, WOOD, H. G., WIGGERT, W. P., AND WERKMAN, C. H The fermentation of phosphate esters by the propionic acid bacteria. Enzymologia, 2, WOOD, H. G., AND WERKMAN, C. H The relationship of bacterial fixation of C02 to succinic acid formation. Biochem. J. (London), 34, WOOD, H. G., LIPsON, N., AND LORBER, V The position of fixed carbon in glucose from rat liver glycogen. J. Biol. Chem., 159, WOOD, H. G., LEAVER, F. W., AND STJERNHOLM, R The metabolism of Propionibacterium arabinosum. Bacteriol. Proceedings 54th Gen. Mtg. 1954, p. 97. WOOD, H. G The significance of alternate pathways in the metabolism of glucose. Physiol. Revs. (accepted for publication). Downloaded from on January 18, 2019 by guest