JOURNAL OF BACTERIOLOGY, Apr., 1965Copyright © 1965 American Society for Microbiology

Vol. 89, No. 4Printed in U.S.A.

Incorporation of C14 From Carbon Dioxide intoSugar Phosphates, Carboxylic Acids, and

Amino Acids by Clostridiumthermoaceticum

LARS LJUNGDAHL AND HARLAND G. WOODDepartment of Biochemistry, Western Reserve University School of Medicine, Cleveland, Ohio

Received for publication 4 December 1964

ABSTRACT

LJUNGDAHL, LARS (Western Reserve University, Cleveland, Ohio), AND HARLAND G.WOOD. Incorporation of C14 from carbon dioxide into sugar phosphates, carboxylicacids, and amino acids by Clostridium thermoaceticum. J. Bacteriol. 89:1055-1064. 1965.-The mechanism of synthesis of acetate from carbon dioxide by Clostridium thermo-aceticum was investigated by incubating cells with glucose or xylose in the presenceof C'402. Sugar phosphates, amino acids, and carboxylic acids were isolated and thespecific radioactivities were determined; the distributions of C'4 were also determinedin some of the compounds. Only fructose-1,6-diphosphate, formate, and lactate hadhigher specific activities than the acetate. The specific activities and distribution ofC'4 in the fructose-6-phosphate and ribose-5-phosphate were such that we concludethat the synthesis of acetate does not occur via a pathway involving the sugar phos-phates as direct intermediates. Likewise, it is shown that pathways including lactate,aspartate, serine, glycine, malate, and succinate are not of importance in the synthesisof acetate from CO2 . The methyl group of free methionine was unlabeled and is not aprecursor of the methyl group of acetate.

Fontaine et al. (1942) isolated Clostridiumthermoaceticum and found that it ferments glu-cose, fructose, and xylose, with acetate as the onlyproduct. Using C1402, Barker and Kamen (1945)found C14 in both carbons of the acetate, andthus demonstrated the existence of a pathway bywhich carbon dioxide is incorporated into themethyl group as well as into the carboxyl groupof acetate. The following mechanism for fermen-tation of glucose was postulated:

C6H1206 + 2H2O 2CH3COOH + 8H + 2CO28H + 2C1402 C14H3C'400H + 2H20

Sum C6H1206 -- 3CH3COOHAccording to these reactions, two-thirds ofthe acetate is formed directly from glucose whileone-third is synthesized from the carbon dioxideformed in the first reaction. Direct evidence ofthe total synthesis of acetate from C"02 wasobtained by Wood (1952a), who demonstratedby mass analysis the formation of C"3H3-C300Hwith a molecular weight 2 mass units greaterthan normal acetate. Fermentations of differenttypes of C14-labeled glucose gave results which

are in agreement with the proposal that carbons1 ,2 and 5,6 of the glucose are directly convertedto acetate, and carbons 3 and 4, to carbon dioxidefrom which the third mole of acetate is produced(Wood, 1952b; Lentz, 1956). It thus appears thatC. thermoaceticum ferments glucose v-ia theEmbden-Meyerhof pathway, although the pos-sibility exists that glucose can be cleaved in otherways leading to formation of the observed typesof labeled acetate.The only known pathway for synthesis of

carbon to carbon bonds from CO2 is v-ia thereductive pentose phosphate cycle which occursin autotrophic and photosynthetic organisms(see review by Elsden, 1962). Fructose-1, 6-diphosphate, fructose-6-phosphate, glucose-6-phosphate, and ribose-5-phosphate were isolated,to evaluate the possibility of synthesis of doublylabeled acetate from C1'402 'ia this cycle. Thespecific radioactivities of the sugars, and alsothe distribution cf C"4 in the different carbons,were determined. Another possible pathway foracetate synthesis is via formate, glycine, serine,pyruvate, and dicarboxylic acids as discussed inthe review by Wood and Stjernholm (1962).

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There is a rapid conversion of CO2 into formateby C. thermoaceticum (Lentz and Wood, 1955),and formate is preferentially inoorporated intothe methyl group of acetate. Therefore, com-pounds which would occur in this pathway wereisolated to investigate their possible role in theformation of acetate from C02 .

MATERIALS AND METHODSFixation of C402 with cell suspensions. C.

thermoaceticum was grown at 55 C under C02 for4 to 6 days in the medium described by Lentz andWood (1955), which was fortified with 45 ml perliter of boiled and filtered tomato juice. In oneof the experiments, xylose (10 g per liter) wassubstituted for glucose. The cells from 30 literswere harvested in a Sharples continuous-flowcentrifuge and suspended under N2 gas in 150 mlof 0.1 M potassium phosphate buffer (pH 7.0) con-taining 0.005 M cysteine. The suspension wastransferred under N2 into a flask stoppered witha serum bottle cap, through which additions weremade by injection with a hypodermic needle. Thecells were incubated together with glucose orxylose for 10 min at 55 C while gassing with N2 .Then the gassing was stopped, and radioactivepotassium bicarbonate was injected; after 5 sec,60% perchloric acid was injected to give a finalconcentration of 6%. The flask was then opened,and the unreacted C'402 was removed by bubblingC1202 through the mixture for 1 hr.

Fractionation of the reaction mixture. The acidicmixture was centrifuged, and the precipitate waswashed three times with a volume of 6% perchloricacid equal to that of the packed cells. The com-bined supernatant solution and washings wereneutralized with potassium hydroxide, and theprecipitated potassium perchlorate was removedby centrifugation. The solution was fractionatedby two methods. In the first procedure, the solu-tion was brought to pH 2 with sulfuric acid andwas then extracted continuously with ether for 42hr. Ether-soluble carboxylic acids were isolatedfrom the extract. The residue of the extractionwas passed through a column of Dowex-50 (Hform), from which amino acids were recovered byelution with 0.2 N ammonium hydroxide. The efflu-ent from the Dowex-50 column was passed througha column of Dowex-1-formate to remove any acidsubstances and thus obtain the neutral compoundsin the effluent. The second procedure was that ofLePage (1957) to obtain the phosphate esters asbarium-water insoluble and barium-alcohol insolu-ble fractions. The barium phosphates were con-verted to free acids by treatment with Dowex-50(H form) and then neutralized with sodiumhydroxide. The individual phosphate esters wereisolated from these solutions as described below.

Purification, identification, and degradation ofcarboxylic acids. The ether-soluble fraction afterneutralization was evaporated to dryness and thenchromatographed on Celite 535 as described bySwim and Krampitz (1954). The radioactivity and

the amount of acid were determined in the frac-tions, and the identity of the acids in the frac-tions was confirmed by paper chromatographywith at least three different solvents.

Acetic acid from the Celite column was steam-distilled and then degraded by the Schmidt pro-cedure (Phares, 1951). Formic acid was steam-distilled and then oxidized to C02 with mercuricchloride according to Piria (1946). Lactic acidwas crystallized to constant radioactivity as itsguanidine salt (Phelps and Palmer, 1917) beforedegradation by the method of Friedemann andGraeser (1933). Succinate was purified by vacuumsublimation at 135 to 145 C.

Purification and identification of sugar phos-phates. The following analytical procedures wereused for assay of sugar phosphates or free sugars:glucose-6-phosphate was assayed with glucose-6-phosphate dehydrogenase (Horecker and Wood,1957); fructose-6-phosphate was also assayed bythis procedure by including hexose phosphateisomerase in the reaction; fructose-1,6-diphos-phate with aldolase and glyceraldehyde phos-phate dehydrogenase (Mandl and Neuberg, 1957);total hexose with anthrone (Koehler, 1952);fructose with resorcinol (Roe, 1934); and pentoseswit.h orcinol (Mejbaum, 1939).

Fructose-6-phosphate, glucose-6-phosphate, andribose-5-phosphate were isolated from thebarium-alcohol insoluble fraction, and fructose-1,6-diphosphate was isolated from -the barium-water insoluble fraction. These fractions wereapplied at pH 7.0 to Dowex 1 X 8 formate columns30 cm long and 1 cm in diameter. The columns werewashed with water, and the monophosphates wereeluted with 0.2 N ammonium formate buffer(pH 4.0) after which the concentration of theformate buffer was gradually increased to elutediphosphates. The elution was followed by de-terminations of radioactivity and total phosphate(Bartlett, 1959). The sugar phosphate fractionswere treated with charcoal (Norit A) and lyo-philized to remove most of the ammonium for-mate. The free sugars were obtained by incubatingthe phosphate esters at 37 C with enough prostatephosphatase in 0.2 N acetate buffer (pH 5.5) toobtain complete hydrolysis within 6 hr. Thehydrolysis was followed by determining the for-mation of inorganic phosphate (Bartlett, 1959).The incubation mixture was desalted by passingit through columns of Dowex-50 and Duolite A-4.The resulting solution was streaked on Whatman3 MM paper and chromatographed with ethylmethyl ketone-acetic acid-40% boric acid inwater (9:1:1; Dickens and Williamson, 1958) asa solvent. A strip of the paper from the edge ofthe chromatogram was sprayed with silver nitrate(Partridge, 1948) to detect the sugars. Strips con-taining the individual sugars were then cut fromthe paper and eluted with water. The concentra-tion of sugar was determined, and then carriersugars were added prior to chromatography on acellulose column according to Schambye, Wood,and Kleiber (1957). Samples of fructose were not

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further purified, but the glucose and ribose were

crystallized from 95% ethyl alcohol before deg-radation. It was found necessary to purify thesugar both as the phosphate ester and as the freesugar to remove contaminating C14-compounds.

Glucose and some samples of fructose were de-graded by fermentation with Leuconostoc mes-

enteroides (Bernstein and Wood, 1957). Duringthis investigation, Busse, Kindel, and Gibbs(1961) published results indicating that therewas some randomization of C14 of fructose by thisprocedure. However, by fermentation of 4- and5-C'4-fructose, it was shown that the results of themicrobiological degradations could be used inspite of this randomization (Ljungdahl, 1962).Moreover, some fructose samples were degradedchemically by the procedure of Brice and Perlin(1957). Ribose was degraded microbiologically(Bernstein and Wood, 1957).Separation and identification of amino acids.

The amino acids were separated on a preparativeAmberlite IR-120 column (1.9 by 150 cm) by themethod of Moore, Spackman, and Stein (1958).A Beckman Spinco amino acid analyzer (model120) was used which was equipped with a dividerfor the eluate, permitting analysis as well as col-lection of fractions. The amino acids were identi-fied by their elution pattern and by paper chroma-tography.

Aspartate was degraded by first converting itto malate from which the a-carboxyl was obtainedby treatment with sulfuric acid (Racusen andAronoff, 1953). In addition, the malate was oxi-dized with potassium permanganate, to obtainCO2 from both carboxyls and acetaldehyde fromcarbons 2 and 3 (Wood et al., 1942). The latterwas oxidized to acetic acid and degraded by theSchmidt reaction (Phares, 1951).Methionine was degraded by the method of

Simmonds et al. (1943), to obtain the methyl groupas tetra methyl ammonium iodide, which wascrystallized.

Radioactivity measurements. All compoundsfrom the degradations were oxidized to CO2 bythe procedure of Van Slyke and Folch (1940), andthe radioactivity of the CO2 was determined witha gas-phase proportional counter (Bernstein andBallentine, 1950). Radioactivity in fractions fromchromatograms was determined by evaporatingsamples to dryness on glass planchets, and bymeasurement with a low-background Nuclear-Chicago counter. The radioactivity of the aminoacids was determined in a Packard Tri Carb liquidscintillation counter. All determinations of radio-activity were corrected to values comparable tothe gas proportional counter.Enzymes and chemicals. Hexose phosphate

isomerase was a gift from Henry Sable. Glucose-6-phosphate dehydrogenase was prepared fromyeast (Kornberg and Horecker, 1955). Triosephos-phate dehydrogenase and aldolase were giftsfrom Joseph Mendicino. Prostate phosphatasewas prepared by the method of Davidson andFishman (1959), from human prostate glands ob-

tained by surgery at Lakeside Hospital, Cleve-land, Ohio. Radioactive potassium bicarbonatewas prepared from BaC403 by acidificationwith perchloric acid, and the liberated C402 wastrapped as KHCO3 in an equivalent amount ofpotassium hydroxide. Cell-free extracts from C.thermoaceticum were prepared from a 25% cell

TABLE 1. Conditions of experiments withcell suspensions

Wet ~~~~~Pre- K.HC140s Incu-Expt WetofGlcoeXyos (count/ KHco, bationno. wctelf Glucose Xylose na min X with

cells ~~~tionle 10-6) C140sg mmoles mmoles min mmoles sec

1 122 10.1 - 10 2,000 1.0 52 90 8.9 - 10 2,000 0.5 53 186 5.5 - 6 2,000 0.5 54t 150 - 13.3 10 1,570 0.39 5

* The cells were preincubated at 55 C under N2together with the glucose or xylose in 0.1 M po-tassium phosphate buffer (pH 7.0) containing0.005 M cysteine before addition of the radio-active bicarbonate.

t Cells were grown on xylose.

TABLE 2. Distribution of C14 in fractions from theperchloric acid extracts of cells of the experiments

listed in Table 1*

Expt 1 Expt 2Ept3 xt4Fraction (glu- (glu- (glucose)(3yElptocose) cose) (lcs) (yoe

Soluble in per-chloric acid... 6,640 5,350 288,000 137,500

Ether-extract-able ......... 3,980 4,140 235,000 116,300

Ether-insoluble. .. 665 520 43,250 21,200Basic sub-

stances, ab-sorbed onDowex-lt. . 18,325

Acid substances,absorbed onDowex-1t 4,510

Neutral sub-stancest 1,280

Barium-water in-soluble 5,390 3,730

Barium-alcoholinsoluble 2,370 3,450

Barium-soluble... 313,500 98,500

* C'4 is expressed as counts per minute X 10-3.The substrate in each experiment is given paren-thetically.

t The basic, acidic, and neutral substanceswere obtained by fractionation of the ether-insoluble fraction.

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suspension in 0.1 M potassium phosphate buffer(pH 7.0) by use of a French press. These extractswere used to test for C1402 fixation via carboxy-dismutase according to methods described byWeissbach, Smyrniotis, and Horecker (1954).

RESULTS

Fractionation of C14-labeled compounds. Table1 presents the conditions used in four experi-ments in which radioactive carbon dioxide wasfixed during fermentation of glucose or xyloseby cell suspensions of C. thermoaceticum. The

cells used in experiment 4 were grown on xylosebecause of the desire to isolate pentose phosphatesfrom this incubation.The distribution of C'4 in the fractions from

these experiments is shown in Table 2. Theperchloric acid extract contained almost all ofthe fixed radioactivity; less than 1% was foundin the cellular debris and precipitated protein.Approximately 0.3% of the added C'4 was fixedin experiments 1 and 2 [experiment 1, (6.64 X10-6/2,000 X 10-6) X 100] and about 10% inexperiments 3 and 4. At present, there is no

TABLE 3. Distribution of C14 in carboxylic acids isolated from ether extracts obtained in the experimentsof Table 1 and 2*

Fraction | Determination Expt 1 Expt 2 Expt 3 Expt 4Fraction (glucose) (glucose) (glucose) (xylose)_ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~I_

Count/min X 10-3Milliequivalents

Count/min X 10-3MmolesCount per minm moleCH3 count per min per ,umoleCOOH count per min per ,umole

Count/min X 10-3MmolesCount per min per,umole

Count/min X 10-3MmolesCount per min per ,umoleCH3 count per min per,moleCHOH count per min per,umoleCOOH count per min per,Amole

Count/min X 10-3MmolesCount per min per,mole

Count/min X 10-3MmolesCount per min per,mole

Count/min X 10-3MmolesCount per min per,umole

Count/min X 10-3MmolesCount per min per jAmole

Count/min X 10-3MmolesCount per min per,umole

Radioactivity recovered (%)

Acid recovered (%)

* The substrate in each experiment is given parenthetically.

Acid

Acetate

Formate

Lactate

Succinate

Malonate

Glycolate

Oxalate

Malate

3,98014.5

3,52011.6

30487

219

4050.38

1,060

2690.17

1,5802127

1,515

0.70.0513.5

5.10.05

101.5

10585

4,14014.3

137.512.5111.98.5

2,4100.39

6,180

218

6790

235,00024.1

65,50019.4

3,375762

2,483

2881

116,30017.8

93,50015.9

5,8902,0303,845

9150.18

5,100

6,5500.52

12,600335645

11,800

150.04

380

820.04

2,045

490.06

805

270.02

1,355

720.02

3,580

8794

-1-1-

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explanation of the differences found in the experi-ments. The major portion of the radioactivity,about 80%, was present in the ether extract inthe form of carboxylic acids. The residue fromthe ether extraction in experiment 4 was dividedinto basic, acidic, and neutral fractions. Thebasic fraction contains amino acids, and con-tained the majority of the remaining radio-activity. Both the acidic and the neutral frac-tions from the residue of ether extraction had alow C14 content and were not investigated further.Barium acetate was added to a sample of theneutralized perchloric acid extracts from experi-ments 3 and 4 to obtain precipitates of phosphateesters as barium salts. A relatively small part ofthe total C14 fixed was found in these fractions.

C14 in carboxylic acids. The results of fractiona-tion of the carboxylic acids on Celite columnsare given in Table 3. Acetate generally containedmost of the C14 and was labeled in both carbons.The carboxyl group contained more C14 thanthe methyl group, which is in accord with theresults of Wood (1952a). Lactate and formatehad higher specific activities than the acetate,and succinate, malonate, glycolate, and oxalatehad lower activities. The C14 of the lactate wasalmost all in the carboxyl group and probablyarises from pyruvate. An exchange between CO2and the carboxyl of pyruvate and between CO2and formate has been shown to be catalyzed byextracts from C. thermoaceticum (Ljungdahl andWood, 1963). The high radioactivity in formateis in agreement with observations by Lentz andWood (1955).

C14 in amino acids. Table 4 shows the specificactivities and the total C14 contents of the freeamino acids from experiment 4 after 5 sec ofincubation with C1402. Less than 50% of theC14 was recovered from the preparative column,but only those compounds which react withninhydrin were determined. Several unidentifiedsubstances reacting with ninhydrin appeared onthe chromatograms, but they were all of lowspecific activity and were not further character-ized. All amino acids except aspartate had a lowerradioactivity than the acetate. It is noteworthythat alanine, glycine, glutamate, serine, andmethionine all had low C14 activities. Aspartate,which may reflect the distribution of C'4 in C4-dicarboxylic acids, was degraded, with the resultslisted in Table 5. Almost all the C14 activityresided in the carboxyl carbons which had equalspecific activities, but the specific activity of thea-carbon was higher than that of the ,3-carbon.

C14 in sugar phosphates. Fructose-1, 6-diphos-phate, fructose-6-phosphate, and glucose-6-phosphate were isolated in experiments 3 and 4

TABLE 4. Distribution of C14 in amino acids iso-lated from cells fermenting xylose, experiment 4

of Tables 1 and 2*

CountCompoundt Amt (umoles) per min Total count/min

per pmole

Basic substances,Table 2...... 18,325,000

Unknown 1. 17.0 648 11,100Unknown 2. 9.85 440 4,450Aspartate ...... 1,240 5,125 6,360,000Threonine...... 16.05 298 4,800Serine ......... 11.1 592 6,560Unknown 3 ... 6.9 920 6,350Glutamate ..... 1,100 225 247,000Proline ........ 71.5 668 47,700Glycine ........ 37.4 514 19,200Alanine ........ 660 488 322,000Unknown 4.... 36.2 102 3,690Valine ......... 86.1 1,388 119,500Methionine .. 9.35Isoleucine ...... 52.3 222 11,650Leucine ........ 80.6 1,646 132,800Tyrosine ....... 10.1 136 1,370

Sum count/min.. 7,298,170

* Per cent radioactivity accounted for, 41.t Compounds listed in order emerging from the

amino acid analyzer.t Methionine was degraded to obtain C14 of the

methyl group, which was unlabeled.

TABLE 5. Distribution of C14 in aspartate from cellsfermenting xylose, experiment 4 of Table 4

CountDetermination per min

per gmole

Total* ........... ............. 5,126a- and 13-carbonst........................ 186a-Carboxyl (COOH) ..................... 2,295a-Carbon (HCNH 2) ...................... 107,B-Carbon (HCH) ........................ 66,3-Carboxyl (COOH) ..................... 2,342Sum of C14 in individual carbons......... 4,810Recovery in degradation, % ............. 94

* Counts per minute per micromole in CO2from combustion of aspartate X 4.

t Counts per minute per micromole in CO2from combustion of acetate of degradation X 2.

and ribose-5-phosphate, in experiment 4. Thedephosphorylated sugars were degraded (Table6). Fructose-i , 6-diphosphate, which was the onlysugar with a higher specific activity than theacetate, had a specific activity 10 times higherthan the fructose-6-phosphate. The latter hadmore C14 activity than the glucose-6-phosphate.More than 90% of the C14 of the sugar phos-phates was located in carbons 3 and 4 of the

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TABLE 6. Distribution of C14 in sugar phosphates obtained from cells fermenting glucose and xylose,experiments S and 4 of Tables 1 and 2a

Expt 3 (glucose) Expt 4b (xylose)Determination

G-6-P F-6-P F-1,6-P G-6 P F-6-Pc F-1,6-Pc R-5-P

Totald 301.5 251.0 3,370 502.0 1,292 13,850 1,970C-i ..3.1 4.5 94.6 4.6 0 160 26.0C-2 ..1.5 1.4 31.0 16.5 13.6 274 19.1C-3 ..30.3 53.5 1,135.0 45.5 183.2 4,510 1,650.0C-4..116.0 105.5 1,750.0 346.0 945.0 6,710 86.7C -5. ........ 35 56 37.5 25.3 43.9 480 42.0C-6... 5. 16.6 13.3 34.8 282

Sum of carbons 1 to 6 154.4 170.5 3,064.7 451.2 1,220.5 12,416 1,823.8Recovery of C14 in degra-

dation, %.526 68e 91 88 95 90 93

a Figures show counts per minute per micromole. G-6-P = glucose-6-phosphate; F-6-P = fructose-6-phosphate; F-1,6-P = fructose-1,6-diphosphate; R-5-P = ribose-5-phosphate. The substrate for eachexperiment is given parenthetically.

b The combined total radioactivity in glucose-6-phosphate, fructose-6-phosphate, and ribose-5-phosphate from experiment 4 was 1,020,000 count/min and in fructose-1,6-diphosphate, 7,135,000count/min.

c Degraded by chemical method.d Count per minute per micromole of C02 from combustion X 6.¢ The glucose-6-phosphate and fructose-6-phosphate were contaminated. The impurity was ribitol,

which probably is not fermented by Leuconostoc mesenteroides, and the degradation should reveal thetrue distribution of C"4.

TABLE 7. Fixation of C1402 by cell-free extracts*

TotalAddition counts

fixed

None ................................. 540Ribose-5-phosphate ...................... 480Glucose-6-phosphate ..................... 600Fructose-6-phosphate ......... ........... 1,540Fructose-1,6-diphosphate ....... ......... 2,020Phosphenol pyruvate ................... 6,890

* The incubation mixture contained extract(22.8 mg of protein), 10 ,umoles of phosphate ester,50 pmoles of MgSO4, 1 ,umole of reduced nico-tinamide adenine dinucleotide 200 ,umoles ofpotassium phosphate (pH 7), 5,umoles of ATP,and 10lAmoles of KHC1"03 (28,000 counts per minper,umole) in a total volume of 3 ml. Incubationwas for 60 min at 45 C in N2 atmosphere.

hexoses and in carbon 3 of the ribose. The dis-tribution of C" between C-1,2,3 and C-4,5,6of the hexoses was highly asymmetric. In experi-ment 4, carbons 4, 5, and 6 of the fructose-1,6-diphosphate contained 60% of the C"4; 84% wasin these carbons of the fructose-6-phosphate and85% was in the glucose-6-phosphate. The resultswere similar in experiment 3.Of the radioactivity in the perchloric acid

extract from experiment 4, about 81% has beenaccounted for in known compounds.

Fixation of C02 by cell-free extracts. Cell-freeextracts of C. thermoaceticum were tested for theability to fix C1402 in the presence of differentsugar phosphates (Table 7). No stimulation ofthe C1402 fixation was observed in the presenceof ribose-5-phosphate and glucose-6-phosphate.Fructose-6-phosphate mxld fructose-1, 6-diphos-phate were slightly stimulatory, and phosphoenolpyruvate caused an increase in the fixation ofC1402. The failure to obtain fixation of C02 withribose-5-phosphate indicates that C02 is notfixed via the photosynthetic cycle involving thecarboxydismutase reaction.

DISCUSSIONThere is convincing evidence based on experi-

ments with labeled glucose that carbons 1, 2, 5,and 6 give rise to acetate and carbons 3 and 4,to C02 in the fermentation by C. thermoaceticum,and that the C02 in turn is converted to acetate.All attempts to demonstrate the carboxydismu-tase reaction have failed, and the synthesis ofthe carbon-to-carbon bond from C02 by a modifi-cation of the Calvin photosynthetic cycle seemsunlikely. Therefore, the possibility of synthesisof an acetate precursor by direct combination oftwo one-carbon compounds seemed worth con-sideration. Krampitz, Suzuki, and Greull (1961)showed that dihydroxyethyl thiamine diphos-phate can be synthesized by addition of two

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equivalents of formaldehyde to thiamine diphos-phate at pH 8.8. Furthermore, Votaw et al.(1963) showed that dihydroxyethyl thiaminediphosphate is cleaved in the presence of phos-phate by phosphoketolase to acetyl phosphate.It seemed possible that this mechanism or onesimilar to it might be involved in the synthesis ofacetate from CO2 by C. thermoaceticum. Dihy-droxyethyl thiamine diphosphate also is anintermediate of the transketolase reaction(Krampitz et al., 1961), and it therefore appearedlikely that if CO2 were utilized via this mecha-nism the pentose phosphates would becomelabeled in C-1 and C-2 from C'402 by C. thermo-aceticum.Experiment 4 with xylose was therefore set up

to test this possibility. This substrate was usedbecause the pool of pentose phosphate might beincreased and thus make it easier to isolate asignificant quantity of pentose phosphate fromthe cells. C. thermoaceticum ferments xylose withthe formation of acetate (Barker, 1944). It isclear (Table 6) that no significant labeling of thepentose occurred in positions 1 and 2, and there-fore the experiment provided no evidence thatacetate is synthesized from C02 via dihydroxy-ethyl thiamine diphosphate.

Surprisingly the fructose-1,6-diphosphate didcontain a very high concentration of C14 incarbons 3 and 4, higher than in the acetate (seeTables 3 and 6); also, the ribose phosphate hadextensive labeling in carbon 3. This raises thequestions of how the fructose-1, 6-diphosphatebecame labeled and whether this pathway playsa part in the formation of doubly labeled acetatefrom CO2. The extensive labeling of carbons 3and 4 of the hexoses is similar to that observedby Bassham et al. (1954) during short-termphotosynthesis and also by Aubert, Milhaud,and Millet (1957) in autotrophic fixation of CO2 .However, this mechanism seems unlikely sincecarboxydismutase seems to be absent in C.thermoaceticum and, in addition, this mechanismshould label the fructose-6-phosphate in posi-tions 3 and 4 and the pentose phosphate inpositions 1, 2, and 3. This would occur becausethe mechanism involves conversion of pentosephosphate and CO2 to phosphoglycerate which isthen converted to triose phosphate. The triosephosphate via the aldolase reaction yields 3- and4-labeled fructose-1, 6-diphosphate which inturn is converted to fructose-6-phosphate. Thelatter, by transketolase and transaldolase reac-tions, is converted to pentose phosphate whichthen undergoes further fixation reactions. Thismechanism also is unlikely because glucose iscontinually being utilized in the fermentation,and the influx of glucose carbon makes it im-

probable statistically that the carbon-to-carboncombinations would both be from CO2 by thismechanism. It is recalled that a mechanism isrequired in which one-third of the molecules ofacetate are formed from 2 molecules of CO2(Wood, 1952a).At the present time, we have no definite ex-

planation of the extensive labeling of the 3- and4-carbons of the fructose-1,6-diphosphate. It isknown that C. thermoaceticum contains a thia-mine-dependent pyruvate carboxylase whichcatalyzes a very rapid exchange of CO2 with thecarboxyl group of pyruvate (Ljungdahl andWood, 1963). The high C14 content found in thecarboxyl group of lactate (Table 3) seems to be areflection of this exchange. The C14 may enter thefructose-1, 6-diphosphate by the following re-versible reactions:

pyruvate + adenosine triphosphatepyruvate kinase

(ATP) ( I phosphate-enolpy-ruvate + adenosine diphosphate (ADP) + Pi

enolasephosphate-enolpyruvate n

2-phosphoglycerate

2-phosphoglycerate phosphoglycerate mutase

3-phosphoglyceratephosphoglycerate kinase

3-phosphoglycerate + ATPk

1 ,3-diphosphoglycerate + ADP113-diphosphoglycerate

+ reduced nicotinamide adenine dinucleotidephosphoglyceraldehyde

dehydrogenase K

glyceraldehyde-3-phosphate+ nicotinamide adenine dinucleotide + Pi

triose phosphateimomerase

glyceraldehyde-3-phosphate (

dihydroxyacetone-phosphateglyceraldehyde-3-phosphate

+ dihydroxyacetone-phosphatealdolase- fructose-i, 6-diphosphate

It might seem unlikely that C'402 would reachfructose-1,6-diphosphate by reversal of such along series of reactions. At present, however, thisseems to be the most likely explanation of thelabeling. The high C14 content of the ribose-5-phosphate in carbon-3 could occur via trans-ketolase exchange between xylulose-5-phosphate

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LJUNGDAHL AND WOOD

and the labeled glyceraldehyde-3-phosphatefollowed by epimerization and isomerization toribose-5-phosphate:

transketolase + xylulose-5-phosphate ±

glyceraldehyde-3-phosphate + trans-

ketolase-dihydroxyethyl thiamine diphosphate

In the same manner, the higher labeling of the4 position of fructose-6-phosphate compared withthe 3 position can be explained by transaldolaseexchange (Ljungdahl et al., 1961). In addition,the fructose-6-phosphate and glucose-6-phosphatemay also become labeled by some dephosphoryla-tion of fructose-1, 6-diphosphate, accounting forthe low labeling in C-3.The question might be raised whether the

doubly labeled acetate may not arise by cleavageof the 3- and 4-labeled fructose-1,6-diphosphateto three acetate molecules. The cleavage of fruc-tose-6-phosphate to acetyl-phosphate and eryth-rose4-phosphate, and also the cleavage of eryth-rose-4-phosphate to two C-2 compounds, wasconsidered by Schramm, Klybas, and Racker(1958). However, doubly labeled acetate isformed from pyruvate and C'302 (Wood, 1952a),and the bacteria have a strong phosphoro-clastic reaction (Ljungdahl and Wood, 1963).Therefore, it is likely that pyruvate is me-tabolized via this reaction and acetate isformed in subsequent steps not involving hexosephosphates.Another pathway which we have examined is

that proposed by Wood and Stjernholm (1962),as illustrated in reactions l to 6:

C1402 HC1400H (1)HC14OOH HC14HO (2)

HC14HO + CH2NH2 = COOH --

C14H20H-CHNH2-COOH (3)

C14H20H-CHNH2-COOH -*

C14H3-CO-COOH + NH3 (4)

C'4H3-CO-COOH + C1402 )

HOOC14-C14H2-CO-COOH (5)

HOOC14-C14H2-CO-COOH --

C14H8-C1400H + HOOC-COOH (6)

Reaction 1 is catalyzed both by resting cells andcell-free extracts of C. thermoaceticum. Reactions2 and 3 involve tetrahydrofolic acid derivativesand have been demonstrated in several clostridia.In reaction 3, ,B-labeled serine is formed which inreaction 4 is converted to methyl-labeled pyru-vate. The pyruvate is then carboxylated in

reaction 5, giving oxaloacetate labeled in carbons3 and 4. Finally, the oxaloacetate is cleaved togive doubly labeled acetate and oxalate. Thisreaction has been observed in Aspergillus niger(Hayaishi et al., 1956). Alternatively, oxalo-acetate is reduced to malate which, after activa-tion to malyl-coenzyme A (CoA) is cleaved toacetyl-CoA and glyoxylate (Tuboi and Kikuchi,1962). Oxalate or glyoxylate may then be re-cycled via glycine. The data from this investiga-tion appear to exclude this pathway. The isolatedfree serine and glycolate had low radioactivitycompared with acetate. Lactate, which reflectsthe labeling of pyruvate, had no C14 in the methylcarbon. Furthermore, aspartate, which is anindicator of the distribution of C14 in malate andoxaloacetate, had almost all its activity in thecarboxyl groups. A cleavage of the latter acidswould therefore yield only carboxyl-labeledacetate.The fact that serine acquired low radioactivity

and the methyl group of methionine was un-labeled indicates that the methyl group of acetateis not formed via compounds containing C-1units in equilibrium with either serine or methio-nine. This is especially interesting in connectionwith the recent findings by Poston, Kuratomi,and Stadtman (1964) that C'4-methyl-B12 givesrise to the methyl group of acetate in extracts ofC. thermoaceticum. Methyl-B12 has also beenfound to be a donor to the methyl group ofmethionine (Guest et al., 1962) in Escherichia coli.If methyl-B12 is an intermediate in the synthesisof methionine and of acetate in C. thermoaceticum,it must exist in two separate pools. Most prob-ably, the methyl-B12 is firmly bound to twodifferent enzymes, one for methionine synthesisand a second for acetate formation.The high radioactivity of formate, and the

finding by Lentz and Wood (1955) that it ispreferentially incorporated into the methyl groupof the acetate, suggests that formate may be aprecursor of methyl-B,2, which may be the im-mediate precursor of the methyl group of theacetate. It seems possible that CO2 combineswith methyl-B,2, forming carboxymethyl-B,2,which is cleaved to yield acetate and B12. Thisis indicated by the finding of Ljungdahl, Irion,and Wood (Federation Proc., in press), who iso-lated from intact cells a B12 derivative in whicha ligand group is formed from C402. The com-pound appears to be a precursor of acetate andis cleaved by light to labeled C02, formaldehyde,methanol, and small amounts of unidentifiedacids.

It is of interest that Butyribacterium rettgeri isable to incorporate C1402 into both carbons ofacetate (Barker, Kamen, and Haas, 1945). Pine

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VOL. 89, 1965 CARBON DIOXIDE FIXATION BY C. THERMOACETICUM

and Barker (1954) found that the formation ofdoubly labeled acetate by this organism does notinvolve intermediates of the di- or tricarboxylicacidl cycles or incorporation of formate into themethyl group of acetate via serine. Wittenbergeran(l Flaxvin (1963) reported recently that thefixation of CO2 by B. rettgeri does not occur bythe carboxvdismutase reaction, nor could thevdetect l)hosphoketolase in extracts from thisorgoanismi.

ACKNOWLEDGMENTS

This iinvestigation was supported by PublicHealth Service grant GMI 11839 from the Divisionof General MIedical Sciences and by contractAT (-30-1)-1320 from the Atomic Energy Com-mission.

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