SYNTHESIS OF CELLULOSE FROM PYRUVATE BY SUCCINATE-GROWN CELLS OF ACETOBACTER XYLINUM

MOSHE BENZIMAN AND H. BURGER-RACHAMIMOV

Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel

Received for publication April 18, 1962

ABSTRACT

BENZIMAN, MOSHE (The Hebrew University ofJerusalem, Jerusalem, Israel) AND H. BURGER-RACHAMIMOV. Synthesis of cellulose from pyru-vate by succinate-grown cells of Acetobacterxylinum. J. Bacteriol. 84:625-630. 1962.-Pyru-vate was converted into cellulose by succinate-grown cells of Acetobacter xylinum. With pyru-vate-1-, 2-, or 3-C14 as substrate, the upper halfof the cellulose monomer mirrored the lower half,both as to total content and distribution of C14.In each case, about 75% of the total radioactivityof the cellulose monomer was found in two carbonatoms (carbon pairs 3:4, 2:5, and 1:6, derivedfrom pyruvate-1-, 2-, and 3-C'4, respectively).The carbonyl carbon of pyruvate contributed 2equivalents to the cellulose monomer, comparedwith 1.4 and 2.8 equivalents contributed by thepyruvate carboxyl and methyl carbons, respec-tively. Cellulose formed in the presence of pyru-vate and C1402 was nonradioactive. The resultssuggest that the carbon chain of the cellulosemonomer is formed in these cells via a condensa-tion involving two molecules of a three-carboncompound. Reactions involving pyruvate whichcould account for the observed distribution ofC'4 in cellulose are discussed.

Whereas washed glucose-grown Acetobacterxylinum cells readily oxidize intermediates of thecitrate cycle, they form no cellulose from them(Schramm, Gromet, and Hestrin, 1957a). Suchsynthesis occurs, however, if the cells have beengrown in the presence of a citrate-cycle inter-mediate (Gromet-Elhanan, 1960).To elucidate the chemical basis for the acquisi-

tion of the cellulose-synthesizing ability, theconversion of pyruvate into cellulose has beenstudied with the aid of specifically C14-labeledcitrate-cycle intermediates. A preliminary reportof this work has appeared (Benziman and Burger-Rachamimov, 1961).

MATERIALS AND METHODS

Cultivation and harvest of bacteria. The strainof A. xylinum was the same as that employed inearlier investigations reported from this labora-tory (Schramm and Hestrin, 1954). Cells grownon succinate were obtained as described byGromet-Elhanan (1960).

Preparation of cell suspension. Cells preparedfrom pellicles harvested at 39 to 40 hr, as des-cribed by Schramm and Hestrin (1954), weresuspended in 15 volumes of 0.04 M potassiumphosphate buffer (pH 6.0), centrifuged in thecold, redispersed in buffer, and used on the sameday. Repeated washing or storage resulted inmarked loss of synthetic activity. Unless other-wise indicated, succinate-grown cells were used.Measurement of synthetic activity. The standard

reaction mixture (10 ml) contained: cells, sub-strate (as potassium salt), and 0.045 M potassiumphosphate buffer (pH 6.0), with oxygen as thegas phase, in a stoppered flask. The incubationmixtures were shaken at 100 oscillations/minin a water bath at 30 C. After the reaction term,the cells and the synthesized cellulose were sedi-mented by centrifugation and repeatedly washed.

Analytical methods. The cellulose samples werecollected and dried on Fiberglas discs. Radio-activity was measured in a gas-flow counter.Values were corrected for self-absorption. Todetermine cellulose chemically, samples wereacetolyzed and hydrolyzed according to themethod of Schramm and Hestrin (1954), and thereleased glucose was determined by the phenol-sulfuric acid method of DuBois et al. (1956).Cellulose present initially in the reaction mixturewas less than 15% of the value found after incuba-tion with the substrate. The endogenous rate ofcellulose synthesis was negligible.

Pyruvate was determined by the direct methodof Friedman and Haugen (1943).

Isolation of glucose from cellulose. Cellulose wasdissolved and hydrolyzed after acetolysis, asdescribed by Schramm and Hestrin (1954).

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Hydrolyzates were freed from sulfate ions byprecipitation with barium acetate, followed byrepeated washings of the precipitate. Acetic acidwas removed from the combined filtrate andwashings by treatment with a mixture of Dowex1 (C03-- form) and Dowex 50 (H+ form). Thefinal solution was concentrated in vacuo to a

small volume.Glucose degradation. Each glucose carbon was

converted specifically to barium carbonate by a

procedure involving fermentation with Leu-conostoc mesenteroides, as detailed by Edelman,Ginsburg, and Hassid (1955), Bernstein andWood (1957), and Abraham and Hassid (1957).The BaCO3 was collected and dried on Fiberglasfilter discs. Counts were corrected for backgroundand self-absorption.

Substrates. Potassium pyruvate was prepared,according to Robertson (1942), from freshlyvacuum-distilled pyruvic acid. Radioactivepyruvates, succinate-2- and -3-C14, and Na2C"403were obtained from The Radiochemical Centre,Amersham, England. Acetate-1-C'4 and acetate-2-C14 were from the Research Specialties Co.,Berkeley, Calif.

RESULTS

Utilization of various substrates for cellulosesynthesis. Both succinate-grown and glucose-

TABLE 1. Formation of cellulose fromvarious substrates by Acetobacter

xylinum

Substrate Celulose Oxygen

Pyruvate .................. 0.16 3.7Succinate.................. 0.04 3.9

Oxaloacetate .............. 0.04 3.5Malate <0.01

Fumarate <0.01

Acetate <0.01

* Cells (10 mg dry wt) were incubated with 0.06M of substrate carbon for 2.5 hr as described inMaterials and Methods. Results are expressed as

,umoles of glucose per mg of cells per hr.t Uptake was followed manometrically, using

2 mg (dry wt) of cells and 20 pmoles of substratein 2.0 ml of 0.045 M phosphate buffer (pH 6.0).Rates were calculated from uptake during first30-min period after adding the substrate. Resultsare expressed as jsmoles of 02 per mg of cells per

hr.

TABLE 2. Contribution of individual carbonatoms of pyruvate to cellulose formed

by Acetobacter xylinum

Expt I Expt II Expt III

Substrate* Specific Specific Specificactivity at activity at activity at

cellulese cellulose cellulose

Pyruvate-1-C'4 2,490 1.3 1,510 1.4 1,610 1.5Pyruvate-2-C14 2,170 2.1 1,850 2.22,120 2.1Pyruvate-3-C'4 1,550 2.61,000 2.81,100 3.0

* Cells (9 mg dry wt) were incubated with200 umoles of radioactive pyruvate for 3 hr asdescribed in Materials and Methods.

t Specific activity of cellulose monomer/specificactivity of pyruvate. Specific activity is in countper min per Amole of pyruvate or cellulosemonomer.

grown A. xylinum cells readily oxidized pyruvateunder standard conditions in a system whichcontained 15 mg (dry wt) of cells and 200,molesof uniformly labeled C14 pyruvate (3.6 X 105count/min). Under these conditions, however,cellulose was not formed from pyruvate byglucose-grown cells, whereas approximately 5%of the total carbon supplied as pyruvate wasconverted into cellulose by the succinate-growncells. Pyruvate gave rise to more cellulose thanany other citrate-cycle intermediate (Table 1).[Gromet-Elhanan (1960), also using succinate-grown cells, obtained an equal yield of cellulosefrom both pyruvate and succinate. The disparitybetween our observations and those of this authorcould be due to differences in the experimentalconditions (cells washed once but not five times;reaction time of 2 hr rather than 5 hr) or to thecircumstance that our cells had a much highersynthesizing activity.] Though no cellulose wasformed by the cells from fumarate or malate,these compounds were nevertheless metabolized,as evidenced by the accumulation of considerableamounts of an unidentified keto acid in themedium. Acetate alone did not produce anycellulose. However, when acetate was suppliedtogether with pyruvate (Table 3) or with glucose(Gromet-Elhanan, 1960), acetate carbons wereincorporated into cellulose. Exogenous C02 failedto serve as a source of cellulose carbon, as in-dicated by the observation that no radioactivitywas detected in the cellulose formed by cell sus-

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CELLULOSE BIOSYNTHESIS BY A. XYLINUM

TABLE 3. Cellulose synthesis by Acetobacter xylinumin the presence of supplementary substrates

Cellu- Effectlose of sup-

Expt Substrate* Supplement formed plement&&umoles (As +of a/As X

glucose) 10O)t

1 Pyruvate 2.5Acetate <0.3Pyruvate-1-C14 Acetate 3.4 134Pyruvate-2-C14 Acetate 105Pyruvate-3-C14 Acetate 106Acetate-i, 2-C14 Pyruvate 1,540

2 Pyruvate - 3.4Succinate - 0.6Pyruvate-l-C'4 Succinate 4.5 102Pyruvate-2-C14 Succinate 100Pyruvate-3-C'4 Succinate 137Succinate-2,3- Pyruvate 162

014

3 Pyruvate 5.0 -

Glucose - 10.7Pyruvate-1, 2,3- Glucose 11.5 15

* Cells (12 mg dry wt) were incubated with0.06 M of each substrate carbon for 2 hr as de-scribed in Materials and Methods.

t As and As + a designate, respectively, radio-activity of cellulose formed from substrate inabsence and presence of supplement.

pensions which were incubated with NaHC'403in the absence or presence of unlabeled pyruvate.

Incorporation of the individual carbon atoms ofpyruvate into cellulose. The succinate-grown cellsincorporated the individual carbons of pyruvateinto cellulose to different extents (see values ofa in Table 2). About 2.8 equivalents of themethyl carbon of pyruvate appeared in the cel-lulose monomer, as compared to 1.4 and 2.1equivalents of the carboxyl and carbonyl carbons,respectively.

Cellulose synthesis in the presence of supple-mentary substrates. When cells were allowed tometabolize pyruvate in the presence of additionalsubstrates (succinate, acetate) whose oxidationmight be expected to yield energy, both the totalmass of cellulose formed and the amount ofpyruvate incorporated into cellulose were foundto increase (Table 3). When glucose served assupplementary substrate, however, the synthesisof cellulose from pyruvate was decreased.

[Gromet-Elhanan (1960) obtained an additiverelationship in cellulose synthesis from a mixtureof pyruvate and glucose.] It should be noted alsothat the presence of pyruvate augmented theincorporation of acetate and succinate carbonsinto cellulose.

In addition, acetate and succinate exertedopposite effects as regards the incorporation ofspecific carbon atoms of pyruvate into cellulose(Table 3, last column).

Distribution of isotope in cellulose monomerformedfrom specifically labeled pyruvate. To clarifythe possible mechanisms by which pyruvatecould serve as a substrate for cellulose synthesis,the distribution of C14 in cellulose formed frompyruvate-1-C'4, 2-C14, and 3-C14 was compared(Table 4). The results demonstrate in each case asymmetrical distribution of the isotope betweenthe upper and lower halves of the anhydroglucosecarbon chain. Although all the carbons of theglucose moiety were found to be radioactive, thebulk of the activity (70 to 75%) was always foundto be evenly shared by only two carbon atoms,namely, carbon pairs 3:4, 2:5, and 1:6 in pyru-vate-1-C'4, 2-C14, and 3-C14, respectively.

Reactions catalyzed by cell-free extracts. Extracts,prepared by disrupting succinate-grown cells in aNossal shaker and assayed as described by

TABLE 4. Isotope distribution in monomer ofcellulose formed by Acetobacter xylinum

from differently labeled pyruvates

Distribution of C14 in cellulosetCarbon in anhydroglucose* Pyruvate Pyruvate- Pyruvate-

-l-C14 2-C4 3-C14

1 4.5 8.0 40.02 9.0 34.0 8.53 34.5 8.5 5.54 38.5 9.0 6.55 9.5 33.0 6.56 4.0 8.0 33.0

Total degradative re- 91 95 92covery of C'4 (%)* Cells (12 mg dry wt) were incubated with

0.02 M pyruvate (800 to 2,000 count per min per,umole) for 3 hr as described in Materials andMethods.

t Values are expressed as percentages of thetotal activity of the monomer. They representaverages of triplicate experiments. The agreementbetween the triplicates was within 5 to 10%.

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Gromet, Schramm, and Hestrin (1957), revealedthe presence of enzyme systems capable of yield-ing: pyruvate and inorganic phosphate from 3-phosphoglycerate, triose phosphate from fructose-1, 6-diphosphate, and hexose-6-phosphate andinorganic phosphate from fructose-i, 6-diphos-phate. The rates of these reactions were similarto those reported by Gromet et al. (1957) forextracts of glucose-grown cells. Assuming thatthese cells also contain the triose phosphatedehydrogenase (Gromet et al., 1957), one can

infer from these findings that the succinate-grown cells possess the complete complement ofenzymes needed for the conversion of pyruvateinto hexose phosphate (Krimsky, 1959a, b).

Tests for the presence in the extracts of a

system capable of equilibrating C1402 with oxalo-acetate in the presence of adenosine triphosphategave negative results. The extracts, therefore,appear to be devoid of phosphoenolpyruvatecarboxykinase activity (Utter and Kurahashi,1954).The extracts of succinate-grown cells were

found to catalyze the following reactions at ratessimilar to those observed with extracts of glucose-grown cells (Gromet-Elhanan, 1960): the oxida-tion of citrate to a-ketoglutarate, the completeoxidation of the latter and of succinate, and theformation of fumarate from malate. These find-ings, in addition to the observed effect of fluoro-acetate in blocking the oxidation of pyruvate or

succinate by whole cells beyond the acetate stage(unpublished data), are compatible with theoperation of a citrate cycle in the succinate-grown cells of A. xylinum (Gromet-Elhanan,1960; King, Kawasaki, and Cheldelin, 1956;Atkinson, 1956; Tanenbaum, 1956).

DISCUSSION

In constructing a scheme for the conversion ofpyruvate into cellulose by A. xylinum, it isnecessary to consider the following observationsmade in this investigation: (i) the symmetricaldistribution of isotope between the upper andlower halves of the anhydroglucose carbon chainformed from singly labeled pyruvate; (ii) thepreferential labeling of anhydroglucose carbonpairs by singly labeled pyruvate (carbon 3:4,2:5, and 1:6, respectively, in the anhydroglucoseformed from pyruvate-1-, 2-, and 3-C14); (iii)the failure of CO2 to serve as a oarbon source forcellulose synthesis in the presence and in the

absence of pyruvate; (iv) the quantitativedifferences observed between the amounts ofradioactivity contributed to cellulose by thedifferent pyruvate carbons (carboxyl < carbonyl< methyl); and (v) the occurrence of radio-activity in all carbons of the cellulose formedfrom singly labeled pyruvate.

In agreement with the suggestion of Bourneand Weigel (1954), based on their findings inA. acetigenum growing on carboxy-C'4 lactate, ourobservations i and ii summarized above indicatethat the anhydroglucose carbon chain of cellulosearises from pyruvate in Acetobacter via a con-densation involving two molecules of a three-carbon compound. The latter could well be triosephosphate (Schramm et al., 1957a; Gromet et al.,1957) formed from phosphoglycerate, which inturn could arise from pyruvate either via phos-phoenolpyruvate or via one or more as yet un-known intermediates (Dickens and Williamson,1959).Two mechanisms for the formation of phos-

phoenolpyruvate from pyruvate have beenproposed: (i) direct phosphorylation of pyruvateby adenosine triphosphate in the presence ofpyruvic kinase (Lardy and Zeigler, 1945; Krimsky1959a, b); and (ii) condensation of pyruvate withCO2 to form malate (Ochoa, Mehler, andKornberg, 1948) or oxaloacetate (Utter andKeech, 1960), followed by phosphorylative de-carboxylation to yield phosphoenolpyruvic acid(Utter and Kurahashi, 1954; Siu, Wood, andStjernholm, 1961). Since malate and oxaloace-tate should rapidly be equilibrated with fumaratein this system (see Results), operation of mecha-nism ii would result in the synthesis of radio-active cellulose by cells incubated with C1402 inthe presence of C12-pyruvate (see review byWeinman, Strisower, and Chaikoff, 1957). How-ever, this was not the case. Moreover, oxalo-acetate was less active than pyruvate as a pre-cursor of cellulose, even though oxaloacetaterapidly entered these cells as evidenced by theobservation that both oxaloacetate and pyruvatewere oxidized at similar rates (Table 1). Thus,it would seem unlikely that oxaloacetate is anintermediate in the pathway from pyruvate tophosphoenolpyruvate. It therefore seems neces-sary to invoke mechanism i (see Hiatte et al.,1958), even though its operation is considered tobe difficult under physiological conditions (Krebs,1954; Krebs and Kornberg, 1957).

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Condensation of two molecules of a three-carbon compound arising from pyruvate shouldyield a product with twice the specific activity ofthe initially labeled pyruvate. Examination of thevalues of a (Table 2) for the different pyruvatecarbons, however, indicates that this was the caseonly for the carbonyl carbon (a = 2.1), whereaslower (a = 1.4) and higher (a = 2.8) values wereobtained for the pyruvate carboxyl and methylcarbons, respectively. A possible way whichwould account for the low contribution of thecarboxyl carbon of pyruvate to cellulose syn-thesis and the high contribution of the methylcarbon is the equilibration of pyruvate (or of athree-carbon unit derived from it) with a sym-metrical four-carbon compound formed by athree-carbon + one-carbon condensation. Inturn, the one-carbon fragment, which is not inequilibrium with exogenous CO2, would arisefrom the methyl carbon of pyruvate. Such anequilibration would result (i) in the loss of halfof the labeling from pyruvate-1-C'4, thus account-ing for the decreased labeling in cellulose formedfrom this substrate compared with that formedfrom pyruvate-2-C'4 or 3-C14; (ii) in an increasedincorporation of the methyl carbon of pyruvateinto cellulose; (iii) in the randomization of isotopeinto positions Cl. C6, C2 C5, and C3 C4 ofanhydroglucose formed from either pyruvate-2-C'4 or -3-C14 but not from pyruvate-1-C14. Inline with this suggestion and assuming that about50% of the pyruvate converted to cellulose wasequilibrated with a symmetrical four-carbon com-pound, the calculated values for the ratio a(Table 2) would be 1.5, 2.0, and 2.5 for pyruvate-1-C'4, 2-C'4, and 3-C'4, respectively, values whichare in close agreement with those found experi-mentally.

If the suggested symmetrical four-carbon inter-mediate were cycled through the citrate cycle,additional randomization of labeling would occurwhen pyruvate-2-C'4 or 3-C'4 is the substrate(Hoberman and D'Adamo, 1960). The appear-ance of radioactivity in all of the carbons of thecellulose formed from pyruvate-1-C'4 can be ac-counted for if one assumes an additional equilibra-tion between triose phosphate and a symmetricalthree-carbon compound like dihydroxyacetone, assuggested by Stjernholm and Wood (1960) forthe propionic acid bacteria.

Although the sum total of the reactions pro-posed explain the differing efficiencies of in-

corporation of the specifically-labeled carbonatoms of pyruvate into cellulose and accountqualitatively for the randomization observed, itshould be noted that the quantitative aspects ofrandomization are not in line with the suggestedover-all scheme. Thus, allowing for the reactionsput forth, pyruvate-3-C'4 should yield the mostrandomized glucose monomer, pyruvate-2-C'4 thenext, and the pyruvate-1-C'4 the least. That thiswas not the case is evident from the data sum-marized in Table 4. The various labeled pyru-vates were similar in that 70 to 75% of the activ-ity of the anhydroglucose was equally shared bythe two carbon atoms which correspond to thelabeled positions of the pyruvate used. The othercarbons of the monomers contained 4 to 9% ofthe activity, with no experimentally significantdifferences in distribution among the glucoseunits derived from the three labeled pyruvates.Schramm, Gromet, and Hestrin (1957b) pre-

sented evidence for the dominance of the pentosecycle in the metabolism of hexoses by glucose-grown cells. With the succinate-grown cells,however, the observed distribution of C'4 in thecellulose formed from the variously labeledpyruvates is not compatible with the participa-tion of a pentose cycle in the oxidative processesof these cells. With the operation of a pentosecycle, the cellulose formed from pyruvate-3-C'4should have a lower specific activity than thecellulose formed from either pyruvate-2-C'4 or1-C14; the data, however, do not agree with sucha prediction. This raises the question as towhether the pentose cycle participates to thesame extent in the succinate-grown cells as in theglucose-grown cells. Alternatively, the possibilityremains that succinate-grown cells do possess anactive pentose cycle, but that kinetic factorsprevailing when pyruvate is a substrate hamperthe flow of material into the pentose cycle.

ACKNOWLEDGMENTS

The authors wish to acknowledge the continu-ous help and interest of the late Shlomo Hestrin.

This investigation was supported by a researchgrant (E-1494) from the National Institute ofAllergy and Infectious Diseases, U.S. PublicHealth Service.

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