metabolism ofsarcina lutea carbohydrate and · metabolism ofsarcina lutea i. carbohydrate oxidation...

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METABOLISM OF SARCINA LUTEA I. CARBOHYDRATE OXIDATION AND TERMINAL RESPIRATION E. A. DAWES AND W. H. HOLMS Department of Biochemistry, University of Glasgow, Scotland Received for publication September 16, 1957 A study of the carbohydrate metabolism of Sarcina lutea was undertaken to elucidate the routes of metabolism available to this strict aerobe which does not metabolize glucose anaer- obically. It was of interest, therefore, to discover whether the organism possesses enzymes charac- teristic of anaerobic fermentation and, if so, their importance in aerobic glucose oxidation. The process of terminal respiration has also been studied. There have been few previous investigations of glucose metabolism by S. lutea. Barron and Friedemann (1941) and Fosdick and Calandra (1945) produced some evidence for the operation of the Embden-Meyerhof pathway in glucose metabolism both with intact cells and cell-free systems. The latter workers, while demonstrating the presence of various enzymes of the Embden- Meyerhof pathway in extracts, did not report on the conditions of aerobiosis employed and stated that the end product of glucose metabolism was primarily acetic acid, although experimental re- sults in support of this statement were not recorded. MATERIALS AND METHODS Growth of the organism. The organism used was a strain originally obtained from Dr. E. F. Gale and was maintained on nutrient agar slants. This strain grew luxuriantly on peptone and glucose-peptone media but did not grow on a glucose-ammonium salt medium. For metabolic studies, cells were grown with forced aeration in 24 or 36 L amounts at 35 C in a medium of the following composition: peptone (Difco), 5 g; KH2PO4, 6 g; NaCl, 0.6 g; per L, adjusted to pH 7.1 with NaOH. At inoculation, a sterile MgSO4 solution was added to a final concentration of 0.04 per cent. After 24 hr growth, cells were checked microscopically for homo- geneity and then harvested on a Sharples super- centrifuge, washed twice with distilled water in a refrigerated centrifuge, and then either used as such or treated as described below. Cells when harvested from the above medium displayed a high endogenous metabolic activity which could be diminished by vigorous aeration of a thick cell suspension at 37 C for a period of 4 to 5 hr (Holms and Dawes, 1955). To avoid complications in the interpretation of results which may be caused by a high endogenous respiration, freshly harvested cells were aerated for 5 hr and the resultant "endogenous-dimin- ished" material lyophilized and stored in bottles at -20 C. Unless otherwise stated, the cells used in the present research were treated in this way; this had the advantage that suspensions of the required density could be prepared rapidly by weighing out the cells and suspending them in water or buffer solution. Lyophilized cells stored at -20 C retained metabolic activities with little impairment over a period of several months. Metabolic experiments requiring the aeration of bacterial suspensions were either carried out in 6 by 1 or 8 by 112 in tubes and the air stream led in via capillary tubing, or in the special ap- paratus described previously (Dawes and Holms, 1956). Analytical methods. Glucose was estimated by the method of Nelson (1944), fructose by the method of Heyrovsky (1956), and pentose by a modification of the Mejbaum (1939) technique. Pyruvic and a-ketoglutaric acids were determined by a modification of the Friedemann and Haugen (1943) method. Mechanical shaking replaced nitrogen-bubbling in the extraction procedures and the total keto acid concentration was ob- tained by a single reading at 431 mg, the isos- bestic point, whereas an additional reading at 390 m,u enabled the relative amounts of each acid to be obtained. The modified method was cali- brated with freshly distilled pyruvic acid and with a-ketoglutaric acid recrystallized from ben- zene. 390 on January 11, 2020 by guest http://jb.asm.org/ Downloaded from

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METABOLISM OF SARCINA LUTEA

I. CARBOHYDRATE OXIDATION AND TERMINAL RESPIRATION

E. A. DAWES AND W. H. HOLMSDepartment of Biochemistry, University of Glasgow, Scotland

Received for publication September 16, 1957

A study of the carbohydrate metabolism ofSarcina lutea was undertaken to elucidate theroutes of metabolism available to this strictaerobe which does not metabolize glucose anaer-obically. It was of interest, therefore, to discoverwhether the organism possesses enzymes charac-teristic of anaerobic fermentation and, if so, theirimportance in aerobic glucose oxidation. Theprocess of terminal respiration has also beenstudied.

There have been few previous investigations ofglucose metabolism by S. lutea. Barron andFriedemann (1941) and Fosdick and Calandra(1945) produced some evidence for the operationof the Embden-Meyerhof pathway in glucosemetabolism both with intact cells and cell-freesystems. The latter workers, while demonstratingthe presence of various enzymes of the Embden-Meyerhof pathway in extracts, did not report onthe conditions of aerobiosis employed and statedthat the end product of glucose metabolism wasprimarily acetic acid, although experimental re-sults in support of this statement were notrecorded.

MATERIALS AND METHODS

Growth of the organism. The organism used wasa strain originally obtained from Dr. E. F. Galeand was maintained on nutrient agar slants.This strain grew luxuriantly on peptone andglucose-peptone media but did not grow on aglucose-ammonium salt medium.For metabolic studies, cells were grown with

forced aeration in 24 or 36 L amounts at 35 C ina medium of the following composition: peptone(Difco), 5 g; KH2PO4, 6 g; NaCl, 0.6 g; per L,adjusted to pH 7.1 with NaOH. At inoculation,a sterile MgSO4 solution was added to a finalconcentration of 0.04 per cent. After 24 hr growth,cells were checked microscopically for homo-geneity and then harvested on a Sharples super-centrifuge, washed twice with distilled water in

a refrigerated centrifuge, and then either usedas such or treated as described below.

Cells when harvested from the above mediumdisplayed a high endogenous metabolic activitywhich could be diminished by vigorous aerationof a thick cell suspension at 37 C for a period of4 to 5 hr (Holms and Dawes, 1955). To avoidcomplications in the interpretation of resultswhich may be caused by a high endogenousrespiration, freshly harvested cells were aeratedfor 5 hr and the resultant "endogenous-dimin-ished" material lyophilized and stored in bottlesat -20 C. Unless otherwise stated, the cells usedin the present research were treated in this way;this had the advantage that suspensions of therequired density could be prepared rapidly byweighing out the cells and suspending them inwater or buffer solution. Lyophilized cells storedat -20 C retained metabolic activities with littleimpairment over a period of several months.

Metabolic experiments requiring the aerationof bacterial suspensions were either carried outin 6 by 1 or 8 by 112 in tubes and the air streamled in via capillary tubing, or in the special ap-paratus described previously (Dawes and Holms,1956).

Analytical methods. Glucose was estimated bythe method of Nelson (1944), fructose by themethod of Heyrovsky (1956), and pentose by amodification of the Mejbaum (1939) technique.Pyruvic and a-ketoglutaric acids were determinedby a modification of the Friedemann and Haugen(1943) method. Mechanical shaking replacednitrogen-bubbling in the extraction proceduresand the total keto acid concentration was ob-tained by a single reading at 431 mg, the isos-bestic point, whereas an additional reading at390 m,u enabled the relative amounts of each acidto be obtained. The modified method was cali-brated with freshly distilled pyruvic acid andwith a-ketoglutaric acid recrystallized from ben-zene.

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METABOLISM OF SARCINA LUTEA

All spectrophotometric measurements weremade in a Hilger Uvispek instrument, fitted witha specially designed thermostatic cell holder,using cuvettes of 1 cm light path.

Protein was determined by the Stickland(1951) method and total nitrogen by the standardmicro-Kjeldahl technique. Oxygen consumptionwas measured by conventional manometric pro-cedures.

Steam-volatile acids were separated by distilla-tion in a Markham still, after adjusting to pH 1by the addition of H2SO4. Six volumes of distillatewere collected and titrated potentiometricallywith 0.02 N NaOH in a stream of C02-free air.For column chromatography the neutralizeddistillates were then concentrated in a continuousvacuum distillation apparatus.

Cell-free extracts. These were prepared by themethods of Mickle (1948) and Lamanna andMallette (1954) using Ballotini no. 12 glass beads(English Glass Company, Leicester).Chromatography. Carbohydrates were chro-

matographed using the solvent systems describedby Norris and Campbell (1949). Phosphorylatedderivatives were run in the solvent of Burrowset al. (1952). Silver nitrate and aniline hydrogenphthalate sprays were used (Partridge, 1946,1949) and for seven carbon sugars, the reagentof Klevstrand and Nordal (1950). Keto acidswere separated as their 2,4-dinitrophenylhydra-zones by the method of Cavallini et al. (1949).Steam-volatile acids were chromatographed oncelite 545 columns, using as liquid phase ethersaturated with 0.5 N H2S04, according to theprocedure of Phares et al. (1952). The acid inthe effluent, after extraction into water, wastitrated to phenol red in a stream of COrfree air.

Isotopic methods. The isotopic techniques em-ployed are described in detail in another paper(Dawes and Holms, 1958).

RESULTS

Effect of conditions of aerobiosis on glucoseutilization. Barron and Friedemann (1941) re-ported that S. lutea does not release CO2 frombicarbonate buffer when incubated with glucoseunder an atmosphere of nitrogen and we haveconfirmed this with our own strain. An experi-ment was also carried out in an open tube,bubbling nitrogen, and oxygen alternatelythrough the cell suspension. Figure 1 reveals themarked effect of the changed conditions of aero-

L.

E 24O

I

a

g '

Time - minutes

Figure 1. Effect of conditions of aerobiosis onglucose utilization. Nitrogen and oxygen bubbledalternately through an open tube containing 5mm glucose and lyophilized cells (10 mg/ml).Temperature, 37 C; rate of gas flow, 850 ml/min.

biosis on glucose consumption. When strictanaerobiosis was achieved in evacuated Thunbergtubes, there was no utilization of glucose inperiods of up to 4 hr.

Oxidation of carbohydrates. The ability offreshly harvested and lyophilized cells to oxidizevarious sugars and sugar derivatives was testedand comparative results are given in table 1.The Qo2 (glucose) value was found to vary, insome cases appreciably, from one batch of cellsto another, but Qo2 values for other compoundsrelative to glucose remained fairly constant andthus enabled the oxidative abilities of differentbatches of cells to be compared on the basis ofQo2 (glucose). Ribose and citrate were bothoxidized at a faster rate by lyophilized cells, whichsuggests that some permeability or transportbarrier has been partially eliminated by thedrying process. Although we reported earlier thatgluconate and 2-ketogluconate were oxidized bythe organism, certain inconsistencies and irrepro-ducible results at a later date led to the discoverythat the original commercial samples of thesecompounds contained impurities which were oxi-dized by the lyophilized cells giving Qo2 valuesof 1.3 and 1.6, respectively. These impuritiesdid not yield pyruvic acid when metabolized inthe presence of arsenite.

Phosphorylated derivatives were not oxidized

1958] 391

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DAWES AND HOLMS

TABLE 1Qo, Values of "endogenous-diminished" Sarcina

lutea for various carbohydratesQO Values for Cells

Substrate

harvesthd Lyophilized

None...................... 3.5 2.3Glucose ...................... 12.7 3.9Glucosamine .................. 9.3 2.0Glucose-6-phosphate .......... 0.0 0.0Glucose-i-phosphate .......... 0.0 0.0Fructose-1, 6-diphosphate ... 0.0 0.0Fructose ...................... 2.5Mannose ..................... 2.1Galactose..................... 1.1Gluconate .................... 0.0 0.02-Ketogluconate .............. 0.0 0.0Ribose ....................... 0.7 1.4Arabinose .................... 0.7Xylose ....................... 0.4Sucrose. 2.02,3,4,6-Tetramethylglucose ... 0.0Citric acid .................... 0.6 1.3a-Glycerophosphate ........... 0.0

Endogenous Qo2 values subtracted from allvalues. Each manometer flask contained: 1.3 ml0.067 M KH2PO4, pH 7.1; 0.5 ml 10 mm substrate(or water for endogenous measurements); 1 mlcell suspension (20 mg/ml); 0.2 ml 20% (w/v)KOH in center well. Air gas phase, temperature37 C, shaking at 140 cycles per min.

by either preparation, thus raising the possibilityof a direct nonphosphorylative pathway forglucose oxidation. Glucose-6-phosphate was notoxidized by protoplasts of the organism whichhad been prepared by lysozyme treatment(Murray, 1956).The importance of phosphate for glucose oxida-

tion was established at the cellular level by thediscovery of "phosphate-deficient" cells. If thesuspending fluid is changed frequently when theendogenous metabolism of cell suspensions isbeing diminished by aeration at 37 C, the result-ing cells utilize and oxidize glucose at a greatlyreduced rate. The addition of inorganic phosphaterestores activity whether measured manometric-ally or by glucose utilization; the latter type ofmeasurement is shown in figure 2. Dialysis ofphosphate-deficient cells further decreased theiractivity which again could be restored by theaddition of inorganic phosphate or arsenate.

None of our cell-free extracts displayed any

oxidative capability towards glucose or othercarbohydrates.The action of inhibitors on glucose oxidation.

When glucose is oxidized, either by fresh or lyo-philized cells, there is an initial rapid rate ofoxygen uptake which falls off when approxi-mately 1.5 ,umoles 02 per ,umole glucose have

TWca/ gAe@a

3-0

No Po,

3.0

il /Time (mins)

Figure 2. Effect of inorganic phosphate on

glucose utilization by an aerated suspension ofcells which had been "endogenous-diminished"with six changes of suspending fluid over a periodof 6 hr. The system consisted of 33 mm tris (hy-droxymethyl)aminomethane (Tris) buffer, pH 7.1,3.34 mm glucose and lyophilized cells, 10 mg/ml.KH2PO4 (pH 7.1) added as indicated to give a finalconcentration of 22 mM.

TABLE 2Action of inhibitors on glucose oxidation by

lyophilized cells

Inhibitor Concentration Inhibition

M X 104 %

Arsenite ................ 20 30.0Azide ................. 40 59.4Cyanide ................ 1 43.0Fluoride ................ 100 0.0Fluoroacetate ........... 8 56.0lodoacetate ............. 1 70.0Malonate* .............. 100 50.0

* At pH 6.0; all others at pH 7.1.In all cases the percentage inhibition is calcu-

lated on the basis of initial rates of oxygen uptakewith 5,pmoles glucose.

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point restores the original rapid rate. The addi-bion of 5 mm arsenite causes the inflection pointbo be clearly defined at 1 ,umole 02 per ,umole4lucose, and a similar result was obtained with-oncentrations of iodoacetate greater than 0.1mM. The action of various inhibitors on glucose)xidation is shown in table 2 where it will beaoticed that arsenite, iodoacetate, cyanide, mal-mnate, azide, and fluoroacetate all inhibit, whereasluoride is without effect.Experiments with 2,4-dinitrophenol (DNP) at

).1-10 mm yielded variable results but, in general,bhe initial rate of oxygen uptake was depressedwhile the total uptake was increased almost tothe theoretical value for complete oxidation ofthe added glucose to CO2 and water. Approxi-nately 50 per cent of the glucose is assimilated in,he absence of DNP and this fact renders doubt-ul the significance of the observed inflectionjoint during glucose oxidation.The inhibitory action of malonate and fluoro-

tcetate indicates the operation of the tricarboxy-ic acid cycle, and the cytochrome system ismplicated by the observed cyanide inhibition.Chromatographic examination for products of

7lucose metabolism. Added glucose (2 mM) rapidlylisappeared from the supernatant of an aerated,ell suspension in water. On chromatograms aairly faint spot, identified as glucose-6-phos-?hate, appeared within 10 min and graduallyntensified until all the glucose had disappeared.klthough a search was made for other carbohy-Irate products of glucose metabolism in these;ystems, using various solvents and spraying-eagents, none was detected even in the presencef inhibitors such as arsenite (1 mM) and fluoride0.22 mM).Following the identification of glucose-6-phos-

)hate on chromatograms, enzymatic tests forhis compound were carried out. Table 3 showshat a small but definite reduction of triphospho-yridine nucleotide (TPN) occurred and in-reased with increasing length of incubation of;he original reaction mixture, thereby confirmingshe chromatographic evidence.Keto acid production in the presence of arsenite.

:n the presence of 5 mm arsenite, pyruvic acidLccumulates at a linear rate from cells whichiave not had their endogenous metabolism di-ninished by aeration. After endogenous diminu-

during glucose oxidation by Sarcina lutea

Time AE340/10 min

min

0 0.0205 0.037

10 0.03730 0.05545 0.05360 0.065

Supernatants obtained from a system contain-ing 32 mm Tris buffer (pH 7.1), 4 mm glucose andlyophilized cells, 8.9 mg per ml.

Glucose-6-phosphate assayed in system: 0.5 ml1 per cent (w/v) NaHCO3, 0.1 ml 0.1 per cent(w/v) Sigma glucose-6-phosphate dehydrogenase,0.1 ml 2 mm TPN, 1.3 ml water, and 1 ml reac-tion supernatant. AE340 read at 10 min.

Time (min)_-, no As,03, 0-0, As,01 (5M)

Endogenous volueS subtracted

Figure S. Production of pyruvic acid fromglucose by aerated cell suspensions in the presenceand absence of arsenite. Freshly harvested cellsuspension aged at 4 C for 24 hr. The system con-tained 26 mm KH2PO4, pH 7.1, 4 mm glucose, 5mM arsenite and bacterial suspension, 7.4 mg/ml.

tion, keto acid production is virtually absent.Keto acids cannot be detected during the oxida-tion of glucose by freshly harvested cells butafter aging or lyophilization small quantities mayappear in the medium; the addition of arsenitecauses a marked accumulation of pyruvic acid.Figure 3 shows that rather more than 1 ,.mole ofpyruvate is produced per MAmole of glucose in thepresence of 5 mm arsenite. In experiments of

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DAWES AND HOLMS

longer duration the pyruvate concentration de-creases after reaching a maximum.The steam-volatile acids produced from glucose

by lyophilized cells in the presence of arsenitewere investigated by column chromatography oncelite (figure 4). Acetic and pyruvic acids were

030 CELITE 545 - ETHER/0-5NH2SO4

* 025 _

~0*20 .u

0o0m15 mz

E

0-05

0 0 40 60 ao0 100 20 40Column effluent (ml)

Figure 4. Column chromatography of steam-volatile distillate from supernatant of glucose me-tabolism in the presence of arsenite.

TABLE 4Dissimilation of glucose-U-C14 by lyophilized cells

in the presence of arsenite

Glucose Rdo Glu- IGlu- Pyu Ratio ofin Radio- cose* coset yr - PyruvateTime inpe activity Assimi- Oxi vate Formed toSuper- in Cells ae ie

Formed Glucosenatant lated dized ~~Oxidizedmm ;Mmoles cpm ,umoles Mmpioles jinoleso 73.75 38.7 58,500 18.8 16.2 16.2 1.0010 16.7 104,250 33.4 23.6 25.2 1.0715 3.1 124,500 39.9 30.7 31.0 1.0130 0.0 123,000 39.4 34.3 36.9 1.08

* Calculated as radioactivity in cells/specificactivity of glucose per ,umole.

t Glucose oxidized = (glucose added)-(glucosein supernatant + glucose assimilated).System contained per 18 ml: 73.7 jAmoles glucose-

U-C'4 (total count 229,900/min), 100 ,umoles ar-senite, 600 mg lyophilized cells; pH 7.1, tempera-ture 37 C, gentle aeration with C02-free air. Atthe indicated times 3 ml samples were withdrawnand centrifuged. The supernatants were assayedfor glucose, pyruvate and radioactivity, and thecells, after washing twice with 10 ml ice-coldwater, were diluted to 25 ml and 0.2 ml portionsassayed.

identified and a third compound, in a positioncorresponding to fumaric acid, was also present.

Dissimilation of uniformly Ci4-labeled glucose inthe presence of arsenite. To obtain more detailedinformation concerning the metabolism of glucosein the presence of arsenite, cell suspensions wereincubated with uniformly C54-labeled glucose andthe fate of the radioactivity was investigated.Data obtained are summarized in table 4. It willbe noticed that a high proportion of the glucoseis assimilated by the cells; one mole of glucoseoxidized yields one mole of pyruvate. After theglucose in the medium had been exhausted, me-tabolism of the assimilated material commenced,as reflected by a decrease of the radioactivity inthe cells.

Experiments with cell-free preparations. Cell-free systems were assayed for various enzymesassociated with different pathways of glucosemetabolism.

(1) Hexokinase:-The phosphorylation of glu-cose can be achieved by hexokinase present inextracts (figure 5). The enzyme could not be

IQ

0-0 Glucose tATP*TPN _- Glucose+TPNArrows indicota further addition of TPN.

Figure 5. Hexokinase activity of cell-freeextract. Spectrophotometer cuvettes contained,where applicable, NaHCO3, 48 /Amoles; MgC92, 40,umoles; glucose, 5 ,umoles; ATP, 12.5 ,umoles;glucose-6-phosphate dehydrogenase, 0.1 ml; andenzyme preparation (1.25 mg protein), 0.2 ml.TPN, 0.2,umole, added as indicated. Total volume,2.8 ml; pH 7.1; temperature, 37 C.

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METABOLISM OF SARCINA LUTEA

0o15

010

000 -0

I010 0 20 30 40 50 60 70 so

Minutes

0-0 Glucose-l-P04+ Extroct; Extroctabon; 0-0 Glucose4Q404

Figure 6. Phosphoglucomutase activity ofcell-free extract. Protocol as for figure 5 exceptthat 5 ,umoles glucose-l-phosphate replacedglucose and ATP was omitted.

detected in some extracts, particularly those pre-

pared by the Lamanna and Mallette (1954) tech-nique, presumably due to the high adenosinetriphosphatase activity of these preparations. Itwill be noticed that the extract used contained a

little glucose-6-phosphate, enabling some reduc-tion of TPN to be obtained in the absence ofadded adenosine triphosphate (ATP).

(2) Phosphoglucomutase:-The conversion ofglucose-i-phosphate to glucose-6-phosphate isshown in figure 6. Here again a small quantity ofglucose-6-phosphate resided in the extract.

(3) Phosphohexose isomerase:-The commer-

cial preparation of fructose-6-phosphate usedcontained some glucose-6-phosphate but theincreased rate of TPN reduction in the presence

of the extract is clear evidence for the presence ofthis enzyme (figure 7).

(4) Phosphofructokinase:-All efforts to detectthis enzyme in extracts have failed.

(5) Aldolase and triosephosphate dehydro-genase:-The formation of 8.5 ,umoles of pyru-vate occurred from 20 ,umoles 3-phosphoglycericacid when 10 gmoles adenosine diphosphate(ADP) were added in the presence of 20 ,umolesarsenite to trap the pyruyate formed and incu-bated for 2 hr. Similar results were obtained withfructose 1,6-diphosphate as the substrate in thepresence of diphosphopyridine nucleotide (DPN),

O.3

0.2

0*I

0-0

O 10 20 50Minutes

0-0 Fructose-6-phospote +Extroct; *-_.Fructose 6-phosphote;0-0 Extract alone

Figure 7. Phosphohexose isomerase activity ofcell-free extract. Protocol as for figure 6 exceptthat 5 ,umoles fructose-6-phosphate replacedglucose.

ADP, and arsenite. These findings indicate thepresence of phosphoglyceromutase, enolase, andpyruvic phosphokinase. Aldolase and triosephos-phate dehydrogenase activity are shown infigure 8.

(6) Glucose-6-phosphate and 6-phosphogluco-nate dehydrogenases:-The first pair of enzymesin the hexosemonophosphate oxidative cycle arereadily demonstrable in cell-free extracts (figure9). This particular preparation gave no TPNreduction with ribose-5-phosphate, although ex-tracts have been obtained which metabolized thiscompound. The activity of glucose-6-phosphatedehydrogenase was greater in glycylglycine thanin phosphate buffer and showed a pH optimumof 7.5. It will be noted that 6-phosphogluconatedehydrogenase was more active than glucose-6-phosphate dehydrogenase in these extracts.

(7) Entner-Doudoroff pathway:-The presenceof a 6-phosphogluconate dehydrase and an aldo-lase specific for 2-keto-3-deoxy-6-phosphoglu-conate, as demonstrated for Pseudomonas sac-charophila by Entner and Doudoroff (1952),could not be shown in our extracts using themethod of Wood and Schwerdt (1954). To date,the Entner-Doudoroff system has been foundonly in gram-negative microorganisms.

Terminal respiration. A comparison was madeof the rates of oxidation of intermediates of the

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396 DAWES AND HOLMS [VOL. 75

~04S

0 00 Jo 0 30 o0 90 520 50 .o0Mi'nutes

Figure 8. Aldolase and triosephosphate dehydrogenase activity of cell-free extract. Spectrophotometercuvettes contained, where applicable, glycylglycine, 24,moles; Tris, 12 pmoles; glutathione, 6,moles;Na arsenate, 25 umoles; fructose-1,6-diphosphate, 5,umoles; and enzyme preparation (2.4 mg protein),0.2 ml. DPN, 0.2,mole, added as indicated. Total volume, 2.5 ml; pH, 7.3; temperature, 37 C. Formeasurement of fructose disappearance the above quantities were doubled and 0.5 ml samples with-drawn at intervals into 2 ml 10 per cent (w/v) trichloracetic acid. After centrifugation, 1 ml portionswere taken for analysis.

o 20 30Tinm (mi)

0C,no subtate; U-U, R-S-P;fO .PGA; 00. 0-6-P.

Figure 9. Glucose-6-phosphate and 6-phospho-gluconate dehydrogenases in cell-free extract.Spectrophotometer cuvettes contained, whereapplicable, glycylglycine, 48 pmoles; substrate,5umoles; MgCl2, 40pmoles; 0.2 ml enzyme prepara-tion (1.6 mg protein), and TPN, 0.2 pmoles. Totalvolume, 2.6 ml; pH 7.5; temperature, 37 C.

tricarboxylic acid cycle by lyophilized cells (table5). Oxalosuccinate was not available for testing.With the exception of cis-aconitate, all the com-pounds were oxidized. Although iso-citrate gavea low rate of oxygen uptake, citrate itself was

rapidly oxidized. However, cell-free extracts werefound to contain aconitase and TPN-dependentiso-citric dehydrogenase activity, as shown infigure 10. Here iso-citric dehydrogenase is therate-limiting step, although some extracts havebeen obtained in which aconitase was not soactive and TPN-reduction was very much morerapid with is-citrate than with citrate or cis-aconitate. The decrease in Eu0 noted in figure 10is due to the reoxidation of reduced TPN (TPNH)which has always been marked in these extracts;further addition of substrate caused rapid reduc-tion once more.The behavior of a-ketoglutarate is particularly

interesting. Freshly harvested cells gave resultssimilar to those of table 5 with the exception thata-ketoglutarate was oxidized at a rate comparableto that of succinate. The lability of the a-keto-glutarate dehydrogenase system is demonstratedby the low rate of oxidation by lyophilized cellsand complete absence of activity in extracts, evenwhen supplemented with coenzyme A, DPN, andglutathione. a-Ketoglutarate accumulates as theproduct of iso-citric dehydrogenase activity inextracts and has been isolated and characterizedas the 2,4-dinitrophenylhydrazone, thus indi-cating that oxalosuccihate is metabolized. In viewof the inhibitory action of malonate and fluoro-acetate, taken in conjunction with the above

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METABOLISM OF SARCINA LUTEA

TABLE 5Qo, values for intermediates of the tricarboxylic acid

cycle using lyophilized "endogenous-diminished" cells

Substrate QO2

Glucose .................... 10.6Pyruvate .................. 8.5Acetate.................... 5.5Citrate.......... 6.3cis-Aconitate ............... 0.0iso-Citrate ................. 0.2a-Ketoglutarate ............ 0.4Succinate .................. 16.4Fumarate .................. 19.8Malate .................... 8.4Oxaloacetate ............... 12.8

Endogenous Qo, values subtracted from allvalues. Protocol as for table 1.

0-3

0-2

0.I

5'amoIe5 .0-6citrote

0-0 * * * *

0 20 40 60

Time (min.)*-4 no substrate; 0--, .e- ketoglutoroteCsO iso-citrote; 4&,citrote; CaO, ct-oconitatc

Figure 10. Aconitase and iso-citric dehydroge-nase activity of cell-free extract. Spectropho-tometer cuvettes contained, where applicable,glycylglycine, 60 pmoles; substrate, 5 ,umoles;MgCl2, 40 Amoles; enzyme preparation (1.4 mg,protein), 0.2 ml; and TPN 0.2 ,umoles. Total vol-ume, 2.6 ml; pH 7.4; temperature, 37 C.

findings, it is concluded that the tricarboxylicacid cycle is operative in S. lutea.Murray and Dawes (1956) showed that the

present organism possesses a cytochrome dif-ference spectrum very similar to that describedby Smith (1954) and, using lysozyme-treatedpreparations, found the cytochrome pigments tobe associated with particulate matter.

DISCUSSION

S. lutea is able to oxidize glucose only underaerobic conditions, and yet extracts of the or-ganism contain all enzymes of the Embden-Meyerhof glycolytic scheme, with the exceptionof phosphofructokinase, an enzyme which isnotoriously labile. It seems quite feasible thatthis enzyme may have been destroyed in thepreparation of the extracts. Certain enzymes ofthe hexosemonophosphate oxidative cycle havealso been found but no evidence for the Entner-Doudoroff system could be obtained.The hexosemonophosphate oxidative cycle re-

sults in the formation of one mole of pyruvic acidwhereas the Embden-Meyerhof and Entner-Doudoroff systems both produce two moles ofpyruvic acid per mole of glucose utilized. In thepresence of arsenite one mole, or rather more,accumulates per mole of glucose utilized and, inaddition, acetate and possibly fumarate arefound. Since acetate is formed by the oxidativedecarboxylation of pyruvate, some of whichmust be escaping the arsenite block, it is clearthat substantially more than one mole of pyru-vate must have arisen per mole of glucose, i. e.,more than can be accounted for solely on thebasis of the hexosemonophosphate oxidativecycle, and the operation of Embden-Meyerhof orEntner-Doudoroff systems must be presumed.Although freshly harvested and lyophilized

cells, and also protoplasts, do not oxidize anyphosphorylated substrate, the importance ofsuch compounds is indicated by the preparationof phosphate-deficient cells with impaired ratesof glucose oxidation, and by the presence in cell-free extracts of hexokinase, other enzymes ofphosphorylative metabolism and also smallamounts of glucose-6-phosphate. A permeabilityor transport barrier toward these compoundsmust therefore reside in the cell membrane.

Support for the operation of the Embden-Meyerhof pathway may be indicated by theobserved iodoacetate inhibition, whereas theapparently conflicting finding that fluoride iswithout effect could be reconciled if the enolaseof this organism were activated by manganese.The impaired rate of oxidation and accumulationof pyruvic acid when glucose is metabolized inthe presence of arsenite suggests that pyruvateoxidation is mediated by a lipoic acid complex.When radioactive glucose is dissimilated in such

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DAWES AND HOLMS

a system more than 50 per cent of the addedglucose is assimilated by the cells.

In the absence of inhibitors the only inter-mediates of glucose metabolism which can bedetected in supernatant liquors are small amountsof glucose-6-phosphate and, in certain casespyruvic acid. No conclusions can be drawn aboutpathways from these findings because, with theexception of the direct oxidative pathways, thesecompounds are common to all known routes ofglucose metabolism.

Thus, studies with cell suspensions and cell-free preparations indicate the possibility thatboth the glycolytic and hexosemonophosphateoxidative pathways of glucose metabolism func-tion in S. lutea, but are operative only underaerobic conditions. The problem of the relativeimportance of the glycolytic sequence underaerobic conditions has been studied isotopicallyand is reported in another paper (Dawes andHolms, 1958). Relevant to this work are theobservations of Hill and Mills (1954). Theyfound that Pasteurella tularensis possesses onlya glycolytic system, yet this is incapable ofanaerobic operation because the organism lacksa DPN-dependent lactic dehydrogenase; anaero-bically reduced DPN cannot be reoxidized. Theaddition of a DPN-dependent mammalian lacticdehydrogenase permitted anaerobic glycolysis tooccur. They reported that they were unable tosecure this effect with S. lutea.

There seems little doubt that terminal respira-tion in this aerobe occurs via the tricarboxylicacid cycle. Glucose oxidation is inhibited byboth malonate and fluoroacetate and cell sus-pensions are able to oxidize all intermediates ofthe cycle with the exception of cis-aconitate. Theposition of the tricarboxylic acids was clarifiedby the detection of powerful aconitase andiso-citric dehydrogenase activity in extracts.Electron transport to oxygen appears to bemediated by the cytochrome system.

ACKNOWLEDGMENTS

Our thanks are due to Dr. Gertrude E. Glockfor a generous gift of 6-phosphogluconate. Weare also greatly indebted to Miss Anne M.Alston, Mr. W. Burns and Mr. J. Smillie fortheir excellent technical assistance at variousstages of this work and to the Medical ResearchCouncil for a grant-in-aid.

SUMMARY

Sarcina lutea does not metabolize glucoseunder anaerobic conditions. Aerobically, thepresent strain oxidized a variety of carbohy-drates, including ribose, but was without actionon phosphorylated compounds, even afterlyophilization. Endogenous metabolism wasdiminished by aeration of cell suspensions forfive hours and if the suspending fluid was changedseveral times during the process the resultingcells had an impaired rate of glucose oxidation;this could be restored to normal by the additionof inorganic phosphate.

Small amounts of glucose-6-phosphate andpyruvic acid are the only extracellular productsof glucose oxidation which can be detected.Glucose metabolism is sensitive to iodoacetateand arsenite but not to fluoride. In the presenceof arsenite, pyruvic and acetic acids accumulate.Enzymes of both the Embden-Meyerhof glyco-lytic sequence and the hexosemonophosphateoxidative cycle have been found in cell-free ex-tracts but no evidence for an Entner-Doudoroffcleavage was obtained. These results suggestthat the glycolytic pathway may operate underaerobic conditions although unable to do soanaerobically.The tricarboxylic acid cycle operates as a

terminal respiration pathway in the organism.

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