ENDOGENOUS RESPIRATION AND OXIDATIVE ASSIMILATION INATOCARDIA CORALLINA

GRAEME G. MIDWINTER AND RICHARD D. BATT

Department of Biochemistry, University of Otago, Medical School, Dunedin, New Zealand

Received for publication May 11, 1959

Nocardia corallina oxidizes a variety of organiccompounds including glucose, acetate, succi-nate, and propionate and, in each case, theamounts of oxygen used and carbon dioxidereleased have been less than the theoreticalvalues for the complete oxidation of the sub-strate. The incubation systems contained noproducts of incomplete oxidation of the sub-strates and, with the results obtained from usinguncoupling agents (sodium azide and 2,4-dinitrophenol), it appeared that the low gas ex-change values were due to concurrent oxidativeassimilation from the substrates.

Oxidative assimilation has been studied withmany bacterial species and the subject has beenreviewed by Clifton (1952, 1957). Barker (1936)studied the oxidation of substrates by the color-less alga Prototheca zopfii, and formulated theproblem of correcting for endogenous respirationwhen studying microbial assimilation, mano-metrically. He stated that "one may take thatmethod of correction to be most basically correctwhich when applied to all vessels gives the mostconstant value of oxygen consumption per unitquantity of substrate decomposed." The validityof subtracting endogenous values in calculatingoxygen uptake figures for substrates removed bycells has been reviewed and discussed by Wilnerand Clifton (1954). In general, the endogenousrespiration does not appear to be suppressedduring exogenous respiration (Moses and Syrett,1955; Cochrane and Gibbs, 1951; Blumenthalet al., 1951; Santer and Ajl, 1954).

This communication (a) considers the rela-tionship between endogenous and exogenousrespiration for N. corallina, (b) describes theeffect of inhibitors on cellular respiration, and(c) includes evidence for oxidative assimilationfrom isotopically labeled substrates.

METHODS

The strain of N. corallina used in the investiga-tion was isolated by enrichment culture using

9

uracil as the main source of carbon and nitrogen(Batt and Woods, 1951). The parent strain waspreserved as a freeze-dried preparation and, forexperimental work, a stock culture was main-tained on glucose (0.75 per cent)-yeast extract(10 per cent)-agar (3 per cent) slopes.Preparation of cell suspensions. Growth media

(200 ml/500 ml conical flask, pH 7.2), containing,per L, a carbon substrate e.g., glucose, acetateor propionate (2 g), ammonium sulfate (1.5 g),thiamine (1.0 mg), inorganic salts (KH2PO4,5 g; MgSO4*7H20, 2 g; and CaCl2, 2 g) and phos-phate buffer (0.1 M) were inoculated from 48-hr-old glucose-yeast extract-agar slopes and in-cubated aerobically at 30 C with slow shaking(60 oscillations per min). The cells were har-vested by centrifugation (2500 X G), washedonce with phosphate buffer (0.1 M; pH 7.2),and suspended in buffer to give a suspensionequivalent to 10 mg dry weight cells per ml. Thesuspensions showed no detectable alteration inmetabolic activity after storing for several daysat 0 to 4 C.

Manometric experiments. (1) With Cl2-sub-strates:-Cell suspension experiments were car-ried out in Warburg manometers using conven-tional procedures (Umbreit et al., 1949) for esti-mating oxygen consumption and carbon dioxideproduction. Each flask contained cells (equiva-lent to 10 mg dry weight), phosphate buffer(0.066 M; pH 7.2), substrate (10 ,imoles); totalvolume, 3.2 ml. The incubation temperature forall experiments was 30 C. The rates of oxygenconsumption (Q02) were expressed as micro-liters of oxygen consumed per hr per mg dryweight cells.

(2) With C@-substrates:-Studies on cellsuspensions oxidizing C'4-labeled substrateswere based on an initial incubation of cells(150 dry weight) with C'4-labeled substrate(200 Amoles) in phosphate buffer (0.033 M;pH 7.2; 30 ml) using large Warburg manometercups (volume, 210 ml). The oxidations proceeded

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to completion and the rate and extent of the reac-tions were followed by measuring oxygen uptake.At the conclusion of the experiment, sulfuric acid(5.0 ml; 1.0 N) was tipped into the system froma side bulb and the flask left to shake for 30 min.The KOH (10 per cent) in the center well wasremoved and, together with the washings andpaper, made up to a final volume of 5.0 ml withCO2-free distilled water for plating. The incuba-tion system was centrifuged to give a super-natant and cells, preparatory to wet combustionand plating.

(3) Release of radioactive CO2 from C'4-labeledcells:-Labeled cells (C14) were prepared byincubation of strain S (grown on a glucose ammo-nium sulfate-thiamine medium; 190 mg dryweight) with a C'4-labeled substrate (200 ,moles:either 3-C'4-propionate, 1-C14-acetate, or U-C'4-glucose) until exogenous oxygen-uptake ceased.The cells were removed, washed and resus-

pended in phosphate buffer (pH 7.2; 0.033 M)to give a final concentration of cells equivalentto 10 mg dry weight/ml. The activity of the cellswas determined by wet combustion of a samplefollowed by plating and counting of the CO2produced.The release of C1402 from the cells during exoge-

nous and endogenous respiration was measuredby incubating the cells (14 mg dry weight) insmall manometer cups (volume, approximately30 ml); half of the samples (7 manometers) inthe absence of added substrate and the remainder(7 manometers) with 100 ,umoles of unlabeledcarbon substrate. At hourly intervals the reac-

tion in a manometer from each group was stoppedby tipping sulfuric acid (0.2 ml; 2 N) from a sidebulb and, after shaking to permit dissolved CO2to be taken up by the center well KOH, the con-

tents of the center well were removed for count-ing.

Radioactivity estimations. Samples containingcarbon solely in the form of carbon dioxide were

plated as BaCO3 on filter paper discs and countedwith an end window Geiger-Muller counter.The counts recorded were corrected for back-ground and selfabsorption. The radioactivity incells or supernatants from incubation systemswas estimated by first oxidizing the organicallybound carbon to carbon dioxide with a chromicacid solution (Van Slyke and Folch, 1940)and then plating the trapped carbon dioxide (in3 N NaOH) as BaCO3.

Chemicals. Sodium propionate (3-C14) wassupplied by Professor H. G. Wood; U-C'4-glucoseand 1-C'4-acetate were obtained from the Radio-chemical Centre, Amersham, England.

Fractionation of cellular constituents. Cells(290 mg dry weight) grown under standard con-ditions in a glucose-ammonium sulfate-thiaminemedium for 48 hr were incubated with 3-C'4-propionate (220 ,moles) in a large Warburgflask until the oxidation of the propionate wascomplete. The cells were centrifuged, washedonce with distilled water, and a sample oxidizedto CO2 to determine the extent of the assimila-tion from propionate (68,700 cpm in 290 mg wetweight cells). The residual cells were dried,ether extracted for 4 hr, and the ether solutionevaporated to dryness; yield of lipid, 83.5 mg.The dried cells were next extracted for 2 hr withboiling chloroform (100 ml); yield of lipid, 15.1mg. The ether and chloroform soluble componentsof the cells therefore constituted 33.8 per centof the dry weight of the cells.

Polysaccharides were separated from the cells(now lipid-free) by treatment with 15 per centKOH (50 ml; 100 C; 2 hr) and the supernatant,containing carbohydrate was prepared by cen-trifugation. The clear solution was neutralizedwith 5 N HCl and further hydrolysis carried outby adding an equal volume of 2 N HCl and heat-ing (100 C; 1 hr). Glucose (10 mg; C"2) wasadded to the hydrolyzate which was extractedthree times with small volumes (5.0 ml) of re-distilled pyridine. The pyridine extracts wereevaporated to dryness and the residue extractedwith water (5.0 ml). To the aqueous solution,2, 4-dinitrophenylhydrazine hydrochloride (100mg) in acetate buffer (5.0 ml; pH 5.0) was addedand the mixture heated on a boiling water-bathfor 1 hr. On cooling a crude crystalline osazonewas obtained; yield, 51.2 mg. Two samples ofthe osazone (10 mg) were oxidized to CO2 andassayed for C14 activity.A sample of glucose (10 mg) subjected to the

same procedures gave a good yield of glucosazone(18 mg).

RESULTS

Endogenous respiration of strain S. Factorsaffecting the endogenous respiration of strain Swere studied, initially, by growing the organismon different carbon substrates and determiningthe respiratory activity of cell suspensions pre-

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TABLE 1Endogenous respiration and acetate oxidation by

various cell suspensions of strain S

Endogenous RespirationCarbon . espiRespiration Aatef Moles 02

Substrate for w Values Oxidation Consumed/CellGrowtb.~~~~~~~~ pMole-llGrowthAcetate

QO0 RQ Qo2 RQ

hr

Acetate 24 12 0.92 69 0.94 0.8948 7 0.91 39 0.91 1.0072 1 1.00 17 0.97 0.9996 0 * 16 0.91 1.01

Propio- 24 14 0.98 66 1.08 0.94nate

48 11 0.89 34 0.93 0.9472 7 1.07 32 0.97 1.0296 0 0.97 13 0.99 1.03

Glucose 24 11 0.94 60 1.06 0.9648 15 1.09 38 0.90 0.9872 11 1.00 27 1.07 1.0096 4 1.00 14 1.00 1.00

* The respiration rate was too low to give asignificant CO2 production value.The experiments were carried out using the

standard manometric conditions (see Methods).Acetate concentration: 10 umoles per manometercup. The Qo2 values were determined from themaximum gradient of the oxygen-uptake curves.The RQ figures were calculated from the total oxy-gen uptake and the CO2 production values after3 hr incubation, i. e., at a time when all of the sub-strate had been degraded by the cells.

pared from inoculated media which had beenincubated aerobically at 30 C for either 24, 48,72, or 96 hr. Each growth medium containedammonium sulfate, thiamine, inorganic salts,and either acetate, propionate, or glucose as thecarbon source (see Methods). The cell suspen-sions were studied manometrically to determinethe rates of endogenous respiration (Qo2E) andthe respiratory quotients for the cells (table 1).The growth curves were determined for the or-ganism growing in media containing the threedifferent carbon substrates; growth was completein 24, 48, and 72 hr for media containing, respec-tively, acetate, propionate, and glucose as thecarbon substrates. Although it is convenient torefer to the period of incubation of the inoculatedmedium (i. e., prior to centrifuging for the prepa-ration of cell suspensions) as the growth time forthe cells, it bears no direct relationship to either

the mean generation time for the organism or tothe time required for maximum cell density to bereached in the growth medium. The growth timerepresents a period of active growth on the sub-strate and, for some of the longer growth times, aperiod (before harvesting of the cells) of incuba-tion of the nonproliferating cells (i. e., in the"resting" state).Although the rates of endogenous respiration

for strain S (table 1) decreased with increasinggrowth times for the cells, the respiratory quo-tients for all of the suspensions tested were in-cluded in the range 0.89 to 1.09. The rate ofendogenous respiration for each suspension wasconstant during the course of the manometryexperiments, i. e., oxygen-uptake curves for allof the suspensions used were linear.

Effect of azide, iodoacetate, and arsenite on theendogenous respiration. With cells grown in a

TABLE 2Effect of azide, iodoacetate, and arsenite on the rate

of endogentous respiration

Per CentAdditions to Incubation System* Qo2 Difference from

Qo2 = 16

Nil 16 0Sodium azide

0.0033 M 24 +500.0016 M 21 +310.0008 M 18 +120.0004 M 17 +6

Sodium iodoacetate0.001 m 12 -250.0005 M 13 -190.0002 M 14 -120.0001 M 15 -6

Sodium arsenite0.0033 M 9 -430.0016 M 9 -430.0008 M 14 -130.0004 M 16 0

* The incubation system contaisied cells (10 ingdry weight) in phosphate buffer (0.066 M; pH 7.2)and the inhibitors were added to give the finalconcentrations shown in the table. The Qos valueswere determined for the interval 60 to 90 min; theeffect of choosing another time interval duringthe manometry experiments was slight since theoxygen-uptake curves were essentially linear.Increasing the final concentrations of sodiumiodoacetate and sodium arsenite above the valuesshown depressed the Qo2 values further.

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glucose-ammonium sulfate-thiamine medium for48 hr, the rates of endogenous respiration wereconsiderably increased in the presence of 0.0033M and 0.0016 M sodium azide (table 2). TheQ02E values were (a) unaffected by azide concen-trations lower than 0.0004 M and (b) re(iucedwith azide at concentrations above 0.01 M.The oxygen-uptake curves were linear at eachconcentration of sodium azide tested.Sodium iodoacetate and sodium arsenite were

tested, each at several different concentrations(table 2). Both substances at the higher concen-trations inhibited the rate of endogenous respira-tion but at no concentration tested was there anyincrease in the Q2E values.

Oxidation of acetate by cell suspensions. Varia-tions in the respiration values for cells of strainS oxidizing acetate were studied using suspen-sions of the organism (a) prepared after differentgrowth times (24, 48, 72, or 96 hr) and (b)grown on different carbon substrates (acetate,propionate, or glucose). The respiration valuesobtained (table 1) were all corrected for the en-dogenous values, the validity of this procedurehaving been deduced first from experiments (re-ported below) on the release of C@4 from labeledcells in the presence and absence of acetate andthen from the observation that the ratios, ,umoles02 consumed per gmole acetate oxidized, shownin table 1, were included in a range of 1.01 to1.92 if no correction for endogenous respirationwas made (range for corrected values, 0.89 to1.03). The rates of oxidation of acetate werelower with cells reaped after the longer growthtimes, e. g., with propionate-grown cells, theQ02acetate figures paralleled more closely thechange in the Qo2E values for the cell suispensions.The relationship between the rates of (a) acetateoxidation and (b) endogenous respiration forcells grown on acetate as the carbon substrateare indicated in the corrected oxygen-uptakecurves for acetate oxidation (figure 1).

Oxidation of propionate and glucose by cellsuspensions. The cell suspensions used to stu(lyacetate oxidation (table 1) were similarly testedwith propionate and glucose as substrates. TheQ02Propionate and Q02glucOse values were lowerwith older cell suspensions; Q02ProPionate forpropionate-grown cells decreased from 90 for24 hr cells to 14 for 96 hr cells and the Q02glucosefor glucose-grown cells decreased from 25 (24-hrcells) to 11 (96-hr cells). These decreases in Q02

24hr,

w

o 100 /B-4hr

72 hr

- - - ~~~~~96hr0 -0 2 3

TIME (hr)

Figure 1. The oxygen uptake curves for cell sus-pensions grown for various times (shown on thegraphs) in an acetate-ammonium sulfate-thiaminemedium and incubated either with acetate (10,umoles; solid lines) or alone (broken lines). Theresults for acetate oxidation have been correctedfor endogenous respiration.

paralleled the observed decreases in the endoge-nous respiration rates of the respective cells.The respiratory quotients for propionate oxida-tion were all within the range 0.78 to, 0.89 anidthe ratios, ,umoles 02 consumed per ,umole pro-pionate oxidized, were in the range 1.43 to 2.00.The corresponding figures for glucose oxidationwere as follows: RQ values, 0.94 to 1.12, and theratios Amoles 02 consumed per Amoles glucoseoxidized, 1.65 to 2.02.

Effect of azide and 2,4-dinitrophenol ont sub-strate oxidations. The oxygen uptake values forthe oxidation of acetate, propionate, or glucoseby suspensions of cells grown under differentconditions (table 1) were all low when comparedwith the theoretical values for the complete oxi-dation of these substances to CO2 and 1120.Theoretical values for the oxidation of the sub-strates (moles 02 required to completely oxidizea mole of substrate) and the observed oxygenuptakes (corrected for endogenous values)when the substances were completely removed by

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cell suspensions of strain S were as follows:acetate, theoretical, 2.0, observed, 0.89 to 1.03;propionate, theoretical, 3.5, observed, 1.43 to2.00; and glucose, theoretical, 6; observed, 1.65to 2.02.The low oxygen uptake values (as compared

with the theoretical values) suggested thatoxidative assimilation from the substrates wasoccurring during the oxidations by the cell sus-pensions. Both 2, 4-dinitrophenol and sodiumazide were tested as uncoupling agents for theoxidations, each over a range of concentrations.The oxygen uptake values for the substrate deg-radations were increased with suitable concen-trations of each inhibitor; the most effective un-coupling concentrations for the quantity of cellsused were for (a) 2,4-dinitrophenol, 0.00016 Mand (b) sodium azide, 0.0033 M. When used at theeffective uncoupling concentrations both sub-stances decreased the rate of substrate oxidation(table 3).

TABLE 3Effect of sodium azide and 2, 4-dinitrophenol on the

oxidation of substrates by cell suspensions

Rate of Oxidation (Qo2) forCell Suspensions with:

Inhibitor addedNo sub- Acetate Propi- Glucosestrate onate

Nil................16 34 27 29Azide (0.0033 M).24 14 3 172, 4-Dinitrophenol

(0.00016 M). 22 14 12 12

Oxygen Uptakes (jumoles 02Consumed/jAmole SubstrateRemoved) for Cells with:

Acetate Propi- Glucoseonate

Nil................ 1.02 1.6 1.8Azide (0.0033 M) 1.81 2.77 2.602, 4-Dinitrophenol

(0.00016 M) ....... 1.52 2.40 2.10

The incubation systems contained cells (grownon a glucose-ammonium sulfate-thiamine mediumfor 48 hr) and phosphate buffer (0.066 M; pH 7.2).Additions to the systems: carbon substrates (10,umoles/manometer cup) and inhibitors to give thefinal concentrations shown in the table. All res-

piration values for the substrates were correctedfor the values obtained in the absence of the sub-strates.

Oxidative assimilation from CJ4-labeled sub-strates. During the oxidation of substrates bycell suspensions of strain S, oxidative assimilationof part of the substrate was assumed to occurbecause of (a) the low over-all oxygen uptakefigures for substrate oxidation and (b) the effectof uncoupling agents on the oxidations. Directevidence for assimilation of carbon by cell sus-pensions from substrates was obtained from ex-periments in which C'4-labeled compounds(3-C'4-propionate, 1-Cl4-acetate, and U-C'4-glucose) were incubated for a defined time withsuspensions of strain S. The incubation systemwas subsequently divided into three fractions,namely, cells, supernatant, and carbon dioxidewhich was released by acidifying the whole sys-tem and trapping the gas in alkali. The fractionswere assayed for C14 activity and the results(table 4) showed that C14 was assimilated fromthese substrates by strain S.

Release of assimilated carbon from cells in thepresence and absence of substrates. Cell suspen-sions, which had been incubated with either 1-C14-acetate, 3-C'4-propionate, or U-C14-glucose,were washed and shaken aerobically with andwithout unlabeled substrates by the proceduredescribed in Methods. The rates of release ofC1402 from the cells were determined and are

TABLE 4Assimilation oj C14 from labeled substrates by cell

suspensions of strain S

Substrates Oxidized by theCell Suspensions

1-C'4- 3-C14-pro- U-C14-acetate pionate glucose

Cpm in the substrateadded.. 20,950 17,650 37,950

FractionationCpm in the respiredCO2..............-.6,490 7,250 10,380

Activity retained incells (cpm) ........ 11,200 6,315 24,825

Activity in the su-pernatant (cpm). 1,880 2,740 2,780

Total activity recov-ered (cpm).19,570 16,305 37,985

Experimental details are given in Methods. Theactivities of the substrates added to incubationsystems are given as the total number of cpm in200,umoles of each substance.

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0 PiO E (+)

2,60w-_J

tn-aj<

U ~~~~~~~~~PROPIONATE(w

40

ACETATE

20 AC TATE

0

0 2 4 6

T I ME (hr)

Figure 2. The release of assimilated C'4 as C1402from cells (14 mg dry weight) which had been incu-bated with either 1-C14-acetate, 3-C'4-propionate,or U-C14-glucose and subsequently incubatedalone (E) or with the same substrate (but un-

labeled) used for the initial assimilation (+).Details of the experimental procedure are given inMethods.

TABLE 5

Distribution of assimilated C14 (from 3-C'4-pro-pionate) in the cellular components

of strain S

Toan Activity ofSample Assayed Sample the OriginalSampleAssayedASsayped Cells (290Asyd

mg Dry Wt)

cPM %

Original cells 68,700 100Ether-extractable lipid

(83.5 mg) 2540 36.8Chloroform-extractable 25,400

lipid (15.1 mg)Osazone 36,000 52.4

In each case, the sample, converted to CO2 bywet combustion, was obtained from 290 mg dryweight of the cell suspension of strain S.

represented graphically in figure 2. Cells whichhad assimilated C14 from 1-C'4-acetate released(a) 18 per cent of the activity in 6 hr while respir-ing alone and (b) 25 per cent of the activity ifthe cells were oxidizing unlabeled sodium acetateduring the same period. When U-C'4-glucose was

the substrate from which assimilation occurred, arelease of 13 per cent of the cellular activity (C'4)occurred in the absence of substrate in a hr and16 per cent was released if the cells were simul-taneously oxidizing unlabeled glucose. With cellswhich had assimilated C14 from 3-C'4-propionate,48 per cent of the activity was released in 6 hrwhen the cells were shaken without added sub-strate while 71 per cent appeared in the sameperiod if the cells were concurrently oxidizingunlabeled propionate.

Distribution of C14, assimilated fromn substrates,in cell fractions. A preliminary fractionation ofthe cellular components, following the incubationof a suspension vith 3-C'4-propionic acid, wascarried out by the procedure described inMethods. Monosaccharide (glucose) obtainedfrom the hydrolysis of cellular polysaccharide wasconverted to the osazone after the addition ofcarrier glucose. From the yield of osazone, aftermaking an allowance for the carrier glucoseused, it has been calculated that not more than20 mg of monosaccharide (glucose) was releasedduring the hydrolysis of 290 mg dry weight cells.The activities of the fractions obtained from

the separation of the cellular components aregiven in table 5.

DISCUSSION

Endogenous respiration. N. corallina strain Sshowed a high rate of endogenous respiration(Qo2E) when prepared from growth media after24 to 48 hr growth (table 1) and this rate wasinvariably lower for cells grown for longer times(72 to 96 hr). The low Q02E values for the oldercells were assumed to be due to the depletion ofthe reserves of cellular components which werethe substrates for endogenous respiration.The endogenous respiratory quotients for all

of the cell suspensions studied (table 1) werewithin the narrow range of 0.89 to 1.00 evenalthough (a) the cells had been grown on dif-ferent carbon substrates and (b) the cells hadbeen reaped from the growth media after dif-ferent growth times. With cells grown on acetateas the carbon source and containing approxi-mately 30 per cent lipid, the respiratory quotientsranged from 0.91 to 1.00 and in no instanceapproached the typical RQ values for cellsoxidizing lipids. Accordingly, it has been con-sidered likely that the immediate substrates forendogenous respiration are carbohydrates; the

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inhibitions of endogenous respiration by sodiumarsenite and sodium iodoacetate are consistentwith this concept (table 2). The endogenousrespiration rates of the various cell suspensionswere increased with those concentrations ofsodium azide and 2, 4-dinitrophenol (table 2)which had been shown to be most effective ininhibiting oxidative assimilation during sub-strate oxidations. The increased Qo2E valueswith azide and 2,4-dinitrophenol are explicableif normal endogenous respiration is associatedwith some resynthesis of the endogenous sub-strate from an intermediate on the catabolicsequence, using energy from the oxidative deg-radations.

+ 0Endogenous substrate 7 i i

+ energy

intermediate CO2 + H20

Cells respiring endogenously in the presence ofthe uncoupling agents would be degrading theendogenous substrates without concurrent re-svnthesis and could lead to the observed increasedrates of endogenous oxygen uptake.

Oxidation of acetate by cell suspensions. TheQ02 values for acetate oxidation by cells grownfor 24 hr were approximately the same for eachcarbon nutrient used (table 1). However the Q02acetate values were much lower for cells grown forlonger periods and the reduction in these valuesfor the older cells paralleled a reduction in theQ,2E values for the same cells. The observedlow respiration values for older cells may be dueto a decrease in the number of metabolicallyactive cells in the older suspensions. However,on this basis cells having a zero rate of endoge-nous respiration should have zero metabolicactivity against acetate. In fact, the cells retaina proportion of their initial metabolic activitywhich is independent of the extent of the en-dogenous respiration of the cells (figure 1; 72and 96-hr-old cells).The oxygen uptake curves for acetate oxida-

tion by cells having no endogenous respirationshowed an initial lag and a subsequent increasein the rate of oxidation (figure 1). The nature ofthe curves suggested that an autocatalyticprocess occurred during acetate oxidation by thecells. In young cells the oxidation of the endoge-nous substrates may be assumed to supply the

citric acid cycle intermediates required for ace-tate oxidation (Santer and Ajl, 1954). However,strain S has been shown to possess isocitritaseactivity and, in cells having no endogenous respi-ration, the glyoxylate by-pass mechanism (Korn-berg and Krebs, 1957) may be considered tosupply citric acid cycle intermediates for theterminal oxidation of the acetate. Since theamounts of the cycle intermediates formed bythis mechanism would increase with increasingrates of acetate oxidation (Wong and Ajl, 1957),the entire process for acetate oxidation couldshow the characteristics of an autocatalyticsystem. If the inflected acetate oxidation curves(figure 1) were, in fact, due to a limited supplyof citric acid cycle intermediates, the inflexionshould be removed if a substance such as succi-nate was added to the cells showing no endoge-nous respiration. In a typical experiment, cells(96-hr-old) oxidizing acetate were comparedwith cells oxidizing a small amount of succinate(2 j,moles) in the presence of acetate (10 ,umoles).The presence of succinate considerably shortenedthe lag on the acetate oxygen uptake curve (aftercorrections had been made for succinate oxida-tion). Cell suspensions which retain metabolicactivity when all endogenous respiration hasceased, constitute a less complicated system andmay prove to be more useful for studying wholecell metabolism than the conventional restingcell suspensions.

Oxidative assimilation was shown to haveoccurred during the oxidation of acetate by cellsuspensions of strain S from the following re-sults: (a) the oxygen uptake values for acetateremoval by the organism were equivalent toapproximately 50 per cent of the theoreticalvalues for complete oxidation (table 1); (b) inthe presence of suitable concentrations of sodiumazide and 2,4-dinitrophenol the oxygen uptakesfor acetate oxidation could be increased (table 3);(c) cell suspensions oxidizing 1-C14-acetateincorporated 57 per cent of the C14 into the cells(table 4). The ability of the cells to assimilatepart of the acetate oxidized was independent of(a) the age and (b) the Q.2E values of the cells(table 1). The processes for acetate oxidation(catabolism) and oxidative assimilation fromacetate (anabolism) appear to be geared together.

Oxidation of propionate and glucose by cellsuspensions. The Q02 values for propionate andglucose oxidations decreased as cells of increasing

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age were used in the incubation systems and therelationship between Q02substrate and Q02E forthe cells was the same as had been observed foracetate oxidation.

Oxidative assimilation occurred during theoxidations of propionate and glucose (table 3);the extent of the assimilation was independentof (a) the growth time for cells used and (b) theQ02E values of the cells. The assimilatory andoxidative mechanisms for these substrates (asfor acetate) appeared to be closely interrelated.

Inhibition of oxidative assimilation. The totaloxygen uptake values for the complete removalof acetate, propionate and glucose by cell sus-pensions of strain S were all increased in thepresence of certain concentrations of sodiumazide or 2,4-dinitrophenol (table 3). In no in-stance however, did the oxygen uptake in thepresence of the inhibitor reach the theoreticalvalue for complete oxidation of the substrate,even although isotope experiments (table 4)had indicated that the low oxygen uptakes weredue to the assimilation of all of the substrate notoxidized. With reference to acetate oxidation bycell suspensions, the oxygen uptake for the com-plete removal of the substrate was equal toapproximately 50 per cent of the theoreticalvalue for complete oxidation (table 1) and theamount of C14 assimilated from 1-C14-acetatewas equal to approximately 50 per cent of thetotal activity of the acetate added to the cells(table 4).Although the inhibition of oxidative assimila-

tion leads to an increased over-all oxygen up-take for a substrate, the rate of oxidation of thesubstrate is usually reduced by the inhibitor.With concentrations of inhibitor capable of com-pletely uncoupling oxidative assimilation (andso giving the theoretical oxygen uptake forcomplete oxidation of the substrate) the rate ofoxidation is usually depressed to such an extentthat the time required for the oxidation to go tocompletion is impractically prolonged. Theexperimental results suggest that the enzymesfor substrate oxidation are more readily inhibitedby the uncoupling agents than the mechanismsfor assimilation from substrates.

Assimilation of C14 from labeled substrates andthe subsequent release of C14 from the cells. Directevidence was obtained for the occurrence oroxidative assimilation in strain S from the ob-servations that during the oxidation of Cl4-

labeled substrates, the isotope was incorporatedinto the cellular constituents (table 4). The ex-tent of the assimilation estimated from the per-centage of the substrate C14 incorporated intothe cells, was approximately the same as theestimates made from the low over-all oxygenuptake figures for the oxidation of the substratesby cell suspensions.The rates of release of C'4 from cells which

had been preincubated with labeled substratesshowed that, if the cells were concurrently oxi-dizing an unlabeled substrate, the rate of releaseof C"4 may be different from the values for thecells respiring endogenously (figure 2). Accord-ingly, no general rule may be made concerningthe validity of correcting the respiration valuesfor substrate oxidation by whole cells for theendogenous respiration figures. The presence orabsence of glucose did not markedly change therate of release of C'4 from cells which had assimi-lated carbon from this substrate. However, withcells which had previously oxidized 3-C'4-pro-pionate, the release of C14 was considerablyincreased in the presence of unlabeled propionateby comparison with the same cells respiring en-dogenously. The incorporation of C14 from pro-pionate appears to lead to the synthesis of a labilecellular constituent which is more rapidly metab-olized in the presence of unlabeled propionatethan during cellular respiration in the absence ofadded substrates. Similar results have been ob-served with other microorganisms (Gibbs andWood, 1952). When Pseudomonas fluorescens wasincubated with C'4-labeled glucose for a shortperiod, the cells showed an increased release ofC402 if unlabeled glucose was added to the cells,by comparison with the cells respiring endoge-nously. Cells grown on U-C'4-glucose, showed thesame rate of release of C'402 irrespective ofwhether or not the cells were oxidizing C12-glucose(Gibbs and Wood, 1952).The preliminary experiments carried out to

determine the distribution of C'" in cells whichhad oxidatively assimilated the isotope fromlabeled substrates (table 5) showed that although37 per cent of the counts were recovered in thelipid fraction (equivalent to approximately 30per cent of the dry cell weight), 52 per cent werepresent in a carbohydrate fraction which con-stituted only a very small percentage of cellularmaterial (less than 8 per cent). The very highactivity of the carbohydrate fraction suggested

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ENDOGENOUS RESPIRATION IN N. CORALLINA

that carbohydrate may be the immediate cellularconstituent on the assimilation pathway.

SUMMARY

The respiratory quotients for the endogenousrespiration of Nocardia corallina strain S, grownfor different times on various carbon substrates,were all in the range 0.89 to 1.00. The rates ofendogenous respiration were increased withcertain concentrations of sodium azide and 2,4-dinitrophenol. The relationship between (a) therate of oxidation of acetate, propionate, andglucose and (b) the rate of endogenous respira-tion of the cells used, was examined. Cells show-ing no endogenous respiration oxidized acetate.Oxidative assimilation was shown to occur duringthe oxidation of _-C'4-acetate, 3-C'4-propionate,and U-C'4-glucose by cell suspensions. The re-lease of C14 from the labeled cells was faster forcells oxidizing unlabeled propionate and acetatethan for cells respiring endogenously. Glucosehad little affect on the release of C1402 from thecells. The distribution of C14 in the constituentsof cells which had oxidized C14-labeled sub-strates was studied.

REFERENCES

BARKER, H. A. 1936 The oxidative metabolismof the colorless alga, Prototheca zopfii. J.Cellular Comp. Physiol., 8, 231-250.

BATT, R. D. AND WOODS, D. D. 1951 The oxida-tion of thymine by an unidentified bacterium.Biochem. J., 49, lxx.

BLUMENTHAL, H. J., KOFFLER, H., AND GOLD-SCHMIDT, E. P. 1951 The effect of glucose oracetate on the rate of endogenous respiration

of Penicillium chrysogenum. Bacteriol. Proc.,1951, 139-140.

CLIFTON, C. E. 1952 The enzymes, Vol. II, Part2, pp. 912-928. Edited by J. B. Sumner and K.MyrbiLck. Academic Press, Inc., New York.

CLIFTON, C. E. 1957 Introduction to bacterialphysiology. McGraw-Hill Book Co., Inc.,New York.

COCHRANE, V. W. AND GIBBS, M. 1951 Themetabolism of species of Streptomyces. IV. Theeffect of substrate on the endogenous respira-tion of Streptomyces coelicolor. J. Bacteriol.,61, 305-367.

GIBBS, M. AND WOOD, W. A. 1952 The effect ofsubstrate on the endogenous respiration ofPseudomonas fluorescens. Bacteriol. Proc.,1952, 137.

KORNBERG, H. L. AND KREBS, H. A. 1957Synthesis of cell constituents from C2-unitsby a modified tricarboxylic acid cycle. Na-ture, 179, 988-991.

MOSES, V. AND SYRETT, P. J. 1955 The endoge-nous respiration of microorganisms. J. Bac-teriol., 70, 201-204.

SANTER, M. AND AJL, S. 1954 Metabolic re-actions of Pasteurella pestis. I. Terminal oxi-dation. J. Bacteriol., 67, 379-386.

UMBREIT, W. W., BURRIS, R. H., AND STAUFFER,J. F. 1949 Manometric techniques and tissuemetabolism. 2nd ed. Burgess Publishing Co.,Minneapolis.

VAN SLYKE, D. D. AND FOLCH, J. 1940 Mano-metric carbon determination. J. Biol. Chem.,136, 509-541.

WILNER, B. AND CLIFTON, C. E. 1954 Oxidativeassimilation by Bacillus subtilis. J. Bac-teriol., 67, 571-575.

WONG, D. T. O. AND AJL, S. J. 1957 Significanceof the malate synthetase reaction in bacteria.Science, 126, 1013-1014.

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