JOURNAL OF BACTERIOLOGY, Nov. 1968, p. 1595-1600Copyright ( 1968 American Society for Microbiology

Vol. 96, No. 5Printed in U.S.A.

Molar Growth Yields as Evidence for OxidativePhosphorylation in Streptococcus faecalis

Strain lOCIhA. J. SMALLEY, P. JAHRLING, AND P. J. VAN DEMARK

Microbiology Section, Division of Biological Sciences, Cornell University, Ithaca, New York 14850

Received for publication 23 August 1968

During the aerobic growth of Streptococcus faecalis strain IOCl, with limitinglevels of glucose as the substrate, a molar growth yield (Y) of 58.2 g (dry weight) permole of glucose was obtained. Under these conditions of growth, glucose was dis-similated primarily to acetate and CO2. The incorporation of 14C-glucose into cellmaterial was no greater under aerobic conditions than during anaerobic growth.Assuming an adenosine triphosphate coefficient of 10.5, the aerobic Y cannot beexplained solely on the basis of substrate phosphorylation and would appear tosubstantiate previous enzymatic evidence for oxidative phosphorylation in this cy-tochromeless species. With mannitol as the substrate, an aerobic Y of 64.6 wasobtained. Extracts of mannitol-grown cells contained a nicotinamide adenine di-nucleotide (NAD)-linked mannitol-1-phosphate (M-1-P) dehydrogenase. The dif-ference in aerobic Y values with mannitol and glucose as substrates would indicatethat the in vivo P/O ratio from the oxidation of reduced NAD generated by theoxidation of M-1-P approximates 0.6. The Y values with pyruvate and glycerol assubstrates under aerobic conditions were 15.5 and 24.7, respectively.

Although Streptococcus faecalis obtains energyanaerobically via glycolysis and substrate phos-phorylation, aerobic growth induces a variety ofchanges in the respiratory mechanisms of thisspecies, including the ability to catalyze oxidativephosphorylation. By using extracts of anaero-bically grown S. faecalis strain lOCI, Dolin (6)was unable to detect oxidative phosphorylationduring reduced nicotinamide adenine dinuclotide(NADH) oxidation. In contrast, Gallin and Van-Demark (8) observed low levels of oxidative phos-phorylation during the oxidation of NADH byextracts of aerobically grown cells of this species.Although the latter study was unique as a demon-stration of oxidative phosphorylation in cyto-chromeless species, M. N. Mickleson (Bacteriol.Proc., p. 139, 1968) recently reported oxidativephosphorylation during NADH oxidation by ex-tracts of S. agalactiae. However, both of thesestudies must be viewed with caution because of

I A preliminary report of this study was presented atthe 67th Annual Meeting of the American Society forMicrobiology; New York, N.Y., 30 April to 4 May,1967. This work was presented in part in a thesis by thesenior author to the Graduate Faculty of Cornell Uni-versity in partial fulfillment of the requirements forthe degree of Master of Science.

the crude enzymatic extracts employed and thelow P/O ratios observed.The molar growth study, based on the adeno-

sine triphosphate (ATP) coefficient [Y (ATP)]concept of Bauchop and Elsden (1), has been use-ful for determining the energy yield of microbialspecies on various substrates. It represents a toolfor the demonstration of oxidative phosphoryla-tion in vivo, as well as a means of determiningP/O ratios more reliable than are obtained withbacterial enzymatic studies. The present report isat comparison of molar growth yields (Y) of S.faecalis strain lOCI during anaerobic and aero-bic growth on various substrates. These resultsappear to confirm previous enzymatic data foroxidative phosphorylation in this species duringaerobic growth.

MATERIALS AND METHODSCulture and growth conditions. S. faecalis strain

lOCi, from the culture collection of the MicrobiologySection of Cornell University, was used in this study.The partially defined medium ofO'Kane and Gunsalus(16), modified by the addition (per liter) of 5 g ofsodium acetate, 100 mg of cysteine, and 0.02 mg oflipoic acid, with various levels and types of substrates,was employed. For studies involving product analyses,sodium acetate was omitted from this medium. The

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SMALLEY, JAHRLING, AND VAN DEMARK

phosphate buffer and substrate were sterilized sepa-rately and added aseptically prior to inoculation. Themedium was inoculated with an 18-hr culture; avolume of inoculum equal to 0.2% of the total growthvolume was used. Aerobic and anaerobic cultureswere obtained as previously described (11). Duringaerobic culture, the conditions of aeration were suffici-ent to maintain an oxygen concentration of 5.6 ,ug/ml.After 24 hr of culture, cells were harvested by centrif-ugation and the resulting cell pellet was washed twicewith distilled water. For dry weight measurements, thewashed pellet was dried in weighing pans at 120 C to aconstant weight. Molar growth yields, expressed asgrams (dry weight) per mole of substrate, were cal-culated according to the method of Bauchop andElsden (1).

Radioactivity measuirements. For studies of glucoseincorporation during growth, solutions containing 5,4C of D-'4C-glucose per ml were sterilized by filtrationand added aseptically to the basal medium. Normalgrowth yields were determined on this medium as

described above. For the determination of glucoseincorporation, cells from 5-ml samples of each culturewere collected on membrane filters (0.45-,um poresize); the filters were washed with distilled water,dried, and glued to planchets. Liquid samples were

prepared for counting by spreading 0.5-ml samplesevenly on planchets, followed by oven-drying at 120C for 18 hr. All samples were counted on a gas flowcounter (Tracerlab, Inc., Waltham, Mass.).

Enzymatic studies. Cell-free extracts were preparedby ultrasonic disruption, as previously described byJacobs and VanDemark (11). Mannitol-1-phosphate(M-1-P) dehydrogenase was assayed spectrophoto-metrically at 340 nm in terms of the oxidation ofNADH by extracts of S. faecalis in evacuated Thun-berg cuvettes with fructose-6-phosphate (F-6-P) as

the hydrogen acceptor.Substrate and product assays. Residual glucose was

determined according to the Microglucostat methodof the Worthington Biochemical Corp. (Freehold,N.J.). Mannitol was measured by the method ofMartinez et al. (12). Lactic acid was assayed accordingto the spectrophotometric method described in theSigma Technical Bulletin No. 825-UV (Sigma Chemi-cal Co., St. Louis, Mo.). Volatile acids were deter-mined by the procedure of Neish (14). Formic acidwas assayed according to the method of Grant (9).Acetic acid was calculated as the difference betweenthe total volatile acids and the level of formatedetected. Acetylmethylcarbinol was estimated by theprocedure of Westerfeld (17).

RESULTS

Figure 1 illustrates the growth yields of S.faecalis grown aerobically and also anaerobicallyon semidefined medium with glucose concentra-tions varying from 0 to 15 mi. The anaerobic Y(glucose) of 21.5 is in close agreement with thatpreviously reported for this species by Bauchopand Elsden (1). The aerobic Y (glucose) of 58.2is greater than expected or explainable solely onthe basis of substrate phosphorylation.

Product analyses were performed to determinethe pathway of glucose dissimilation during aero-

bic growth. As illustrated in Table 1, acetate isthe primary product during aerobic growth, withlow levels of lactate, formate, and acetylmethyl-carbinol also produced. These analyses are inclose agreement with the studies of O'Kane (15)with resting cells of this strain, and indicate thatglucose is dissimilated aerobically primarily toacetate and CO2.

Figure 2 illustrates the difference in the Y value

Substrate Concentration(Mmoles/m I.)

FIG. 1. Aerobic and anaerobic growth yields of S.faecalis with glucose as substrate. Molar growth yieldswere determined according to the method of Bauchopand Elsden (1). Error flags representt tlhe stanzdarddeviation of six determinationis.

TABLE 1. Products and carbon balanice ofS. faecalisIOCI grown aerobically on glucosea

Glucose used(jumoles/ml)

Products

9.97 59.82

Lactate......................... 1.830 5.49Acetylmethylcarbinol........... 0.248 0.99Acetate......................... 16.980 33.96Formate........................ 0.020 0.02CO2 (estimated from AMC)..... 0.496 0.50CO2 (estimated from acetate) ... 16.980 16.98

a Results expressed as micromoles of carbonper milliliter. (Total of all products was 57.94,umoles of carbon per ml.) Carbon recovery(57.94/59.82) X 100 = 96.9%.

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OXIDATIVE PHOSPHORYLATION IN S. FAECALIS

observed and that expected from substrate phos-phorylation when glucose is dissimilated to acetate-and CO2. One would expect that, during the aero-bic dissimilation of glucose by S. faecalis, twoATP molecules via substrate phosphorylationwould result from the dissimilation of one mole-cule of glucose to two molecules of pyruvate, andtwo additional ATP molecules would be formed-during the subsequent degradation ofthe pyruvateto acetate and CO2. Assuming a Y (ATP) of 10.5,as predicted by Bauchop and Elsden (1), onewould expect a theoretical Y value of 42 -ti 8 ifonly substrate phosphorylation were involved.Therefore, the difference in this calculated yieldand the observed Y value of 58.2 would appear tobe due to an energy-producing reaction other thansubstrate phosphorylation, i.e., oxidative phos-phorylation.

Greater incorporation of the substrate (glucose)into cellular material during aerobic growth is analternative possibility for the greater cell yield.Therefore, the incorporation of "IC-glucose intocellular material during growth was determined.As outlined in Table 2, the incorporation of glu-cose into aerobic-grown cells is less than thatunder anaerobic conditions. Thus, the greatercell yield cannot be accounted for by a greateraerobic incorporation of glucose into cellularmaterial. We therefore interpret these data to sub-stantiate previous enzymatic data for oxidativephosphorylation in aerobically grown cells of this*cytochromeless species.One may calculate an in vivo P/O ratio from

these data by dividing the difference in the ob-served aerobic Y (glucose) and the theoretical Yvalue if only substrate phosphorylation were in-volved, i.e., 16.2 divided by the Y (ATP) of 10.5times 4 (since 4 pairs of electrons are transportedto oxygen during the oxidation of a glucose mole-cule). This P/O ratio resulting from the aerobicdissimilation of glucose by S. faecalis approxi-mates 0.4. However, this calculation assumes aY (ATP) of 10.5. If, under aerobic conditions, the

Y (ATP) is less than 10.5, as suggested by thestudies of Hernandez and Johnson (10), this P/Oratio could approach 1.The determination of Y value of S. faecalis on

other substrates provides further evidence for invivo oxidative phosphorylation in this strain. TheY values obtained with mannitol, sodium pyru-vate, and glycerol as substrates are illustrated inTable 3. The average aerobic Y (mannitol) was65.5, or 7.3 greater than the aerobic Y (glucose).The pathway of mannitol degradation in this

species appears to occur via a nicotinamideadenine dinucleotide (NAD)-linked M-1-P dehy-drogenase. NADH oxidation by cell-free extractsof mannitol-grown S. feacalis occurs with F-6-Pbut not fructose as the electron acceptor (Fig. 3).The products of aerobic mannitol catabolism byS. faecalis were primarily acetate and CO2 (Table4), as was the case during the aerobic dissimilationof glucose (Table 1). Therefore, the difference of7.3 between aerobic Y (mannitol) and aerobic Y(glucose) represents the energy obtained duringthe transport of two hydrogens to oxygen via anNAD-linked respiratory pathway.One may calculate an in vivo P/O ratio from

these data by dividing the difference in the aerobicY (mannitol and the aerobic Y (glucose), i.e., 7.3,by the Y (ATP) of 10.5. This P/O ratio resulting

2-P'+4H++4e- 2-P+4H ++4e-

GLUCOSE 2 PYRUVATE -.2 ACETATE

+ 2Co2

Observed Yaerobic = 58

Theoretical Y by SubstratePhosphorylation(4xlO.5) = 42

Difference= Yoxphos = 16

FIG. 2. Calculation of energy production from molargrowth yields ofS. faecalis grown aerobically on glucose.

TABLE 2. Glucose incorporation in S. faecalis IOCI

Glucose added Cells formed Per-cent of

Growth condition Glucose Atiyb Cell .. cell carbonGlucose cabn At At"o lcoseit Dry wt b t*" Actsvity" foh [ ^ ~~Cmole crbon -Aciiy ofj5gluc>omse ofcellS cabn Activity fcle from(jsmoles/(media) of cells c~~iarons c(sfcells glucoseMl) (jatoms/ (ei) carbon (Ag/Im)i aors(el) carbon

Anaerobic 5 30 60,564 2,019 188 7.8 2,901 372 18.410 60 119,840 1,997 276 11.5 5,729 498 24.9

Aerobic 5 30 59,409 1,980 276 11.5 2,892 252 12.710 60 120,584 2,010 604 25.2 4,652 185 9.2

a Expressed as counts per minute per milliliter.b Expressed as counts per minute per microatom.

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SMALLEY, JAHRLING, AND VAN DEMARK

from NADH oxidation by S. faecalis approxi-mates 0.6 to 0.7 (Fig. 4).The observed anaerobic Y (pyruvate) of 6 is

in agreement with previous evidence that anaero-bically S. faecalis catalyzes the dismutation ofpyruvate to lactate, acetate, and C02 (A. K.Miller, Ph.D. Thesis, Cornell University, 1942).Assuming one substrate phosphorylation per

pyruvate molecule under aerobic conditions, theobserved aerobic Y (pyruvate) of 15.5 is greaterthan one can account for by substrate phosphory-lation alone. This difference in molar growthyield apparently results from the transport of twoelectrons to oxygen during the oxidation of eachpyruvate molecule. Thus, by dividing this addi-tional growth yield of 5 by Y (ATP) of 10.5, asin the calculations of Fig. 4, the P/O ratio of thisoxidation approximates 0.5.The aerobic Y (glycerol) of 24.7 is somewhat

lower than might have been predicted in view ofthe aerobic Y (glucose), Y (mannitol), and Y(pyruvate) values obtained. Jacobs and Van-Demark (11) demonstrated that glycerol oxida-

TABLE 3. Molar growth yields of S. faecalis IOCIwith mannitol, pyruvate, and

glycerol as substrates

Substrate Y (g per mole)

Mannitol (aerobic) ............... 64.6Pyruvate (anaerobic) ............. 6.0Pyruvate (aerobic) ............... 15.5Glycerol (aerobic) ............... 24.7

90

C1 00-0

z-Q

Fructose

0.2 A

Fructose-6-P

10 20TIME (SECONDS)

FIG. 3. Mannitol-l-phosphate/NAD oxiiactivity of S. faecalis. Assays were run isThunberg cuvettes containing 3.0 ml of reac

cuvette contained 200 ,umoles of sodium a6.0), cell extract (2.0 mg of protein), 50fructose or F-6-P, and 1.5 limoles of NA)from the side arm to start the reaction).

TABLE 4. Products and carbon balance ofS. faecalisIOCI grown aerobically on mannitola

Mannitol used(nAoles/ml)

Products

9.750 58.50

Lactate........................ 1.620 4.86Acetylmethylcarbinol............ 0.162 0.64Acetate........................ 17.865 35.73Formate........................ 0.035 0.04CO2 (estimated from AMC)..... 0.324 0.32C02 (estimated from acetate) .... 17.830 17.83

a Results expressed as micromoles of carbonper milliliter. (Total of all products was 59.43,umoles of carbon per ml.) Carbon recovery =(59.43/58.50) X 100= 101.6%.

NAD NADH+ H

Mannitol-1-PQ4 Fructose-6- PO4

Y aeobk = 64.6

Yaerobic = 58.2Difference=YNADH0= 6.4

4105 = 0.6

FIG. 4. Calculation of energy production and P/Oratio from aerobic molar growth yields with mannitoland glucose as substrates.

tion by this strain involves the oxidation of a-glycerol phosphate by a flavine-adenine dinucleo-tide-linked oxidase not involving NAD with theformation of dihydroxy-acetone phosphate andH202. The relatively low aerobic Y (glycerol)probably indicates that this nonpyridine nucleo-tide-linked oxidative step is not a site of oxidativephosphorylation. Furthermore, during this oxida-tion, it would appear that the potential energy of1 mole of NADH is dissipated during the NAD-linked peroxidation of H202 formed during theoxidation of glycerol phosphate.

DIscussIoNDespite the general acceptance of the observa-

tion of Bauchop and Elsden (1) that the Y (glu-cose) of S. faecalis approximates 22 anaerobically,some workers have reported somewhat higher

doreductatsed anaerobic Y (glucose) values with this species.evacuatedh Forrest and Walker (7) have reported an anaero-

cetate (pH bic Y (glucose) of 32 at glucose concentrations ofImoles of less than 5 ,umoles/ml. In contrast, in the presentDH (tipped study, employing a semidefined medium with glu-

cose levels between 2.5 and 12.5 ,umoles/ml, the

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OXIDATIVE PHOSPHORYLATION IN S. FAECALIS

anaerobic Y (glucose) is 21.5 and in agreementwith the results of Bauchop and Elsden (1). For-rest and Walker employed a complex mediumwhich may have had a level of alternate hydrogenacceptors sufficient to replace pyruvate as a hydro-gen acceptor at low glucose concentrations, there-by allowing the pyruvate to serve as a source ofadditional energy via dismutation. Interpretationof the results of Forrest and Walker is also com-plicated by the fact that their culture medium con-tained 2% sodium citrate, which, as shown byCampbell and Gunsalus (4), can serve as anenergy source for this species. Because the pro-ducts of citrate dissimilation by S. faecalis includeacetate and formate (4), the presence of this sub-strate in their medium may explain the high per-centage of volatile acids observed among the prod-ucts.Beck and Shugart (3) also reported higher

growth yields with S. faecalis than those observedby Bauchop and Elsden. However, since the stud-ies of Beck and Shugart involved the use of 100-ml quantities of media in 250-ml flasks, it islikely that the conditions of culture were semi-anaerobic. Thus, the availability of some oxygenas a hydrogen acceptor may explain their in-creased cell yields.

In contrast to the aerobic Y (glucose) of 58.2observed in the present study, Deibel and Kvetkas(5) reported an aerobic Y (glucose) with S.faecalis strain FB82 of 43.8. These workers alsodemonstrated that fumarate would substitute forpyruvate as a hydrogen acceptor in the anaerobicdissimilation of glucose. During anaerobicgrowth, Y of 47.1 was obtained with glucose asthe substrate and the fumarate as an alternatehydrogen acceptor. Their products analyses indi-cate that, during growth under these conditions,lactate accounts for over 50% of the glucosecarbon utilized. One would expect the molargrowth yield of the glucose dissimilated to lactateto approximate 22. When the observed growthyield with glucose plus fumarate is corrected forthe lactate produced, the growth yield from thedissimilation of glucose to acetate and CO2 isfound to be considerably higher than can be ac-counted for on the basis of substrate phosphoryla-tion. Thus, this portion of the Deibel and Kvetkasinvestigation would appear to substantiate thepresent study.The low P/O ratios calculated in the present

study may indicate that only one site of oxidativephosphorylation is involved in the respiration ofS. faecalis. Based on our present knowledge ofthe respiratory chain of S. faecalis, this site ofoxidative phosphorylation could occur either atthe NADH-flavoprotein (6) or the flavine-naph-thoquinone level (2, 6). If our interpretation of

the data of Deibel and Kvetkas is correct, theability of fumarate to substitute for oxygen in thissystem of phosphorylation would implicate theNADH-flavine site, since fumarate reduction hasbeen shown to be flavine mediated in this strain(11).The observation that these in vivo P/O ratios

are less than unity may mean that the Y (ATP) isless than the accepted value of 10.5 (1). Thestudies of Hernandez and Johnson (10) wouldindicate that the Y (ATP) is not constant underall environmental conditions and may be less than10.5 under aerobic conditions. Whereas the Y(ATP) was considered to be 10.5 in the presentstudy, a lower Y (ATP) obtained aerobicallywould increase the energy yield and the P/Oratios one can calculate from the data in thepresent studies.However, if the Y (ATP) does approximate

10.5 under aerobic conditions (13), these frac-tional P/O ratios may be explained on the basisof a coupled oxidase-peroxidase reaction duringNADH oxidation (6), in which either the oxidaseor peroxidase activity is energy-yielding.Whereas the present investigations have used

molar growth yields in the demonstration of invivo oxidative phosphorylation, an extension ofsuch methods can be of value in the determinationof the site as well as the mechanism of oxidativephosphorylation in S. faecalis. Thus, furtherinvestigations with this species are underway,including the determination of molar growth andoxygen yields on other substrates as well as astudy of the effects of inhibitors and uncouplersof oxidative phosphorylation on these molargrowth yields.

LITERATURE CITED

1. Bauchop, T., and S. R. Elsden. 1960. The growthof microorganisms in relation to their energysupply. J. Gen. Microbiol. 23:457-469.

2. Baum, R. H., and M. I. Dolin. 1965. Isolation of2-solanesyl-1,4-naphthoquinone from Strepto-coccus faecalis lOCI. J. Biol. Chem. 240:3425-3433.

3. Beck, R. W., and L. R. Shugart. 1966. Molargrowth yields in Streptococcus faecalis var.liquefaciens. J. Bacteriol. 92:802-803.

4. Campbell, J. J. R., and I. C. Gunsalus. 1944.Citric acid fermentation by streptococci andlactobacilli. J. Bacteriol. 48:71-76.

5. Deibel, R. H., and M. J. Kvetkas. 1964. Fumaratereduction and its role in the diversion of glucosefermentation by Streptococcus faecalis. J.Bacteriol. 88:858-864.

6. Dolin, M. I. 1955. The DPNH-oxidizing enzymesof Streptococcus faecalis. It. The enzymesutilizing oxygen, cytochrome c, peroxide, and2,6-dichlorophenolindophenol or ferricyanide

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as oxidants. Arch. Biochem. Biophys. 55:415-435.

7. Forrest, W. W., and D. J. Walker. 1965. Synthesisof reserve materials for endogenous metabolismin Streptococcus faecalis. J. Bacteriol. 89:1448-1452.

8. Gallin, J. I., and P. J. VanDemark. 1964. Evidencefor oxidative phosphorylation in Streptococcusfaecalis. Biochem. Biophys. Res. Commun. 17:630-635.

9. Grant, W. M. 1948. Colorimetric microdetermina-tion of formic acid based on reduction to form-aldehyde. Anal. Chem. 20:267-269.

10. Hernandez, E., and M. J. Johnson. 1967. Anaero-bic growth yields of Aerobacter cloacae andEscherichia coli. J. Bacteriol. 94:991-995.

11. Jacobs, N. J., and P. J. VanDemark. 1960. Com-parison of the mechanism of glycerol oxidationin aerobically and anaerobically grown Strepto-coccus faecalis. J. Bacteriol. 79:532-538.

12. Martinez, G., H. A. Barker, and B. L. Horecker.

1963. A specific mannitol dehydrogenase fromLactobacillus brevis. J. Biol. Chem. 238:1598-1603.

13. Mayberry, W. R., G. J. Prochazka, and W. J.Payne. 1968. Factors derived from studies ofaerobic growth in minimal media. J. Bacteriol.96:1424-1426.

14. Neish, A. C. 1952. Analytical methods for bac-terial fermentations. National Research Councilof Canada Rept. No. 46-8-3, 2nd ed.

15. O'Kane, D. J. 1950. Influence of the pyruvateoxidation factor on the oxidative metabolism ofglucose by Streptococcus faecalis. J. Bacteriol.60:449-458.

16. O'Kane, D. J., and I. C. Gunsalus. 1948. Pyruvicacid metabolism. A factor required for oxida-tion by Streptococcus faecalis. J. Bacteriol.56:499-506.

17. Westerfeld, W. W. 1945. A colorimetric deter-mination of blood acetoin. J. Biol. Chem. 161:495-502.

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