JOURNAL OF BACTERIOLOGY, July 1967, p. 184-191Copyright © 1967 American Society for Microbiology

Vol. 94, No. 1Printed in U.S.A.

Aerobic Metabolism of Streptococcus agalactiaeM. N. MICKELSON

National Animal Disease Laboratory, Animal Disease and Parasite Research Division, Agricultural ResearchService, U.S. Department of Agriculture, Ames, Iowa 50010

Received for publication 2 May 1967

Streptococcus agalactiae cultures possess an aerobic pathway for glucose oxida-tion that is strongly inhibited by cyanide. The products of glucose oxidation byaerobically grown cells of S. agalactiae 50 are lactic and acetic acids, acetylmethyl-carbinol, and carbon dioxide. Glucose degradation products by aerobically growncells, as percentage of glucose carbon, were 52 to 61% lactic acid, 20 to 23% aceticacid, 5.5 to 6.5% acetylmethylcarbinol, and 14 to 16% carbon dioxide There was noevidence for a pentose cycle or a tricarboxylic acid cycle. Crude cell-free extracts ofS. agalactiae 50 possessed a strong reduced nicotinamide adenine dinucleotide(NADH2) oxidase that is also cyanide-sensitive. Dialysis or ultrafiltration of thecrude, cell-free extract resulted in loss of NADH2 oxidase activity. Oxidase ac-tivity was restored to the inactive extract by addition of the ultrafiltrate or by addi-tion of menadione or K3Fe(CN)6 . Noncytochrome iron-containing pigments werepresent in cell-free extracts of S. agalactiae. The possible participation of these pig-ments in the respiration of S. agalactiae is presently being studied.

Glucose metabolism by streptococci generallyhas been regarded as fermentative, and, with theexception of certain strains of Streptococcusfiaecalis (11, 13), the streptococci have not beennoted for their oxidative capabilities. S. mastiditis,a Lancefield group B organism, was shown byGunsalus and Wood (5) to possess dehydrogen-ases for a number of alcohols, with methyleneblue as a hydrogen acceptor; Griesen and Gun-salus (4) studied an ethyl alcohol oxidizing sys-tem in S. mastiditis that used oxygen without hy-drogen peroxide accumulation. Cultures of S.agalactiae consume oxygen at an appreciablerate with glucose as a substrate (10), and oxida-tive capabilities are enhanced when the culturesare grown with aeration. The proportion of thetotal flavin occurring as flavin adenine dinucleo-tide was also increased in cells that were grownunder aeration. The respiration of the cell suspen-sions was strongly inhibited by cyanide. This re-port deals with some of the characteristics of theaerobic metabolism of glucose by S. agalactiae.

MATERIALS AND METHODS

Cultures. Seven cultures of S. agalactiae, 50, 80,142, 356, 383, 660-1, and Cornell 48, were used. Thelatter was obtained from G. E. Morse, School ofVeterinary Medicine, Univ. of Pennsylvania, Phila-delphia. For a comparison culture, S. pyogenes Rich-ards was used in some experiments.

Culture medium and growth conditions. The mediumfor growth of the organisms was composed of 1%

yeast extract (Difco), 0.4% Casitone (Difco), 0.8%glucose, and 0.1 M phosphate buffer (pH 7.0). Thephosphate buffer was sterilized separately as a 1 Msolution and was added to the medium after autoclav-ing and cooling. Aerated cultures were incubated in50 ml of medium in a loosely capped 125-ml Erlen-meyer flask on a New Brunswick gyrotary model S-3shaker, operated at 120 rev/min. Nonaerated cultureswere treated identically except that they were notshaken. For cell-free extracts, cultures were grown ona shaker in 250 ml of medium in 1-liter flasks. Allcultures were incubated at 37 C for 10 to 15 hr. Thecells were harvested by centrifugation, washed oncewith distilled water, and used immediately or storedat -60 C. No appreciable loss in respiratory activityresulted from storage for 2 to 3 weeks. Endogenousrespiration was never greater than 5 to 6 ,,liters of02 per mg (dry weight) of cells per hr.

Cell-free extracts. Cells harvested from shakercultures were washed once with 0.02 M NaHCO3,mixed with an equal weight of Alumina 305 (AlcoaCo., Pittsburgh, Pa.), and frozen at -60 C on a pieceof glassine paper. Active extracts were obtained fromcells held for several weeks at -60 C. The frozen cell-Alumina mixture was placed in a chilled mortar in anice bath and was ground for no longer than 5 min.The ground mixture was transferred to a centrifugetube by successive additions of cold 0.05 M phosphatebuffer (pH 7.5), with 1 ml for each 50 to 60 mg (dryweight) of cells. The cellular debris and Alumina weresedimented by centrifugation at 20,000 X g in aServall centrifuge. The extract was held in an ice bathduring use or, when stored, was held at -60 C for aslong as 3 weeks and still remained active.

Extracts were dialyzed or ultrafiltered in Visking

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tubing (8 mm) at 4 C to separate diffusible componentsfrom the crude reduced nicotinamide adenine di-nucleotide (NADH2) oxidase. The extract was dialyzedagainst 0.05 M phosphate buffer (pH 7) that was stirredon a magnetic stirrer and changed each 24 hr. Dialysisfor 48 to 72 hr was sufficient to reduce NADH2 oxidaseactivity 90% or more. When not used immediately,dialyzed extracts were stored frozen at -60 C forseveral days. Ultrafiltration was performed at 125 mmof pressure until the volume was reduced 95%. Thefiltered enzyme was diluted back to its original volumewith 0.05 M phosphate buffer and was combined withthe ultrafiltrate for assaying NADH2 oxidase activity.

Protein content of the cell-free extracts was deter-mined by the method of Stadtman, Novelli, and Lip-mann (14).

Manometric studies. All experiments were done at37 C by use of conventional manometric proceduresdescribed by Umbreit, Burris, and Stauffer (15). Whencyanide was used as a respiratory inhibitor, 1 M KCNrather than KOH was used in the center well of theWarburg flasks.When the contents of the flask were to be analyzed,

0.2 ml of 4 N H2SO4 was added from a side arm to stopthe reaction. The reactants were carefully washed intoa centrifuge tube, neutralized topH 7 with 4 N NaOHand bromothymol blue, deproteinized with 1 ml of0.3 N Ba(OH)2 and 1 ml of 5% ZnSO4*7H20, andmade up to 10 ml. After centrifugation, samples ofthe clarified supernatant fluid were used for analysis.

Glucose and lactic acid determinations were donewith the anthrone and Barker-Summerson procedures,respectively, as described by Umbreit, Brnis, andStauffer (15).

Acetic acid was determined by steam distillation ofthe volatile acids and titration of the distillate with0.005 N NaOH after removal of CO2 by aeration withC02-free air. Identification of the acid was done bygas chromatography. (We are grateful to Milton J.Allison for gas chromatographing the volatile acidsamples.)

Carbon dioxide was determined by collection of thegas produced in the respirometer vessel in C02-freeNaOH, carefully transferring the filter-paper wickand alkali from the center well into a 10-ml volumetricflask by washing with C02-free distilled water, fol-lowed by manometric measurement of the CO2 inportions of the sample.To test for the existence of the pentose cycle path-

way of glucose degradation, cell suspensions ofaerobically grown S. agalactiae 50 were allowed tooxidize glucose-1-14C and glucose-6-14C (Volk Radio-chemical Co., Skokie, Ill.) in Warburg vessels. (Weare grateful to Donald Robertson for supplyingthe labeled sugars and for determining the activity inthe CO2 samples.) To 40 uAmoles of glucose was added0.5 ,Amole of glucose-1-_4C or glucose-6-14C containingI ,uc of radioactivity per ,umole. Carbon dioxide wascollected in C02-free NaOH, quantitatively washedinto a 10-ml beaker with C02-free distilled water, andwas precipitated as BaCO3 with 0.5 ml of a mixture of0.5 N BaCl2 and 0.4 N NH4Cl. The BaCO3 was filteredon a 20-mm membrane filter (Millipore Corp., Bed-ford, Mass.), washed with a solvent mixture of 1 part

ethyl ether to 3 parts ethyl alcohol, and dried. Radio-activity was measured by transferring the entireBaCO3 sample or a sample of cell suspension or cellsupematant fluid to a counting vial containing athixotropic gel [4% Packard Cab-O-Sil in XDCscintillator fluid of G. A. Bruno and J. E. Christian(1)]. Radioactivity was measured with a Packardautomatic refrigerated scintillation counter (model3145; Packard Instrument Co., Inc., LaGrange, Ill.).A control with unlabeled glucose was run to determinethe extent of oxidation by measuring 02 uptake,glucose consumption, and carbon dioxide formation.Enzyme activity. NADH2 oxidase activity in cell-

free extracts was measured by a decrease in absorptionat 340 m, because of oxidation of NADH2. It wasalso measured manometrically to establish oxygen asthe terminal electron acceptor. NADH2 was estimatedfrom the extinction of 6.22 X 106 (cm2 per mole) (7).Triosephosphate dehydrogenase activity was measuredon dialyzed extracts by an increase in absorption at340 mjs because of reduction of NAD with 3-phos-phoglyceraldehyde as the substrate (8, 16). Lacticdehydrogenase activity was measured in dialyzed ex-tracts as a decrease in absorption at 340 m,u becauseof oxidation of NADH2 in the presence of pyruvate.Pyruvate disappearance and lactic acid formationwere confirmed by chemical analysis of the contentsof the cuvette. Pyruvate analysis was by the method ofKoepsell and Sharpe (7). Reduced nicotinamideadenine dinucleotide phosphate (NADPH2) oxidaseactivity was measured in the same manner as forNADH2. Glucose-6-phosphate and 6-phosphoglu-conate dehydrogenases were measured in dialyzedextracts with unreduced NADPH2 (NADP) and un-reduced NADH2 (NAD) as electron acceptors.Amytal was used to inhibit flavoprotein reductionby NADH2 (la, 9), and antimycin A and cyanide wereused to inhibit reoxidation of recuced flavoprotein(9). Decrease in absorption at 450 m, was used as ameasurement of flavin reduction. A sample reducedwith sodium hydrosulfite served as a control.

Absorption measurements were made with a Beck-man DU-2 spectrophotometer.

Menadione was used as an aqueous suspension of1 ,umole/ml.NADH2 (P-L Biochemicals, Inc.) and NAD

(Boehringer), 6-phosphogluconic acid (barium salt),and glucose-6-phosphate (sodium salt) (Boehringer)were purchased from Calbiochem, Los Angeles, Calif.DL-Glyceraldehyde-3-phosphate (barium salt) andantimycin A were purchased from Nutritional Bio-chemicals Corp., Cleveland, Ohio, and sodium amytalwas a gift from Eli Lilly & Co., Indianapolis, Ind.

RESULTSOxidative activity of cell suspensions. Some cul-

tures of S. agalactiae were shown (10) to have anappreciable rate of respiration with glucose as thesubstrate. The respiratory activity was enhancedwhen the cell suspension was produced in aeratedcultures. Variation in oxidative capabilities of S.agalactiae cultures is shown in Fig. 1. The timeof harvesting the cells from shaker cultures was

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FIG. 1. Oxidation of glucose by cell suspensions ofvarious cultures ofStreptococcus agalactiae. Reactants:0.1 Mphosphate, pH 7; 0.02 M glucose; and washed cellsof;S. agalactiae. There were 3 to 5 mg (dry weight ofcells perflask.

important for the demonstration of aerobic be-havior. Cultures numbered 660-1, 80, and Cornell48 exhibited aerobic tendencies only when har-vested after 6 to 8 hr of incubation or when only30 to 60% of the glucose had disappeared fromthe medium. For other cultures, i.e., 50, 383, 356,active cell suspensions were obtained after as longas 10 to 15 hr of growth, when nearly all of theglucose had disappeared.A culture of S. pyogenes grown under the same

conditions showed a respiratory rate similar to aculture of S. agalactiae 80 and was included forcomparison. The inhibition of the respiration ofS. agalactiae by cyanide at concentrations or-dinarily used to inhibit cytochrome-mediatedrespiration in other systems is shown in Fig. 2.When cell suspensions of S. agalactiae 50 were

allowed to degrade glucose aerobically, the prod-ucts were lactic and acetic acids, acetylmethyl-carbinol, and carbon dioxide. Sometimes traces ofpyruvic acid were found, but always accountingfor less than 1% of the carbon of the substrateused. About 52 to 61% of the glucose carbon wasfound as lactic acid, 20 to 23% as acetic acid, 5.5to 6.5% as acetylmethylcarbinol, and 14 to 16%as carbon dioxide (Table 1).When pyruvate was the substrate, the products

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FIG. 2. Cyanide inhibition of respiration on glucoseof a cell suspension of Streptococcus agalactiae 50.Reactants: 0.1 M phosphate buffer, pH 7; 0.02 M glu-cose; 5.6 mg (dry weight) of cells per flask (1) Noinhibitor; (2) 0.001 m KCN.

of degradation were lactic and acetic acid, acetyl-methylcarbinol, and carbon dioxide (Table 1).About 16% of the pyruvate carbon was found aslactic acid, 41% as acetic acid, 8.5% as acetyl-methylcarbinol, and 30% as carbon dioxide.There was no oxygen consumed by cell suspen-

sions on any of the tricarboxylic acid cycle com-pounds, except pyruvate and oxalacetate. Acetatewas not attacked.To assess the possible existence of a pentose

pathway, S. agalactiae 50 cells were allowed tooxidize glucose-1-14C and glucose-6-14C in thepresence of 38.3 ,moles of unlabeled glucose.The carbon dioxide was collected and assayed forradioactivity. Oxygen consumption values (21.2and 21.0 ,umoles for glucose labeled at 1 and 6carbons, respectively) were identical to that of anunlabeled glucose control (21.8 .smoles). Lacticacid and carbon dioxide production were deter-mined in the control (50.0 and 27.3 Mimoles, re-spectively), and the results were similar to thosein Table 1. There was insignificant labeling in thecarbon dioxide from either of the labeled sugars(Table 2); 90% ofthe labeled carbon with glucose-

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TABLE 1. Degradation of glucose and pyruvate by cell suspensions of Streptococcus agalactiaegrown in shaker cultures"

Amt of Carbon Carbon re-Substrate substrate Oxygen Lactic acid Acetic acid AMCb Pyruvic acid dioxide covery (%)

glucose 39. 5e 23 41.8 27.4 3.85 0.68 33.8 98.039.7 23.4 41.8 27.4 3.85 0.45 35.5 98.5

pyruvate 35.8 9.2 6.88 21.8 2.28 _ 31.5 97.6

- Reactants: 0.02 M glucose or pyruvate; 0.1 M phosphate, pH 7; and 4 mg (dry weight) of cells perflask. Volume, 2.0 ml.

b Acetylmethylcarbinol.c Results, except for last column, are in micromoles.

TABLE 2. Aerobic degradation of labeled glucoseby Streptococcus agalactiae SOa

Recovery of labeled carbon (dpm)Portion

Glucose-1-14C Glucose-6-14C

Carbon dioxide 370 (0 .52)b 290 (0.46)Cells 90,000 (12.7) 86,800 (13.8)Supernatant

fluidc 631,050 (89.5) 475,000 (75.7)Total recovery 721,420 562,090Percentage of

recovery 102 90

a Reactants: 0.1 M phosphate (pH 7); 7.5 mg(dry weight) of cells per flask. Volume, 2.0 ml.Substrates: 40 ,umoles of unlabeled glucose; 40,Amoles of unlabeled glucose + 0.5,umole of glu-cose-1-14C (707,000 dpm); and 40 ,umoles of un-labeled glucose + 0.5 ,umole of glucose-6-'4C(627,000 dpm).

b Percentage of total counts added.c Contains lactic and acetic acids and unused

substrate.

1-'4C and 76% of the labeled carbon from glu-cose-6-"4C were found in the cell supernatantfluid containing lactic and acetic acids. The re-mainder of the label was found in the cells, givingrecoveries of 102 and 90% of the labeled mate-rials, respectively.

Cell-free extracts of S. agalactiae 50-crude ex-tract. Crude (undialyzed), cell-free extracts had astrong NADH2 oxidase activity with oxygen asthe electron acceptor. The oxidase was inhibitedstrongly by 0.001 M cyanide, about 15% byamytal, and 30% by antimycin A (Fig. 2a).Sodium azide was required in higher concentra-tion than cyanide; 0.005 M caused a 65% inhibi-tion of NADH2 oxidase activity in cell-freeextracts. Manometric experiments with NADH2as substrate showed 0.5 mole of oxygen consumedfor each mole of NADH2 (Table 3). Hydrogenperoxide was not found as a product of the reac-

TABLE 3. Oxidatioln ofNADH2 by cell-free extractsof Streptococcus agalactiae 50a

NADH2 oxidized Oxygen consumed(umoles) (umoles) Oxygen/NADH,

2.16 1.29 0.592.16 1.13 0.521.08 0.65 0.602.06 0.93 0.45

a Reactants: 0.1 M phosphate, pH 7; 0.5 ml ofcell-free extract containing 1.9 to 2.9 mg of proteinper ml; and 1 or 2 Omoles of NADH2 per flask.Volume, 2.0 ml.

tion, nor did this extract destroy hydrogenperoxide by catalase action. NADPH2 was notoxidized by the crude extract. There was noreduction of either NAD or NADP by crudecell-free extracts when glucose-6-phosphate or 6-phosphogluconic acid was the substrate, andcyanide was present to prevent the NADH2oxidase reaction. Cell suspensions did not oxidizeNADH2 .The cell extracts showed a strong absorption at

420 m, (Fig. 3). However, there was no evidencethat this absorption was due to cytochromes, be-cause there was no appearance of reduced cyto-chrome bands in the region of 530 to 560 mA whensodium hydrosulfite was added. Furthermore,dense cell suspensions were examined with aspectroscope and a strong light source, and nocytochrome absorption bands were found. Underthe same conditions, the absorption bands at 532and 555 mA could be seen in suspensions ofbaker's yeast and an unidentified pseudomonad.

Dialyzed cell-free extracts. When the crude ex-tract was dialyzed for 48 hr against 0.05 M phos-phate, the NADH2 oxidase activity was lost. Theoxidase activity could be restored to 50 to 80% ofits original level by the addition of exogenouselectron acceptors such as menadione or K3Fe-(CN)6, but not with hydrogen peroxide (Fig. 4).A component or components lost from the

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FIG. 2a. Effect of cyanide, antimycin A, and amytalon NADH2 oxidase in crude cell-free extracts ofStrep-tococcus agalactiae 50. Reactants: (1) 0.22 Mtmole ofNADH2; 150 ,umoles of phosphate buffer, pH 7; 100Ag of extract protein; (2) same as (1) except 0.001 MKCN with enzyme extract, S min before addition ofNADH2; (3) same as (2) except 0.5,g ofantimycin Ainstead of cyanide; (4) same as (2) except 0.005 M Naamytal instead ofcyanide.

crude extract upon dialysis were necessary forlinking electron transport to oxygen. The NADH2oxidase activity of crude bacterial extracts couldalso be reduced by ultrafiltration. Activity was re-stored by addition of the ultrafiltrate to the inac-tivated enzyme, with restoration of NADH2 oxi-dase activity equal to the level of that obtainedwith 0.4 ,umole of menadione or K3Fe(CN)6 (Fig.5). Cyanide inhibited the NADH2 oxidation thatwas restored by addition of the ultrafiltrate.Two enzymes of the glycolytic cycle were meas-

ured in dialyzed cell-free extracts of S. agalactiae50. In crude extracts, their activity was obscuredby the strong NADH2 oxidase, and they couldnot be assayed spectrophotometrically. When theNADH2 oxidase was reduced by dialysis of theextract, the addition of pyruvate resulted in arapid oxidation of NADH2 (Fig. 6A). The disap-pearance of pyruvate and formation of lactic acidby lactic dehydrogenase were confirmed by anal-ysis of the contents of the cuvette. NAD was

FIG. 3. Absorption spectra of cell-free extract ofStreptococcus agalactiae 50 and reduced cytochrome c.Reactants: cell-free extract from 50 mg of lyophilizedcells per cuvette, plus a few crystals of Na2S204, vol-ume 3.0 ml; cytochrome c, 0.75 mg, plus a few crystalsof Na2S204, volume 3.0 ml.

MINUTESFIG. 4. Inactivation of NADH2 oxidase by dialysis

of cell-free extract of Streptococcus agalactiae 50 andreactivation with menadione and K3Fe(CN)r,. Re-actants: 150 ,umoles of phosphate buffer, pH 7; 0.22,umoles of NADH2, dialyzed or crude extract; 100 Mugofprotein, volume 3.0 ml. (1) Cell-free extract dialyzed48 hr; (2) crude, cell-free extract; (3) same as (1) plus0.4 Mmole oj K3Fe(CN)6 ; and (4) same as (1) plus 0.4,umole of menadione.

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reduced with 3-glyceraldehydephosphate as a sub-strate that demonstrated the presence of triose-phosphatedehydrogenase (Fig. 6B). Dehydro-genase activity for glucose-6-phosphate and

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FIG. 5. Reactivation of NADH2 oxidase of Strep-tococcus agalactiae 50 with ultrafiltrate of the crudeenzyme extract. Reactants: 0.22 ,umole of NADH2;150 ,moles ofphosphate buffer, pH 7, volume 3.0 ml.(1) 100 Ag of ultrafiltered cell-free extract protein; (2)same as (1) plus 0.5 ml of ultrafiltrate (equivalent to0.5 ml of original crude extract); (3) same as (2) plus0.001 M cyanide; (4) same as (1) plus 0.4 umole ofmenadione.

.50 A. 2LACTIC DEHYDROGENASE

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6-phosphogluconate was absent in the dialyzedextract.

Attempts to demonstrate the reduction of flavinas the first step in NADH2 oxidation by crudecell-free extracts were made with amytal, anti-mycin A, and cyanide as inhibitors. Results ofsuch an experiment are shown in Fig. 7.

DISCussIoNr Except for S. faecalis, the oxidative metabolismof streptococci has received little attention. Thefinding of an active respiratory pathway in S.agalactiae that was inhibited by cyanide was unex-pected since cytochrome-cytochrome oxidaseterminal respiration has not been reported in thestreptococci. Dolin (2, 3) has shown that NADH2oxidation in S. faecalis is the result of the activityof two enzymes, a flavoprotein NADH2 oxidaseand a flavoprotein peroxidase. This system al-lowed electron transport to oxygen without hy-drogen peroxide accumulation and was not in-hibited by cyanide.With purified enzyme preparations from S.

faecalis, Dolin (3) reported that NADH2 oxida-tion proceeded about 1/200th as fast in oxygen-saturated solutions as in the presence of hydrogenperoxide. The rate of NADH2 oxidation by crudecell-free extracts of S. agalactiae was unchangedby the addition of hydrogen peroxide. If it was aperoxidatic system, one might expect an increasein rate when the natural electron acceptor wassupplied in excess. NADH2 oxidase in a cell-free

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FIG. 6. Lactic and triosephosphate dehydrogenases in dialyzed cell-free extracts ofStreptococcus agalactiae 50.Reactants for lactic dehydrogenase: 30 MAmoles ofpyruvate; 0.22 ,Amole ofNADH2; 150 Mmoles ofphosphate, pH7.0; and 0.5 ml ofdialyzed cell-free extract containing 0.75 mg ofprotein per ml, volume 3 ml. (1) NADH2 oxidaseactivity ofthe dialyzed extract; (2) same as (1) except 30 ,Imoles ofpyruvate were added. Reactantsfor triosephos-phate dehydrogenase: 180 t,moles ofNa4P407 , pH 8.4; 150 ,Amoles ofNaHAsO4 , pH 8.4; 12 ,umoles ofcysteine;18,moles of 3-DL-glyceraldehyde-3-phosphate; 0.85 ,umole ofNAD; and 0.5 ml of dialyzed cell-free extract con-taining 0.75 mg ofprotein per ml.

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FIG. 7. Flavin reduction in extracts ofStreptococcusagalactiae during NADH2 oxidation. Reactants: 150,umoles of phosphate buffer (pH 7); 0.22 ,umole ofNADH2; 0.2 ml of cell-free extract containing 1.87mg ofprotein per ml. Volume 3.0 ml. (1) No inhibitor;(2) few crystals ofNa2S204; (3) 0.5 ,ug ofantimycin A;(4) 0.001 M KCN; and (5) 0.005 M Na amytal. Inhibitorsincubated 5 min with extract at room temperature beforeadding NADH2 to start reaction.

extract from a Lancefield group D Streptococcuswas increased about 50% by the addition of hy-drogen peroxide (data of T. J. McDonald of thislaboratory). When catalase was added to NADH2oxidase system of S. agalactiae 50, there was nochange in rate. If hydrogen peroxide was theterminal electron acceptor, one might expectcatalase to compete with peroxidase for the per-oxide and reduce the rate of NADH2 oxidation.

Cell suspensions of S. agalactiae 50 have beenshown to decompose hydrogen peroxide whenglucose was present (10). The activity was notstrong, and evidence that it functions in theNADH2 oxidase of cell-free extracts is inconclu-sive.

In view of these observations, it appears un-likely that the NADH2 oxidase in extracts of S.agalactiae is similar to the oxidase-peroxidase sys-tem of S. faecalis.The inhibition of respiration of glucose by cel

suspensions of S. agalactiae and the inhibition ofNADH2 oxidation in cell-free extracts by cyanidesuggests that a metallocomponent is involved inelectron transport to oxygen. The absorption at420 m, by cell-free extracts suggests the presenceof an iron-containing chromophore. Under cer-tain conditions of growth, a brownish-red pig-ment was produced by S. agalactiae 50. The pig-ment was partially extractable with water or

phosphate buffer at neutral or slightly alkalinereactions. Upon dialysis, the cell-free extracts lostsome of the brownish color. It may be one of thecomponents responsible for loss of NADH2 oxi-dase activity in the dialyzed cell-free extracts.

There was no labeling of the carbon dioxideformed when cell suspensions degraded glucoselabeled in the 1 and 6 carbons.The absence of a pentose pathway and the

presence of lactic and triosephosphate dehydro-genase suggest a classical fermentative pathwayof glucose degradation to pyruvate. NADH2 oxi-dase, by removing the reductant (electron donor)for the lactic dehydrogenase reaction, leavespyruvate to be oxidized to acetic acid and carbondioxide. Lactic acid, acetic acid, acetylmethyl-carbinol, and carbon dioxide accounted for all ofthe carbon of the glucose that was degraded bycell suspensions of S. agalactiae under aerobicconditions. If the degradation proceeds as pro-posed, there should be 1 mole of oxygen con-sumed for each mole of acetic acid and carbondioxide formed. When 2 moles of carbon dioxidewere subtracted from the total carbon dioxide foreach mole of acetylmethylcarbinol formed, thenumber of micromoles of oxygen consumed andof acetic acid and carbon dioxide formed with theglucose substrate (Table 1) were 23, 27.4, 26.1,respectively, in one experiment and 23.4, 27.4,27.8, respectively, in the other. When 0.001 MKCN was present to inhibit oxidation, the degra-dation was diverted from an oxidative to a fer-mentative one, and more of the glucose degradedwas recovered as lactic acid (10).When pyruvate is the substrate, there should be

one-half mole of oxygen consumed for each moleof pyruvate oxidized to acetic acid and carbondioxide. When the pyruvic acid converted to lacticacid and acetylmethylcarbinol was subtractedfrom the total pyruvic acid used and when thecarbon dioxide value was corrected for carbondioxide arising through the formation of acetyl-methylcarbinol, the molar ratio between oxygenand pyruvate consumed and products formed was25.4 pyruvate, 9.2 oxygen, 21.8 acetate, and 26.9carbon dioxide (Table 1).The agreement between oxygen consumed and

products formed, though not exact, leaves littledoubt that the aerobic degradation of glucose andpyruvate occurs as suggested. The data fromfermentation analyses suggest that the oxidativemetabolism is not a major energy pathway forthis organism. Evidence for a Krebs' cycle orpentose cycle is lacking. There is about 0.5 moleof oxygen consu.med in converting 1 mole ofpyruvate to acetic acid and to carbon dioxidewhen correction is applied for the carbon dioxideequivalent to the acetylmethylcarbinol which isformed. The oxygen to acetic acid to carbon di-oxide ratios for the two glucose experiments ofTable 1 are 0.85/1.0/0.96 and 0.86/1.0/0.85.

Although there is no direct evidence for in-

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AEROBIC METABOLISM OF S. AGALACTIAE

volvement of a quinone compound, menadionereactivated NADH2 oxidase in dialyzed extracts.Antimycin A inhibited the reduction of cyto-chromes by reduced quinone in Agrobacteriumtumefaciens (9) and in beef heart mitochondria(11). In certain extracts of S. agalactiae, antimy-cin A evidently inhibited reoxidation of reducedflavoprotein that resulted in a decrease in opticaldensity at 450 m,u equal to reduction of the flavinwith Na2S204. Cyanide, though an effective in-hibitor of NADH2 oxidation in crude cell-freeextracts, was not as effective as antimycin A indemonstrating flavin reduction. The evidence forinvolvement of an iron-containing component inthe transport of electrons from NADH2 to oxygenis circumstantial. It is based primarily on cyanidesensitivity of the system and on the presence ofiron-containing pigments in extracts of the cells.(W. G. McCullough has established that iron isthe metal in the pigment of S. agalactiae.) Thepigments under study are not cytochromes. Whenthe NADH2 oxidase of cell-free extracts was in-activated by dialysis or ultraffiltration, the enzymecould be reactivated by addition of the ultra-filtrate. The reactivated enzyme was inhibited bycyanide. That oxygen is the terminal acceptorwas shown by the consumption of 0.5 mole ofoxygen for each mole of NADH2 oxidized. It istempting to suggest that the iron-containing pig-ments function in electron transport between thereduced flavoprotein and oxygen, though evidenceavailable at this time does not permit this conclu-sion.

S. pyogenes cultures had only a slow respira-tion that was unchanged whether or not the cellswere grown with aeration, and their respirationwas not inhibited by cyanide (10). The NADH2oxidase in crude cell-free extracts of S. pyogeneswas only about 0.1 as active as that of S. agalac-tiae, and it was also insensitive to cyanide.The function of the iron-containing compounds

in the respiration of S. agalactiae is under study.LITERATURE CITED

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la. CHANCE, B. 1961. Generation of reducing powerin mitochondria by energy-linked DPN reduc-tion. V. Molecular structure and biochemicalreactions. Proc. Robert A. Welch Found. Conf.Chem. Res., Houston, Tex., 1960, p. 93-101.

2. DoLIN, M. I. 1955. The diphosphopyridinenucleotide (DPNH) oxidizing enzymes of S.

faecalis. II. The enzymes utilizing oxygen,cytochrome, peroxide and 2,6-dichlorophenol-indophenol or ferricyanide as oxidants. Arch.Biochem. Biophys. 55:415-435.

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4. GRIESEN, E. C., AND I. C. GUNSALUS. 1944. Analcohol oxidation system in streptococci whichfunctions without hydrogen peroxide accumula-tion. J. Bacteriol. 48:515-525.

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6. HORECKER, B. L., AND A. KORNBERG. 1948. Theextinction coefficients of the reduced band ofpyridine nucleotides. J. Biol. Chem. 175:385-390.

7. KOEPSELL, H. J., AND E. S. SHARPE. 1952. Micro-determination of pyruvic and a-oxoglutaricacids. Arch. Biochem. Biophys. 38:443-449.

8. KREBS, E. G. 1955. Glyceraldehyde-3-phosphatedehydrogenase from yeast, p. 407-411. In S. P.Colowick and N. 0. Kaplan [ed.], Methods inenzymology, vol. 1. Academic Press, Inc., NewYork.

9. KURUP, C. K. R., C. S. VAIDYANATHAN, AND T.RAMASARMA. 1966. NADH oxidase system ofAgrobacterium tumefaciens. Arch. Biochem.Biophys. 113:548-553.

10. MICKELSON, M. N. 1966. Effect of lactoperoxidaseand thiocyanate on the growth of Streptococcuspyogenes and Streptococcus agalactiae in achemically defined culture medium. J. Gen.Microbiol. 43:31-44.

11. O'KANE, D. J. 1950. Influence of the pyruvateoxidation factor on the oxidative metabolismof glucose by Streptococcus faecalis. J. Bac-teriol. 60:449-458.

12. RAMASARMA, T., AND R. C. LESTER. 1960. Studieson the electron transport system. XXIV. Reduc-tion and oxidation of exogenous coenzyme Q.J. Biol. Chem. 235:3309-3314.

13. SEELEY, H. W., AND P. J. VANDEMARK. 1951. Anadaptive peroxidation by Streptococcus faecalis.J. Bacteriol. 61:27-35.

14. STADTMAN, E. R., G. D. NOVELLI, AND F. Lip-MANN. 1951. Coenzyme A function in acetyltransfer by the phosphotransacetylase system.J. Biol. Chem. 191:365-376.

15. UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF-FER. 1957. Manometric techniques, 3rd ed.Burgess Publishing Co., Minneapolis.

16. VELICK, S. F. 1955. Glyceraldehyde-3-phosphatedehydrogenase from muscle, p. 401-406. InS. P. Colowick and N. 0. Kaplan [ed.], Meth-ods in enzymology, vol. 1. Academic Press,Inc., New York.

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