JOURNAL OF BACTERIOLOGY, Jan., 1967, p. 278-285Copyright © 1967 American Society for Microbiology

Effects of Oxygen and Heme on the Developmentof a Microbial Respiratory System

N. J. JACOBS, E. R. MACLOSKY, AND S. F. CONTI'Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire

Received for publication 20 August 1966

ABSTRACT

The effect of adding hemin to anaerobically grown cells of a strain of Staphylococ-cus epidermidis, which was heme-deficient due to anaerobic growth, has been ex-amined. Cells grown anaerobically in media containing hemin exhibited a markedincrease in several oxidative activities as compared with cells grown anaerobicallywithout hemin. The respiratory activity of whole cells and a cyanide-sensitive re-

duced nicotinamide adenine dinucleotide oxidase activity of cell-free extracts wereincreased fourfold. The content of enzymatically reducible pigments which exhibitdifference spectra similar to cytochromes bi and o was also markedly increased.These pigments are mostly sedimented at 100,000 X g (1 hr). Hemin also caused amarked increase in respiratory activity when added directly to the anaerobic cultureafter the period of growth, but did not cause a similar increase in respiration whenadded to washed, resting-cell suspensions. Under the latter conditions, heme pig-ments were formed which exhibited difference spectra similar to, but not identicalwith, the spectra of pigments found in anaerobic cells grown in the presenceof hemin. When resting suspensions of cells grown anaerobically without hemin wereexposed to air, a rapid fourfold increase in respiratory activity and a limited increasein cytochrome-like pigments occurred. The presence of the heme precursor A-amino-levulinic acid during this aeration resulted in a rapid and marked increase in hemepigments, but only a slight stimulation of respiratory activity. The possible implica-tions of these results for the roles which heme and oxygen play in the developmentof the respiratory system of this organism are discussed.

In some microorganisms which can grow bothaerobically and anaerobically, such as yeast andPasteurella pestis, the content of respiratory en-zymes and cytochrome pigments is markedlydiminished by growth under conditions whereoxygen is not available (4, 6, 17, 18). However,the role which oxygen plays in the developmentof the respiratory system in these facultativeanaerobes is not known. With the yeast Saccha-romyces cerevisiae, it has been established thatoxygen has a marked effect on the formation ofthe respiratory system; exposure of resting sus-pensions of anaerobically grown cells to oxygeninduces the formation of respiratory enzymes,cytochrome pigments, and even mitochondrialstructures (15, 18, 20).Our previous investigation with a strain of

Staphylococcus epidermidis established that thisorganism also exhibited a much decreased oxida-tive activity and cytochrome content as a result

1 Present address: University of Kentucky, Lexing-ton.

of growth under anaerobic conditions. However,if anaerobic growth took place in media whichhad been supplemented with hemin, the resultingcells exhibited a much higher respiratory rate(with glucose as substrate) and also containedlarge quantities of pigments resembling cyto-chromes bi and o, as compared with cells grownanaerobically without hemin. This addition ofhemin also resulted in a markedly increased abil-ity of anaerobic cultures to reduce nitrate. Thus,some functional cytochromes seemed to beformed in the absence of oxygen if heme wassupplied (10, 11). These observations suggestedthat one of the important functions of oxygen inthe development of the respiratory system in thismicroorganism was as a requirement for the bio-synthesis of heme. Goldfine and Bloch (7) havesuggested that the lack of hemoprotein synthesisduring anaerobic growth of some facultativeanaerobes may perhaps be ascribed to a require-ment for oxygen in the synthesis of the prostheticgroup, heme. A probable role for oxygen in heme

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biosynthesis is suggested by the observation that,in rat liver mitochondria, one of the terminalreactions in the biosynthesis of heme (the con-version of coproporphyrinogen to protoporphy-rin) requires molecular oxygen (16). S. epidermi-dis forms large quantities of coproporphyrinwhen grown anaerobically (9). The observationthat other bacteria, such as Escherichia coli,form cytochromes during anaerobic growth sug-gests that not all bacteria exhibit an oxygen re-quirement for heme synthesis (8).

In the present investigation, the electron trans-port system in extracts of S. epidermidis whichhad been grown anaerobically in the presence ofhemin was characterized more completely andwas compared with the electron transport systemfound in aerobically grown cells. We also investi-gated the effect of adding hemin to resting sus-pensions of cells which had been grown anaerobi-cally without hemin, and the ability of oxygen toinduce the development of the respiratory systemin resting suspensions of these cells.

MATERIALS AND METHODSThe organism used, the conditions of growth, the

preparation of cell suspensions, and the spectrophoto-metric techniques have been described previously (10).Carbon monoxide difference spectra were obtained byadding hydrosulfite to both sample and referencecuvettes and carbon monoxide to the sample cuvette.Cell suspensions were disrupted by means ofa BransonSonifier (Branson Instruments, Danbury, Conn.) orby the "x" press (5). Reduced nicotinamide adenine-dinucleotide (NADH2) oxidase activity was deter-mined by following the rate of decrease in opticaldensity at 340 m, in the presence of 0.025 M potassiumphosphate buffer (pH 7.0), 400 ,ug of NADH2 per 3ml, and various concentrations of extract. Protein wasdetermined by the method of Lowry et al. (14), A-Aminolevulinic acid hydrochloride was purchasedfrom Sigma Chemical Co. (St. Louis, Mo.).

RESULTS

Localization ofpigments in cells grown anaero-bically with hemin. To characterize further thecytochrome-like pigments in cells grown anaero-bically with hemin, the distribution of these pig-ments was determined after fractionation of cell-free extracts by use of an ultracentrifuge. Asuspension of cells grown anaerobically in mediacontaining added hemin (10) was broken in the"x" press and was sedimented at 10,000 X g for20 min to remove intact cells and large fragments.A portion of this supernatnat fluid (13.5 ml) wasthen sedimented at 100,000 X g for 1 hr. Theresulting pellet was resuspended in the originalvolume (13.5 ml), and the difference spectra ofthese various fractions were recorded (Fig. 1).An examination of the heights of the peaks inthese spectra revealed that more than half of the

EE 0.02- I00,00 A -pellet | supernatantz

w- 0.0

a: ;Or .

z

500 550 600

WAVELENGTH - m,UFIG. 1. Reduced minus oxidized diffierence spectra of

extracts of cells grown anaerobically with hemin andsubjected to diffierential centrifugation. All extractssuspended in 0.025 M phosphate buffper (pH 7.0), andspectra were recorded after addition of a few crystals ofsodium hydrosulfite to the extract in the sample cuvetteand aeration of the extract in the reference cuvette.

cytochrome b1-like component with a peak at 558mu was sedimented by centrifugation at 100,000X g for 1 hr. This observation is consistent withthe view that this pigment resembles a typicalcytochrome as it is associated with a particulateportion of the cell.

Comparison ofNADH2 oxidation in extracts ofcells grown aerobically and anaerobically in thepresence and absence of hemin. We previouslyreported that whole cells grown anaerobicallywith hemin exhibit a much higher respiratoryrate (with glucose as substrate) than cells grownanaerobically without hemin. This respiratoryrate was about 60% of the rate exhibited byaerobically grown cells. To characterize furtherthe respiratory system of these cells, we examinedthe NADH2 oxidase activity of cell-free extracts.This activity was about four times higher in cellsgrown anaerobically with hemin than in cellsgrown anaerobically without hemin (Table 1).This is in accord with our previous conclusion,that anaerobic growth in the presence of heminresults in a marked increase in respiratory en-zymes. It is interesting that cells grown anaero-bically in the presence of hemin, which containpigments resembling cytochromes b1 and o, exhibitonly about 40% of the NADH2 oxidase activityof the cells grown aerobically, which containcytochromes oftheeb,o, anda type (10). Furtherwork is needed before the difference in oxidativecapacity of these two types of cells can be under-

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TABLE 1. NADH2 oxidase activity in extracts ofcells grown under various conditions

Amt (,imoles) of NADHGrowth condition oxidized per min per

mg of protein&

Aerobic. . 0.0480Anaerobic.................. 0.0046Anaerobic + hemin ......... 0.0180

a Cell-free extracts were prepared by disruptingcell suspensions in the sonic oscillator and thenremoving the remaining whole cells and cell debrisby centrifugation at 2,000 X g for 20 min.

stood. The NADH2 oxidase activity of cellsgrown anaerobically in the presence of hemin wasinhibited approximately 65% by 10-3 M cyanide.This is the same degree of inhibition noted byTaber and Morrison (19) for the NADH2oxidase activity in extracts of aerobically grownS. aureus.

Effect of addition of hemin to washed, restingsuspensions of cells grown anaerobically withouthemin on cytochrome content and respiratorycapacity. Since our previous studies indicatedthat the presence of added hemin during the entireperiod of anaerobic growth caused a marked in-crease in respiratory catalysts, it was importantto establish whether a similar effect of hemincould be observed when hemin was added aftergrowth to resting suspensions of cells grownanaerobically without hemin. A washed suspen-sion of these cells [8 mg (dry weight) per ml]prepared as previously described (10) was incu-bated in the presence of hemin (30 ,g/ml) and0.033 M phosphate buffer (pH 7.0) for 60 minunder an atmosphere of nitrogen gas. The sus-pension was then washed five times by centrifuga-tion in 0.85% saline (0 C) to remove looselybound hemin. The difference spectra of the result-ing cells are shown in Fig. 2 and 3, along withthe spectra of cells which had not been treatedwith hemin. The spectra of anaerobic cells grownin the presence of hemin is included for compari-son. The respiratory rate of these cell suspensions,and of a suspension of aerobically grown cells,was also examined (Table 2). As described previ-ously, cells grown anaerobically in the presence ofhemin exhibit about 55% of the respiratory ac-tivity of aerobically grown cells. They also con-tained pigments resembling cytochrome b, in thereduced minus oxidized spectrum (peaks at 558,524, and 427 mA), and cytochrome o in the car-bon monoxide spectrum (peak at 417 m,u, andtrough at 435 m,u). The cells which were grownanaerobically without hemin, but which wereincubated with hemin under resting-cell condi-tions, also exhibited pigments. Although these

E

-0.04

z

w

z 0.02

z

cr

0Ocir

hemin added after growth

\\ grown with hemin

_no hemin added

4 50 550 650WAVELENGTH - mjj

FIG. 2. Reduced minus oxidized difference spectraof cell suspensions exposed to hemin during the growthperiod or after the growth period under resting-cell con-ditions. All suspensions contained 2.35 mg of cells (dryweight) per ml, and were prepared as described in thetext or previously (10). The sample cuvette was reducedwith a few crystals of hydrosulfite, and the referencecuvette was aerated.

E

0.02z

LLJ ..grown with herina

LLJ~~~~~~~~~~~~~~~

O - I; t < anohemin added

O - I z h e m n added after growth

450 550 650

WAVELENGTH - m,u

FIG. 3. Carbon monoxide difference spectra of cellsuspensions prepared as in Fig. 2. All suspensions con-tained 2.35 mg of cells (dry weight) per ml, and wereprepared as described in Fig. 2.

TABLE 2. Respiration of cells exposed to heminunder growth or resting-cell conditions

Amt of 02Condition ~~consumedCondition per hr per

mg of cells"

pAlitersCells grown anaerobically withouthemin.............................. 10

Cells grown anaerobically withouthemin; hemin added after growth... 13

Cells grownL anaerobically withaddedhemin.37

Cells grown aerobically............... 67

a Respiration rate was determined manometri-cally with glucose as substrate, as described previ-ously (10).

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pigments were grossly similar to those present incells grown with hemin, there were some differ-ences. For instance, in the reduced minus oxidizedspectra, prominent peaks appeared at 560 and427 m,u in the cells incubated with hemin underresting conditions, and at 558 and 427 m,u in thecells grown in hemin. In addition, the ratio of theheight of the Soret peak to the a peak was muchsmaller in cells grown in the presence of hemin.In the carbon monoxide spectra, the cells grownwith hemin exhibited a Soret peak at 418 mp,whereas those incubated with hemin under restingconditions exhibited a peak at 421 m,u. As de-scribed previously (10), the pigments in cellsgrown anaerobically with hemin could be reducedeither with hydrosulfite or by endogenous sub-strates. With cells to which hemin had been addedunder resting-cell conditions, reduction of pig-ments by endogenous substrate was not readilyobtained, and rapid reduction was only observedupon the addition of hydrosulfite. However, it ispossible that the inability of these pigments to bereduced rapidly by physiological substrates is aconsequence of the low respiratory capacity ofthese cells (see below). It seems likely that mostof the pigments formed on addition of hemin towashed, re4ting suspensions of cells grown anaero-bically without hemin are not cytochromes, butmay rather be hemochromogens formed by com-bination of heme with some nitrogenous constitu-ents of the cell other than the apoenzymes of thecytochromes.The addition of hemin to these resting cells

also caused no substantial increase in respiratoryrate (with glucose as substrate) over anaerobicallygrown cells which had never been incubated inhemin (Table 2). This does not appear to becaused by the inability of hemin to penetrate thecell, since cell-free extracts (prepared by sonicoscillation) of washed suspensions which hiadbeen incubated in hemin exhibited hemochromo-gen pigments spectrally similar to those of thewhole cells. These extracts were prepared fromcells which had been extensively washed, and thepigments remained in the supernatant solutionafter removal of whole cells and cell debris bycentrifugation at 10,000 X g for 20 min. There-fore, we tentatively concluded that the pigmentsare not bound to the cell wall and that hemin can

penetrate the cell under the incubation conditionsdescribed.Thus, we were not able to demonstrate any sig-

nificant effect of hemin on respiratory activitywhen it was added to washed, resting suspensionsin phosphate buffer of cells grown anaerobicallywithout hemin, nor did the heme pigments whichwere formed under these conditions have the

properties of cytochromes. This is in sharp con-trast to the effect which hemin exerts when it ispresent during the growth of this organism underanaerobic conditions. The cells which are thusobtained exhibit a much higher respiratory ratethan those which have not been in contact withhemin, and their pigments do have the propertiesof cytochromes.

Effect of adding hemin directly to the cultureafter the period ofanaerobic growth. Our previousexperiments (10) reported a marked effect ofhemin on the respiratory capacity of anaerobicallygrown cells when hemin was added to the growthmedium at the time of inoculation and was, there-fore, present during the entire period of anaerobicgrowth. The experiments reported above indicatedthat a similar effect of hemin could not be readilydemonstrated when hemin was added to washed,resting suspensions of cells grown anaerobicallywithout hemin. In an attempt to gain further in-formation regarding this phenomenon, we deter-mined the effect of hemin when it was addeddirectly to cultures after anaerobic growth in theabsence of hemin.Anaerobic cultures were grown under the same

conditions as previously described (10), exceptthat the gluose concentration in the growthmedium was reduced from 0.5 to 0.25%. After agrowth period of 16 hr, a small volume of heminsolution was added directly to the culture flask,to give a final concentration of 0.3 pAg/ml, andprecautions were taken to introduce as little air aspossible. (Preliminary growth experiments re-vealed that cultures were near the end of thelogarithmic phase of growth after 16 hr.) A simi-lar volume of 0.01 NKOH solution (KOH is usedto dissolve the hemin) was added to a separateanaerobic culture to serve as a control. Incuba-tion of both flasks was continued for 1 hr at 37 Cunder anaerobic conditions. Both flasks werethen chilled to 0 C, and the cells were removedby centrifugation, washed, and resuspended inphosphate buffer. The two suspensions werethen tested manometrically for respiratory activitywith glucose as substrate as described previously(10). The results of several experiments revealedthat cultures to which hemin had been addedyielded cells which exhibited an averageQo2 of 22,whereas those to which hemin had not beenadded exhibited a Qo, of 9. Thus, hemin had adefinite and marked effect in causing an increasein respiratory activity under these conditions.Studies are in progress to define further the con-ditions under which this effect of hemin can beobserved.

Effect ofaeration on the development ofrespira-tory activity and cytochrome content of cells

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grown anaerobically without hemin. When restingsuspensions of cells grown anaerobically withouthemin were aerated in the presence of buffer andglucose, they developed an increased respiratoryrate when tested with glucose as substrate. Mostof this increase occurred within 1 hr after expo-sure to air (Table 3). The maximal Qo2 achievedunder these conditions varied between 24 and 33.This usually represented about a fourfold in-crease over cells which had not been exposed toair. Buring the aeration period, no significantincrease in density of the suspensions could bedetected. Additional aeration beyond a 2-hrperiod did not result in an increase in Qo2 even ifmore glucose was added to the aerated suspen-sions. The respiratory rates achieved under theseconditions are considerably lower than those ex-hibited by cells grown aerobically (Qo2 of 66) andalso somewhat lower than those exhibited bycells grown anaerobically in the presence ofhemin (Qo2 of approximately 44) (10). The differ-ence spectra of the anaerobic cells aerated for 2 hrare shown in Fig. 4 and 5, and are comparedwith the spectra of cells which were not aerated.A small but definite increase in pigments resem-bling cytochrome bi (peaks at 558 and 428 m, inreduced minus oxidized spectra) and cytochromeo (peak at 416 and trough at 435 m,u in the car-bon monoxide spectra) was caused by aeration.It is significant that the amounts of these pig-ments (as indicated by the height of the peaks) inthe anaerobic cells aerated for 2 hr were only a

TABLE 3. Respiratory rate of cells grownanaerobically after various periods

of aeration

Period for which cells Amt of 02 consumed perwere aerated hr per mg of cellse

min plitersNone 630 1260 1890 21120 24240 21

a The cell suspensions used were prepared asfollows. Suspensions of cells grown anaerobicallywithout hemin were prepared as previously de-scribed (10). These cells (which had been washedonce in cold saline) were adjusted to a concentra-tion of approximately 5 mg/ml (dry weight) in0.025 M potassium phosphate (pH 7.0) and 0.008 Mglucose. A 50-ml amount of this suspension wasplaced in a 500-ml Erlenmeyer flask, on a rotaryshaker at 37 C. Sampleswere removed at the timeintervals indicated, cooled to 0 C, and testedmanometrically for respiratory activity as indi-cated previously (10).

E

. 0.02zw

0.01

z0

uz

0enm

4

cell suspensionoerated for 2 h r.

, cel suspension before% exposure to oir

400 450 500 550 600 650

WAVELENGTH - mp

FIG. 4. Reduced minus oxidized difference spectra ofcells grown anaerobically and exposed to air for 2 hr.Cell suspensions (prepared as described in Table 4)were adjusted to a concentration of 7 mg/ml in 0.05 Mphosphate buffer, pH 7.0. A few crystals of sodiumhydrosulfite were added to the sample cuvette, and thereference cuvette was aerated vigorously.

EI-

Z 0.02w

2a: 0.01

z

0

zmCE0

CDcr

cell suspension(aeroted for 2 hr.

O'

\.cell suspension beforeexposure to oir

400 450 500 550 600 650WAVELENGTH - mp

FIG. 5. Carbon monoxide difference spectra of cellsgrown anaerobically and exposed to air for 2 hr. Cellsuspensions as in Fig. 4.

fraction of those obtained in aerobically growncells. For instance, the same density of aerobicallygrown cells would have revealed peaks at 556 and428 m,u in the reduced minus oxidized spectrafour to five times higher than those shown inFig. 5 (10). These results indicate that a rapid,but limited, increase in Qo2 occurred when restingsuspensions of anaerobic cells were exposed to airat 37 C. This increase was also accompanied bya limited increase in pigments resembling cyto-chromes. Experiments are in progress to deter-mine whether these changes in respiratory activityare dependent upon the synthesis of new protein.

Effect of the heme precursor A-aminolevulinicacid on the development ofrespiratory activity andpigment content during the aeration of anaero-bically grown cells. When A-amninolevulinic acid(ALA) was added to resting suspensions whenthey were exposed to air for short periods of

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time, the resulting cells exhibited a marked in-crease in pigment content as indicated by thedifference spectra (Fig. 6 and 7). The appearanceof these spectra suggests that ALA causes atleast a threefold increase in the content of thesepigments, which are doubtlessly some kind ofheme compound. Further experiments indicatedthat the formation of large quantities of hemecompounds in the presence ofALA did not occurif the cells were exposed to air at 0 C or if theywere held under anaerobic conditions at 37 C.Whether or not these heme pigments are truecytochromes or merely hemochromogens is notknown. In Table 4, the respiratory rate of thesecells is shown. As indicated, the addition ofALAcaused only a slight stimulation in the Qo2 of theresulting cells. (This stimulation, although small,was noted repeatedly in several different experi-ments.) Thus, although ALA caused a marked(at least threefold) increase in heme biosynthesis,

'E J A cells exposed to oir.| in the presence of ALA

0.01 cells exposed to

zoir without

0oddition of

z

a) cells~ not exposed

or to air

CI

400 450 500 550 6oo 650

WAVELENGTH - m,

FIG. 6. Reduced minus oxidized difference spectra ofanaerobically grown cells exposed to air in the presenceand absence ofA-aminolevulinic acid (ALA). These sus-

pensions were prepared as follows: 70 ml ofa suspensionofanaerobically grown cells (prepared in the presence ofglucose and phosphate buffer as in Fig. 5) was placed ina 500-ml Erlenmeyer flask and was incubated at 37 Cfor 45 min (without shaking). Where indicated, ALAwas added to the incubation mixture at a level of10 mgper 70 ml. After 45 min, the flasks were chilled to 0 C,and the cells were removed by centrifugation, washed incold saline, and resuspended in 0.05 M phosphate buffer,pH 7.0. The spectra were recorded at a cell density of4.7 mg/ml (dry weight) after addition of hydrosulfite tothe sample cuvette and aeration ofthe reference cuvette.Cells not exposed to air were held at 0 C and did not re-ceive ALA.

E

5~-

Ia

LuJ

Ur

z4tmcr0.05o.

0

41 n

exposed to oir inpresence of ALA

cells exposed to oir *

without addition of ALA

i1s not exposed 'to oir

650

WAVELENGTH - mju

FIG. 7. Carbon monoxide difference spectra ofanaerobically grown cells exposed to air in the presenceand absence of A-aminolevulinic acid. Suspensionsprepared as in Fig. 6.

TABLE 4. Respiratory rate of cells grown anaero-bically and exposed to air in the presence and

absence of A-aminolevulinic acid (ALA)

Amt of OsCondition consumed per hr

per mg of cells

,sliersCells not exposed to air..... 9.5Cells exposed to air without addi-

tion ofALA......... 17.0Cells exposed to air in the presence

ofALA. 19.5

a Cell suspensions were prepared as in Fig. 6.Respiratory activity was determined as in Table 3.

it only caused a slight stimulation of respiratoryadaptation.

DISCUSSIONGrowth of this strain of S. epidermidis under

anaerobic rather than aerobic conditions causesa marked decrease in cytochrome content andrespiratory activity. However, our results indicatethat the addition of hemin to the anaerobicgrowth medium causes a marked increase incytochrome-like pigments and oxidative activitieswhich appear similar to those formed duringaerobic growth. For instance, cells grown anaero-bically in the presence of added hemin contain

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pigments which exhibit difference spectra identicalto the cytochromes of the bi and o type presentin aerobically grown cells. (However, no cyto-chrome of the a type was detected in anaerobicallygrown cells.) These pigments are readily reducibleby endogenous substrates and are apparently as-sociated with the particulate portion of the cell(partly sedimented at 100,000 X g for 1 hr). Inaddition, cells grown anaerobically in the presenceof added hemin exhibit a marked increase inrespiratory activity as compared with thosegrown anaerobically without hemin. This is indi-cated by a sixfold increase in Qo, of whole cells(with glucose as substrate) and a fourfold in-crease in the cyanide-sensitive NADH2 oxidaseactivity of cell-free extracts. Thus, the addition ofhemin to the medium in which anaerobic growthoccurs causes a marked increase in a respiratorysystem which has at least some of the propertiesof the normal respiratory system found in aero-bically grown cells. The addition of hemin toaerobically grown cultures causes no increase inthe high level of respiratory activity and cyto-chrome content of the resulting cells (10, unpub-lished observations).Although this effect of hemin is obtained when

hemin is present during the entire period of an-aerobic growth, a similar effect was not observedwhen hemin was added to washed, resting suspen-sions of cells grown anaerobically in the absenceof hemin. Under these conditions, hemin does notcause a marked increase in respiratory activity,and the hemochromogen pigments formed do nothave the properties of typical cytochromes. How-ever, a marked effect of hemin on the respiratorycapacity of anaerobic cells was observed when itwas added directly to the culture medium afteranaerobic growth. We do not yet know why theeffect of hemin can be observed under these latterconditions, but not when it is added to washed,resting suspensions. If we assume that the apo-enzymes of the cytochromes, minus the hemeprosthetic group, are present in cells grown an-aerobically without hemin, our results wouldsuggest that, when in the growth medium, cellsare more capable of reactions required to couplehemin to preformed apoenzymes. On the otherhand, this effect of hemin may require the synthe-sis of new apoenzyme, and this process wouldalso be favored by keeping the cells in the growthmedium. Another possibility is that componentsof the respiratory chain other than hemoproteins,such as vitamin K2, are missing from anaero-bically grown cells (1). The addition of hemin tofully grown cultures while the cells remain in thegrowth medium may also cause an increased syn-thesis of these components. A further investiga-

tion of the effect of hemin under different condi-tions should yield significant information regard-ing the mechanism by which hemin exerts itseffect.

Regarding the question of the presence or ab-sence of the apoenzymes of the cytochromes incells grown anaerobically without hemin, it isinteresting to compare our results with restingcells with previous results obtained with a mutantstrain of S. aureus, which is heme-deficient be-cause of a genetic block in heme biosynthesis.With this organism, the addition of hemin aftergrowth to resting suspensions grown withouthemin does cause a restoration of respiratory andcatalase activity (12). Chang and Lascelles (3) ob-served the formation of cytochrome bi whenhemin was added to cell-free preparations ofparticles of this mutant grown in the absence ofhemin. These results would suggest that this mu-tant can form the apoenzymes of the cytochromesduring growth in the absence of hemin. It is notyet possible to interpret our own results withanaerobically grown cells of S. epidermidis interms of whether or not the apoenzymes of thecytochromes are formed during anaerobic growthin the absence of hemin. Perhaps the formation ofthe apoenzymes of the hemoproteins is dependentupon the presence of the prosthetic group, heme,during a period when protein synthesis is occur-ring. Recent studies with mammalian hemoglobinsynthesizing systems indicate that heme doeshave an enhancing effect on globin formation (2,13; C. L. Hammel and S. P. Bessman, FederationProc. 23:317). Of course, in the case of S. epi-dermidis, heme may be playing a more indirectrole in producing the observed increases in therespiratory system of anaerobically grown cells.Our experiments in which resting suspensions

of cells grown anaerobically without hemin wereexposed to air indicate that this exposure resultsin a rapid, but limited, fourfold increase in respir-atory capacity of whole cells, as determined withglucose as substrate. There is also a limited in-crease in pigments resembling cytochromes dur-ing this period of aeration. Although their exactsignificance must await further investigation,these results suggest that oxygen is capable of in-ducing some changes in the respiratory systemunder resting-cell conditions. One of the goals offuture work will be to determine whether thiseffect of oxygen is primarily to allow heme syn-thesis, or whether oxygen plays another role inde-pendent of its proposed role in heme synthesis ininducing the formation of the components of therespiratory system.The effect of the heme precursor A-aminolevu-

linic acid during this exposure to oxygen was of

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interest, since it demonstrated that these anaero-bically grown cells, although they are deficient inheme compounds, can rapidly synthesize largeamounts of heme if they are exposed to oxygenand an appropriate heme precursor. The factthat this increased synthesis of heme has a slightstimulatory effect on the development of respira-tory capacity may have some implications regard-ing the role of heme in the formation of therespiratory system. Certainly, it is clear that dur-ing the period when resting suspensions areexposed to air, the marked increase in heme for-mation brought about by the addition of A-amino-levulinic acid is not accompanied by a markedincrease in rate of development of the respiratorysystem. Further studies are in progress to answersome of the questions raised by these observa-tions.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService grants AI-06344 from the National Instituteof Allergy and Infectious Diseases and GM 08565from the National Institute of General MedicalSciences, and by Career Program Award I-K3-GM-9716 to S.F.C.

LITERATURE CITED

1. BISHOP, D. H. L., K. P. PANDYA, AND H. K.KING. 1962. Ubiquinone and vitamin K in bac-teria. Biochem. J. 83:606-614.

2. BRUNS, G. P., AND I. M. LONDON. 1965. The effectof hemin on the synthesis of globin. Biochem.Biophys. Res. Commun. 18:236-242.

3. CHANG, J. P., AND J. LASCELLES. 1963. Nitrate re-ductase in cell-free extracts of a hemin-requir-ing strain of Staphylococcus aureus. Biochem. J.89:503-510.

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