JOURNAL OF BACTERIOLOGY, Sept. 1968, p. 665-671 Vol. 96, No. 3Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Electron Transport System of theProtoheme-requiring AnaerobeBacteroides melaninogenicus

VICTOR RIZZA, PETER R. SINCLAIR, DAVID C. WHITE, AND PAUL R. CUORANTDepartment ofBiochemistry, University ofKentucky, Medical Center, Lexington, Kentucky 40506

Received for publication 4 June 1968

Protoheme is essential for the growth of some strains of Bacteroides melanino-genicus. At low concentrations in the growth medium, protoheme determines thedoubling time, total cell yield, and amount of cytochrome per bacterium. At highprotoheme concentrations, the doubling time, total cell yield, and amount of en-zymatically reducible cytochrome appear to remain nearly constant, and proto-heme is accumulated by the cell. The accumulated protoheme can support thegrowth of the bacterium for at least eight generations in a protoheme-free medium.When growth and cytochrome content are proportional during growth at low pro-toheme concentrations, the bacteria incorporate 10 to 20% of the total availableprotoheme into a membrane-bound respiratory system. This respiratory systemincludes cytochrome c, a carbon monoxide-binding pigment, and possibly flavo-proteins. The pigments can be reversibly reduced by reduced nicotinamide adeninedinucleotide or endogenous metabolism and can be oxidized anaerobically byfumarate or by shaking in air. Electron transport is inhibited by 2-n-nonyl-4-hy-droxy-quinoline-N-oxide.

Many strains of Bacteroides melaninogenicusrequire heme and vitamin K for growth (4, 5, 8).The fact that both heme and vitamin K are re-quired for growth of this obligate anaerobe sug-gests that an electron transport system may beinvolved in its metabolism. This study was initi-ated to determine whether a respiratory systemis present in B. melaninogenicus and whether thelevel of heme supplementation in the growth me-dium affects the formation of the respiratory sys-tem.The strain of B. melaninogenicus used in this

study, strain CRA, required protoheme forgrowth. Growth of this strain in complex mediumwas independent of the presence of carbohydrate.Large amounts of ammonia, acetate, propionate,lactate, and butyrate are the major products ofmetabolism of B. melaninogenicus (10). In thisstudy, we demonstrated the presence of a mem-brane-bound respiratory system involving flavo-proteins, cytochrome c, and a carbon monoxide-binding pigment. These pigments were reversiblyreduced by reduced nicotinamide adenine dinu-cleotide (NADH) and were oxidized by fumarate.The growth rate, final cell yield, and concentra-

1Present address: 18 North Road, ChelmsfordCentre, Mass.

tion of respiratory pigments were dependent onthe concentration of protoheme supplied in themedium. Even though the growth of the organ-ism was limited by the concentration of proto-heme in the medium, only 10 to 20% of the addedprotoheme was found in the enzymatically reduci-ble cytochrome c.

MATERIALS AND METHODSOrganism. B. melaninogenicus strain CR2A was a

gift from R. J. Gibbons. Cultures were preserved bythe addition of 15% (v/v) sterile glycerol during thelate log phase of growth and were kept at -60 Cin an atmosphere of CO2-H2. The criteria used tojudge the purity of the cultures included phase-con-trast microscopic examination of the living cells,Gram stain, and colonial morphology. The organismused in these experiments was a typical gram-negative,nonmotile cocco-bacillary form that gave small,glistening, smooth colonies which turned black in 6to 7 days when incubated anaerobically on blood agarplates. Concomitant with pigment production, therewas a zone of clearing around each colony on theblood agar plate.

Media. The growth medium consisted of 2.7%(v/v) Trypticase (BBL), 0.3% yeast extract (Difco),34.5 mM NaCl, 14.6 mM K2HP04, and 14.7 mMK2COs. The medium was autoclaved for 15 mmin at121 C, cooled to room temperature, equilibrated withH2-C02, and stored inside Brewerjars. The stock sol-

665

RIZZA ET AL.

ution of protoheme was prepared by dissolving 20 mgof protoheme in 0.5 ml of pyridine. The volume ofthe protoheme solution was brought to 5 ml with 0.5M NH40H and was stored in the dark at -16 C. Thefinal concentration of protoheme added to the mediumis indicated in the text. The organism was streaked onblood agar which was prepared with 4.0% (w/v) HeartInfusion Agar (Difco) and 0.5% agar (BBL) that wasautoclaved and cooled to 55 C prior to the addition of10% (v/v) sterile defribrinated horse blood (ColoradoSerum Co., Denver, Colo.).

Growth of the organism. Bacteria were grown at 37C in flasks or screw-cap centrifuge tubes inside Brewerjars in an atmosphere of 5% CO2 and 95% H2 (v/v).A red-hot nichrome wire was used to catalyze waterformation from residual oxygen in the Brewer jars.The wire was kept hot for at least 30 min prior toincubation. All media were stored in these jars andwere equilibrated with the C02-H2 atmosphere beforeuse. Unless a sufficiently large inoculum was used,it was difficult to obtain reproducible bacterial growth.In these experiments, the inoculum used was 10% ofthe final volume of the culture. The inoculum wasprepared by transferring the cells to medium withoutadded protoheme and allowing the cells to grow to amaximal absorbance. This was repeated until nofurther growth was observed. Under these conditions,the inoculum used in the third transfer would not growunless protoheme was present in the medium.

Preparation of cell suspensions. Cultures wereharvested in the late log phase of growth by centrifuga-tion at 14,000 X g for 30 rnin. The bacterial pelletwas washed and suspended in 50 mm phosphate buffer(pH 7.6) that contained 1 mm dithiothreitol and 10.4mm sodium thioglycolate. The buffer was deoxy-genated with nitrogen or CO2.

Determination of the doubling time. The doublingtime was defined as the time necessary for the absorb-ance to double between bacterial densities of 0.07and 0.35 mg (dry weight)/ml. The absorbance wasmeasured in round tubes, 13 mm in diameter, with aSpectronic-20 colorimeter (Bausch & Lomb,Rochester, N.Y.) at 750 nm.

Protein. Protein was determined colorimetrically(9) with bovine serum albumin as the standard.

Difference spectra. Samples from the same bacterialsuspension having respiratory pigments at two dif-ferent oxidation levels were compared by differencespectroscopy. Difference spectra were measured witha Cary 14 CM spectrophotometer as described pre-viously (14, 16). To obtain reduced minus oxidizeddifference spectra, the respiratory pigments in one partof a bacterial suspension were allowed to reduce byendogenous metabolism. In another portion of thesame bacterial suspension, the respiratory pigmentswere oxidized before measurement by vigorouslyshaking in air or by the anaerobic addition of fuma-rate. The measurement of reduced minus oxidizeddifference spectra was established as an accuratemeasure of cytochrome content by comparing differ-ence spectra to the absolute spectrum of bacteria.The absolute spectrum was obtained by measuringthe bacteria with endogenously reduced pigmentsagainst ground glass of appropriate light scattering

properties (14). The carbon monoxide-reduced minusreduced difference spectra were measured in the fol-lowing way. The respiratory pigments in a bacterialsuspension were allowed to reduce by endogenousmetabolism. A portion of the suspension was saturatedwith carbon monoxide and was compared to a secondportion of this suspension.

Cytochrome c was measured as the absorbanceincrement between the a maximum at 555 nm and thepoint at 555 nm on a base line connecting points at600 and 537 nm in the reduced minus oxidized differ-ence spectrum (unless otherwise stated). Substratessuch as NADH, succinate, or formate were added toanaerobic suspensions in Thunberg cuvettes or underscrubbed nitrogen as described (16). Spectra at thetemperature of liquid nitrogen were measured essen-tially as described by Estabrook (1), in an apparatusemploying Lucite cuvettes with a 2-mm path length.This apparatus was designed by T. Orr.

Pyridine hemochrome. Pyridine hemochromes weremeasured as described by Falk (2).

Extraction ofhemes. To extract protoheme from thebacteria, cold acetone, water and concentrated HCI(50:10:2.5, v/v) were added to the bacterial pellet,and the suspension was homogenized with a Teflonhomogenizer and then centrifuged in polypropylenetubes. Approximately 40 ml of acid acetone was usedper g (dry weight). The residue was suspended inwater, and the heme c was determined by preparingthe pyridine hemochrome. The supernatant portionof the acid acetone extraction was then extracted withether, and the ether was washed with 0.27 M HCl. Theaqueous layer was then extracted again with moreether. The porphyrin was separated from the proto-heme by extracting the combined ether phases with 4.5M HCI. The porphyrin was then taken up by anotherportion of ether by adjusting the HCI to pH 5 withNaHCO3, and the absorbance was measured afterextraction with 1.5 M HCI. The porphyrin spectrumhad absorption maxima at 601, 556, and 409 nm, witha ratio of the maxima at 409 to 556 nm of 19.4 [theratio expected for protoporphyrin is 19.3 (2)]. Theconcentration of protoporphyrin was calculated withe4w = 262 X 10 per mole per cm (2). The etherlayer containing protoheme was washed with twoportions of 0.27 M HCl, and the ether was evaporatedto dryness in vacuo. The reduced pryidine hemo-chrome was then prepared.

Materials. Materials were the best grade com-mercially available, as reported previously (15).

RESULTS

Protoheme requirement for growth. The com-plex medium necessary for growth of B. melanino-genicus contained less than 3.7 pmoles of proto-heme/ml, as measured with the medium at 10times the usual concentration. In this medium, B.melaninogenicus was unable to grow unless proto-heme was added, provided that the bacteria weredepleted of heme by incubation in the absence ofheme until growth stopped. Bacteria that weredepleted of heme showed an increase in both the

666 J. BACTERIOL.

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final cell yield and in the doubling time when theprotoheme supplementation of the medium wasincreased between <3.7 and 615 pmoles/ml. Atprotoheme concentrations above 615 pmoles/ml,the total cell yield remained near 0.9 mg (dryweight) /ml and the doubling time between 3 and4 hr (Fig 1).

Cytochrome system. The respiratory pigmentsof a suspension of B. melaninogenicus can beoxidized by shaking the bacteria in air or by theanaerobic addition of fumarate in a Thunbergcuvette. The reduced minus oxidized differencespectrum is shown in Fig 2. The spectrum wasthe same whether oxidation was achieved byshaking in air or by the anaerobic addition offumarate. In the suspension in which the respira-tory pigments were oxidized by shaking in air, thepigments were reduced in 20 min by the endoge-nous metabolism of the bacteria at room tempera-ture. A cytochrome with maxima at 555, 528, and422 nm could be detected in the reduced minusoxidized difference spectrum. A minimum at 450nm in this spectrum suggested the involvement offlavoproteins and possibly of nonheme iron pig-ments in this respiratory system. A carbon mon-oxide-binding pigment, with maxima at 578, 540,and 416 nm and with minima at 553 and 425 nm,could be detected in the carbon monoxide minusreduced difference spectra. The respiratory pig-ments were reduced by the endogenous metabo-lism of the bacteria. In bacteria grown in thepresence of less than 0.60 nmole of protoheme/ml, in which the respiratory pigments were re-duced by endogenous metabolism, there was nofurther reduction of the cytochrome system whenNa2S204 was added.

Localization of the cytochrome system. B. mela-

.6 -14

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FIG. 1. Doubling time and final yield ofBacteroidesmelaninogenicus with different levels ofprotoheme. Theinocula consisted of cells depleted ofprotoheme. Dryweight and doubling time were determined as describedin Materials and Methods.

WAVELENGTH (ml.)420 460 500

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500 540 580 620 660WAVELENGTH mp

FIG. 2. Difference spectra of Bacteroides melanino-genicus. Bacteria were suspended at a density of 6.1mg of bacterial protein/ml in 50 mM phosphate buffer(pH 7.6) containing I mm dithiothreitol, 10.4 mm sodiumthioglycolate, and 15% glycerol. The buffer was deoxy-genated with nitrogen. Symbols: Solid line, bacteriawith pigments reduced by endogenous metabolism com-

pared to bacterialpigments oxidized by vigorous shakingin air; dashed line, bacteria with pigments reduced byendogenous metabolism and saturated with carbon mon-

oxide compared to bacteria with pigments reduced byendogenous metabolism.

ninogenicus could be ruptured, as determined byphase-contrast microscopy, by exposure to ultra-sonic vibration for 4 min. This exposure was per-

formed under nitrogen and the temperature was

kept below 10 C. The cell-free preparation was

then centrifuged at 25,000 X g for 30 min, thesupernatant portion was decanted, and the pelletwas resuspended in the phosphate-thioglycolatebuffer. The resuspended particulate preparationcontained 99% of the cytochromes present in theintact bacteria. Less than 0.1% of the cyto-chromes could be detected in the supernatantfluid.

Respiratory system. The respiratory pigmentsof the resuspended particulate preparation couldbe completely reduced in the presence of 5 mmNADH. The pigments were not reduced in thepresence of succinate, formate, DL-lactate, or

hydrogen. In the presence of 1 AM NADH, theanaerobic addition of 20 mi fumarate resulted inoxidation of the pigments. The reduction of therespiratory pigments in the presence of NADHwas completely inhibited in the presence of 2 mm2-n-nonyl-4 hydroxyquinoline N-oxide. The re-duced minus oxidized difference spectrum of themembrane preparation at room temperature andat -190 C is illustrated in Fig. 3. At -190 C, themaxima of the cytochrome were at 550, 520,

z

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667VOL. 96, 1968

RIZZA ET AL.

.06

.04F

.02E

Z .00

z -.02

z

C .02

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400 440 480 520 560 600

WAVELENGTH (mg)

FIG. 3. Difference spectrum ofa cell-free membranepreparation of Bacteriodes melaninogenicus. Pigmentsin membrane preparations reduced in the presence of5mM NADH were compared to pigments in membranepreparations oxidized in the presence of 20 nmfumarate. The membranes were suspended in the bufferdescribed in Fig. 2 at a concentration of 10.3 mg (dryweight)/ml. Upper curve indicates the difference spec-trum measured at -190 C in Lucite cuvettes of 2-mmpath length. Lower curve indicates a difference spectrumofa part of the same preparation measured at 25 C incuvettes of10-mm path length.

and 428 nm. There was no evidence of splittingof the a maximum.

Identification of the hemes of the respiratorysystem. B. melaninogenicus was grown to a densityof 0.45 mg (dry weight) /ml in the presence of 77pmoles of protoheme/ml. The low yield of cellsindicates that growth was limited by this proto-heme concentration (Fig. 1). In another experi-ment, the bacteria were grown to a density of 0.9mg (dry weight) /ml in 1,550 pmoles of proto-heme/ml. In this case, the protoheme concentra-tion did not limit growth. The bacteria wereharvested and extracted with acid acetone. Theprotoheme and protoporphyrin in the acid ace-tone extract and the heme c in the residue fromthe extraction were measured (Table 1). The hemec in the residue, measured as the reduced pyridinehemochrome, was equivalent to the heme c cal-culated from the difference spectrum of wholecells. In both experiments, more heme was foundin the reduced pyridine hemochrome of the intactbacteria than was enzymatically reducible as cyto-chrome c. This indicates that there is protohemein the bacteria that is not enzymatically reducible.

TABLE 1. Hemes in Bacteroides melaninogenicusgrown in the presence ofprotoheme

Amt of protohemebacteria grown with

Heme77 1,550

pmoles/ml pmoles/ml

Enzymatically reduciblecytochrome ca".. ..........23.6180.0

Total heme in bacteria.. 30.6 143.0Protoheme extracted... 0.05 89.0Protoporphyrin ex-

tracted ............... 31.3 73.2Heme c in residue afterextraction.. 22.3 78.0

a Enzymatically reducible cytochrome c wasdetermined from the reduced minus oxidized dif-ference spectra, as in Fig. 2, and was calculatedwith Ew = 20.0 X 103 per mole per cm. Themeasurement of hemes and porphyrins is describedin Methods and Materials. Total heme in thebacteria represents the heme measured as thereduced pyridine hemochrome of the intact bac-teria. This measurement includes both heme c andprotoheme.

6 Amount of heme, expressed as nanomoles pergram (dry weight).

Effect ofprotoheme concentration on cytochromeformation. The effect of different levels of proto-heme in the medium on the concentration ofenzymatically reducible cytochrome c is shown inFig 4. The cytochromes were determined afterthe bacteria had reached their maximal celldensity. The concentration of enzymatically re-ducible cytochrome c and of the carbon mon-oxide-binding pigment increased as the protohemeconcentration increased to approximately 0.5Ag of protoheme/ml. At this concentration, amaximal level of enzymatically reducible cyto-chrome was found to be reproducible, althoughthe absolute concentration of pigments variedsomewhat. At higher protoheme concentrations,the level of enzymatically reducible cytochromedecreased slightly. The enzymatically reduciblecytochrome formed with various levels of proto-heme supplementation paralleled the changes indoubling times and final cell yields, as illustratedin Fig 1.

Growth with excess protoheme. When B. mela-ninogenicus was grown in the presence of 1.5nmoles of protoheme/ml for several generationsand then was transferred to medium that did notcontain protoheme, the bacteria were capable ofat least eight divisions before growth stopped.When protoheme was added to this culture,growth began immediately.As the protoheme content of the medium was

668 J. BAXCTERIOL.

t,

BACTEROIDES MELANINOGENICUS

I \

II~~~~ -

( % PROTOHEME IN-, .CYTOCHROME C

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N MOLES PROTOHEME/mi CULTURE FLUID

FIG. 4. Cytochrome content ofBacteroides melanino-genicus grown with increasing concentrations ofproto-heme in the medium. The cytochromes were measuredfrom the reduced minus oxidized difference spectra as inFig. 2. Symbols: A, the absorbance increment betweenthe a maxima at 555 nm and the minimum at 539 nm;*, the absorbance increment between the y maximumat 416 nm and the trough at 450 nm in the cells withpigments reduced by endogenous metabolism versus cellswith pigments oxidized in air; 0, the absorbance incre-ment between the y maximum at 416 nm and the troughat 425 nm in the difference spectrum of a suspensionwith pigments reduced by metabolism and saturatedwith carbon monoxide compared to a suspensionwith pigments metabolically reduced. All results areplotted per milligram, dry weight. In the lower portionof the figure, the percentage of the protoheme in themedium at the beginning of the experiment that wassubsequently found as enzymatically reducible cyto-chrome c is shown (A). The right hand axis refers tothis percentage.

increased, the cells examined after centrifugationbecome darker in color. Cells grown on bloodagar plates (containing approximately 0.2 ,umoleof protoheme/ml) formed the characteristicshiny black colonies which give the bacteria itsname. The black color has been known to be dueto protoheme for a long time (11). Black colonies

formed during an 8-day incubation period werescraped from the blood agar plates and werewashed twice with 50 mm phosphate buffer (pH7.). The concentration of protoheme was deter-mined by preparing the reduced pyridine hemo-chrome of a suspension of the washed pellet inthe buffer. About 43% of the dry weight of thebacteria was protoheme. The viability of the cellsin the colonies decreased as the cell mass becamedarker.

Efficiency of protoheme utilization. When thegrowth rate, final cell yield, and cytochrome con-tent were proportional to the protoheme con-centration in the growth medium, between 10and 20% of the available protoheme was foundas enzymatically reducible cytochrome c (lowerpart of Fig 4). Less than 0.01% of the initialprotoheme could be detected in the medium aftera bacterial growth cycle. At protoheme concen-trations greater than 761 pmoles/ml, a smallerproportion of the total protoheme in the growthmedium was found as enzymatically reduciblecytochrome c and a progressively larger propor-tion was found as nonenzymatically reducibleprotoheme.During growth, B. melaninogenicus produces

an agent that can destroy protoheme. This activ-ity can be measured by adding protoheme to thespent medium after the cells have been removedand monitoring the decrease in protoheme withtime; the protoheme is determined as the reducedpyridine hemochrome. The protoheme-destroyingactivity can be eliminated by boiling the medium,lowering the pH to 2.0, or vigorously aerating themedium before adding the protoheme. In ourexperiment the protoheme-destroying activitydecreased proportionately with dilution of themedium. In an experiment in which 90% of theadded protoheme had disappeared, we were ableto recover approximately 25% of the protohemewhen the medium was vigorously aerated beforethe pyridine hemochrome was prepared. Thissuggested that part of the protoheme is reducedto a nondetectable compound, which becomesdetectable after oxidation in air. Measured asultraviolet fluorescence after extraction withethyl acetate and acetic acid (3:1, v/v) (2), noprophyrins could be detected in the medium afterthe heme had disappeared.

DISCUSSION

The metabolism of the obligate anaerobe B.melaninogenicus appears to require the presenceof a functional membrane-bound electron trans-port system. The formation of this electron trans-port system is dependent on the availability ofprotoheme in the growth medium. Once the or-

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RIZZA ET AL.

ganism has depleted its internal stores of heme.growth is dependent on added protoheme. Thegrowth rate, total cell yield, and the level of bothcytochrome c and the carbon monoxide-bindingpigment are dependent on the protoheme con-centration supplied in the medium. In three heme-requiring species of Haemophilus (13), the totalcell yield, cytochrome b, cytochrome oxidase o,and NADH oxidase flavoprotein are dependenton the protoheme concentration supplied in thegrowth medium.The function of the electron transport system

in the metabolism of B. melaninogenicus remainsobscure. Both NADH, a reductant, and fumarate,an electron acceptor, have thus far been shownto react with the particulate electron transportsystem. Oxygen acts as an electron acceptor but islethal to these organisms. Another heme-requiringanaerobe, B. ruminicola, contains a membrane-bound electron transport system that is reducedin the presence of NADH and oxidized in thepresence of fumarate (17). The metabolic proc-esses in B. melaninogenicus by which NADH isgenerated may result from the metabolism ofamino acids. In preliminary experiments, theaddition of carbohydrates to the growth mediumdid not result in faster growth rates, and verylittle of the carbohydrate was utilized duringgrowth. The organism produced large amounts ofammonia and 2, 3, and 4 carbon acids duringgrowth. A very active collagenase is also releasedduring the growth cycle (6). These findings areconsistent with the hypothesis that amino acidsare important in the metabolism of this organism.The enzymatically reducible cytochrome c can

be quantitatively accounted for as the heme c thatremains in the residue after acid acetone extrac-tion of the bacteria. When the bacteria are grownin protoheme concentrations that limit growth,protoporphyrin is found in the bacteria. Some ofthis protoporphyrin may represent protohemethat was deironed in the acid acetone extractionprocess. The conditions of extraction of thehemes, which are acid and highly reducing, favordeironing of the hemes (2). Some protohemeexists in the cells, even when they are grown atlow protoheme concentrations, as the total hememeasured as the reduced pyridine hemochromeexceeds the heme c. The protoheme could repre-sent the prosthetic group of the carbon monoxide-binding pigment, which has the spectral propertiesof cytochrome oxidase o (12). This pigment isthought to contain protoheme as its prostheticgroup. Since the a maxima of cytochrome o andcytochrome c in the reduced minus oxidized differ-ence spectra almost completely overlap and theheme c is equivalent to the enzymatically reduciblecytochrome c, it is unlikely that there is much

cytochrome oxidase o in this organism. Since theconcentration of the carbon monoxide-bindingpigment closely parallels that of the cytochrome c(Fig. 4), there is a strong possibility that the cyto-chrome c reacts with carbon monoxide. Otherbacterial cytochrome c pigments have been re-ported to react with carbon monoxide (7, 18).There is no evidence for enzymatically reduciblecytochrome b in this strain of B. melaninogenicus.When the bacteria were grown at high protohemeconcentrations, protoheme could be recoveredfrom the bacteria. This protoheme was not enzy-matically reducible and did not combine withcarbon monoxide unless it was chemically reduced.Strain CR2A of B. melaninogenicus differs some-what from one strain described earlier by C. A.Reddy and M. P. Bryant (Bacteriol. Proc., p.40, 1967). These investigators found that onestrain of B. melaninogenicus contained cyto-chrome c and a second strain contained cyto-chromes b and o.During growth, B. melaninogenicus generates

a substance that destroys protoheme that is addedto the spent medium. This substance has theproperties of a powerful reducing agent. Part ofthe protoheme that is lost can be recovered byaerating the medium, and the products of theprotoheme-destroying agent are apparently notporphyrins. The protoheme-destroying agentprobably accounts for the fact that only 10 to20% of the protoheme available in the mediumat the beginning of the growth cycle can be re-covered in the cytochrome c when the growth ofthe bacteria is limited by the concentration ofprotoheme added to the medium.The highest concentration of cytochrome c

found in B. melaninogenicus, 0.11 nmole/mg (dryweight), corresponds to three times the concen-tration of cytochrome b plus cytochrome o foundin the heme-requiring anaerobe B. ruminicola (17)and is equal to the level of these cytochromesfound in aerobically growing Staphylococcusaureus (3). The level of cytochrome c in B.melaninogenicus is 10% of that found in anaero-bically growing H. parainfluenzae.We have previously reported (P. R. Courant

and D. C. White, Bacteriol. Proc., p. 105, 1967)that strain BE-1 is capable of growth in theabsence of added protoheme. The bacteria usedin this study contained a slightly larger gram-negative cocco-bacillary organism which ap-parently supplied minimal protoheme to strainBE-1. This organism had a very long doublingtime and apparently limited the growth of strainBE-1 to this very slow rate. All attempts to isolatea pure culture of strain BE-1 failed, so the strainwas discarded. The present study was performedwith strain CR2A. Strain CR2A gave no evidence

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of variation and was absolutely dependent onprotoheme in the medium, as documented in thispaper.

ACKNOWLEDGMENTS

This investigation was supported by grant GB-4795from the National Science Foundation, by PublicHealth Service grant GM-10285 from the NationalInstitute of General Medical Sciences, and by a con-tract with the Agricultural Research Service, U.S.Department of Agriculture, administered by theEastem Utilization Research and DevelopmentDivision, Philadelphia, Pa.

LITERATURE CITED

1. Estabrook, R. W. 1961. Spectrophotometricstudies of cytochromes cooled in liquid nitro-gen, p. 436-459. In J. E. Falk, R. Lemberg, andR. K. Morton (ed.), Haematin enzymes.Pergamon Press, New York.

2. Falk, J. E. 1964. Porphyrins and metalloporphy-rins. Elsevier Publishing Co., New York.

3. Frerman, F. E., and D. C. White. 1967. Membranelipid changes during formation of a functionalelectron transport system in Staphylococcusaureus. J. Bacteriol. 94:1868-1874.

4. Gibbons, R. J., and L. P. Engle. 1964. Vitamin Kcompounds in bacteria that are obligateaerobes. Science 146:1307-1309.

5. Gibbons, R. J., and J. B. MacDonald. 1960.Hemin and vitamin K compounds as requiredfactors for the cultivation of certain strains ofBacteroides melaninogenicus. J. Bacteriol.80:164-170.

6. Gibbons, R. J., and J. B. MacDonald. 1961.Degradation of collagenous substrates by Bac-teroides melaninogenicus. J. Bacteriol. 81:614-621.

7. Henderson, R. W., and D. D. Nankiville. 1966.Electrophoretic and other studies on haem pig-

ments from Rhodopseudomonas palustris: cyto-chrome 552 and cytochrome c. Biochem. J.98:587-593.

8. Lev, M. 1958. Apparent requirement for vitaminK of rumen strains of Fusiformis nigrescens.Nature 181:203-204.

9. Lowry, 0. H., N. J. Rosenbough, A. L. Farr,and R. J. Randall. 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem.193:265-275.

10. Sawyer, S. J., J. B. MacDonald, and R. J.Gibbons. 1962. Biochemical characteristics ofBacteroides melaninogenicus. Arch. Oral. Biol.7:685-691.

11. Schwabacher, H., D. R. Lucas, and C. Rimington.1947. Bacterium melaninogenicum-a misnomer.J. Gen. Microbiol. 1:109-120.

12. Smith, L. 1954. Bacterial cytochromes. Bacteriol.Rev. 18:106-130.

13. White, D. C. 1963. Respiratory systems in thehemin-requiring Haemophilus species. J. Bac-teriol. 85:84-96.

14. White, D. C., 1965. Synthesis of 2-demethylvitamin K2 and the cytochrome system ofHaemophilus. J. Bacteriol. 89:299-305.

15. White, D. C. 1966. The obligatory involvement ofthe electron transport system in the catabolicmetabolism of Hemophilus parainfluenza. An-tonie van Leeuwenhoek. J. Microbiol. Serol.32:139-158.

16. White, D. C. 1967. Effect of glucose on the forma-tion of the membrane-bound electron transportsystem in Haemophilus parainfluenza. J. Bac-teriol. 93:567-573.

17. White, D. C., M. P. Bryant, and D. R. Caldwell.1962. Cytochrome-linked fermentation in Bac-teroides ruminicola. J. Bacteriol. 84:822-828.

18. Yamanaka, R., H. De Klerk, and M. D. Kamen.1967. Highly purified cytochrome c derivedfrom the diatom Navicula pelliculosa. Biochim.Biophys. Acta 143:416-424.

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