JOURNAL OF BACTERIOLOGY, May, 1965Copyright © 1965 American Society for Microbiology

Vol. 89, No. 5Printed in U.S.A.

Location of Chlorophyll in Rhodospirillumrubrum

STANLEY C. HOLT' AND ALLEN G. MARRDepartment of Bacteriology, University of California, Davis, California

Received for publication 18 January 1965

ABSTRACTHOLT, STANLEY C. (University of California, Davis), AND ALLEN G. MARR. Location

of chlorophyll in Rhodospirillum rubrum. J. Bacteriol. 89:1402-1412. 1965.-If cells ofRhodospirillum rubrum are broken by sonic and ballistic disruption, the chlorophyll isnot found in discrete cytoplasmic structures, but is located in a more extensive struc-ture of the cell, the intracytoplasmic membrane. Direct electron microscopy of sonicallydisrupted cells of R. rubrum and stereo-electron microscopy of osmotically shockedcells reveal the presence of a tubular network of internal membranes originating fromthe periphery of the cell.

Schachman, Pardee, and Stanier (1952) foundthat the photosynthetic pigments released fromRhodospirillum rubrum by sonic treatment andabrasion were sedimentable in the ultracentrifuge.A fraction was purified by differential centrifuga-tion; this fraction contained particles which were600 to 1,000 A in diameter, had a sedimentationconstant of 190S, and contained the entire pig-ment system of the cells. These particles werenamed chromatophores.

Vatter and Wolfe (1958) initiated the study ofthe ultrastructure of the photosynthetic bacteriaby examining thin sections of R. rubrum, Rhodo-pseudomonas spheroides, and Chromatium. The cy-toplasm of cells grown photosynthetically con-tained circular profiles approximately 500 to 1,000A in diameter surrounding regions of low electrondensity. Cells grown heterotrophically did notcontain these structures. Vatter and Wolfe (1958)concluded that these circular profiles were sec-tions of spherical particles identical with thechromatophores as isolated by Schachman andco-workers (1952).

Tuttle and Gest (1959) found that all of thepigment in lysates of R. rubrum prepared by os-motic shock of protoplasts was sedimented at lowcentrifugal force, whereas chromatophores iso-lated after mechanical disruption required muchlarger centrifugal forces for sedimentation. Marr(1960) found that osmotic shock of R. rubrumreleased most of the nucleic acid but only 8% ofthe chlorophyll. These results could be explainedeither by the size of the chromatophores relative

1 Present address: Department of Microbiology,Dartmouth Medical School, Hanover, N.H.

to the size of the opening in the cell produced byosmotic shock or by the location of the photosyn-thetic apparatus in a membranous continuum.Marr (1960) and Stainier (1963) postulated thatthe chromatophore, as isolated by Schachmanand co-workers (1952), is the result of mechanicaldisruption of the intracytoplasmic membrane.

This paper presents evidence that the principallocus of the photosynthetic pigments in R. rubrumis a system of membranes which originate fromthe peripheral membrane and which form a tubu-lar, branched network in the cytoplasm.

MATERIALS AND METHODS

Growth of bacteria. R. rubrum (strain S-1) wascultured in liquid medium containing 0.1% NH4Cl,0.02% MgSO4c7H2O, 0.002% CaC12, 0-05%K2HPO4, 0.1% DL-malate, 0.1% monosodiUm L-glutamate, 0.5% biotin, and 0.13 mg/100 ml of themixture of trace elements of Aaronson and Baker(1959). The pH was adjusted to 7.2 to 7.4 withNaOH before autoclaving.

Cultures were grown phototrophically in testtubes (2.8 X 20.0 cm) or in flat Roux bottles (5 X10 X 25 cm) in a water bath at 30 C in an enclosurewhich reduced the ambient light intensity. Thecultures were sparged with a mixture of 95% N2and 5% Co2, at a rate of approximately 100ml/min. Illumination was provided by 375-w Syl-vania R-32 movie lights adjusted to give a uniformlight intensity at the culture vessel. The voltagewas stabilized with a constant voltage transformerand set at 62 v a-c with an autotransformer. Thelight intensity was varied by adjustment of thedistance from the lamp to the culture or by inter-posing wire screen rather than by varying thevoltage. A photronic cell (Weston Master IV)

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LOCATION OF CHLOROPHYLL IN R. RUBRUM

with a diffusing cone was used to measure theintensity of light incident on the culture vessel.

Cultures were grown chemotrophically in thedark in similar culture vessels which were wrappedin aluminum foil. The cultures were incubated at30 C and sparged with 95% air-5% CO2 .

All cultures were growing exponentially at thetime of harvest. As evidence of a steady-state,the differential rate of chlorophyll synthesis wasmeasured periodically and was constant for atleast two generations prior to harvesting cellsfrom the cultures. The concentration of bacteriawas always less than 0.2 mg (dry weight) of cellsper milliliter at the time of harvest. At higherdensities the "self-shading" increases the differ-ential rate of chlorophyll synthesis.The cells were harvested by centrifugation at

4 C and washed by three successive centrifugationsfrom cold deionized water at 3,000 X g for 20 min.Chlorophyll was determined by the procedure ofCohen-Bazire, Sistrom, and Stanier (1957). Tur-bidity was determined by measuring the opticaldensity in a 1-cm absorption cell with use of aBeckman DU spectrophotometer at 680 mru.

Counting of intact cells. Cells were counted witha Coulter electronic counter (model B) with a30-,A aperture; samples were diluted in 0.85%NaCl-0.1% formalin which had been filteredthrough a membrane filter with a 0.45 tu averagepore-size. An aperture current of 1/0.707, an am-plification setting of 1/4, and a lower thresholdsetting of 10 were used.

Electron microscopy. Samples were fixed, de-hydrated, and embedded as described by Ryterand Kellenberger (1958). The agar blocks weredehydrated in aqueous ethyl alcohol (v/v) ac-cording to the following schedule: 20%, 50%, 75%ethyl alcohol for 30 min each, 95% ethyl alcoholfor 60 min, and two successive dehydrations inabsolute ethyl alcohol for 60 min. Alcohol wasremoved with propylene oxide, and the sampleswere embedded in a mixture of 35% Araldite 6005and 12% Epon 812.

Sections were cut on a Porter-Blum ultramicro-tome with use of glass knife, mounted on Formvar-coated 100- or 200-mesh copper grids, and stainedwith lead hydroxide according to the procedure ofMillonig (1961). Preparations to be shadowed weredried on 100- or 200-mesh Formvar-coated gridsand shadowed with uranium at an angle of 110.Samples were negatively stained with 2% phos-photungstic acid adjusted to pH 7.0, accordingto the procedure of Huxley and Zubay (1960).

Stereo-electron micrographs were made withuse of relatively thick sections in an RCA stereoholder. All samples for electron microscopy wereexamined in an RCA EMU-3E or 3G electronmicroscope.

Sonic treatment. A pellet of washed cells, har-vested from a culture grown at a light intensityof 1 ft-c, was suspended in cold 0.05 M phosphatebuffer (pH 7.0) to give a concentration of 5 mg(dry weight) per milliliter. An amount (40 ml) wastransferred to the cup of the transducer of a Ray-

theon 10-kc sonic oscillator. The transducer cupwas flushed with H2, and the temperature of thesamples was maintained at less than 5 C duringtreatment. The oscillator was operating at fullelectrical output (approximately 80 acoustical).At intervals, 2.0-ml samples were removed and2.0 ml of cold 0.05 M phosphate buffer were addedto the transducer cup. An appropriate correctionwas made for the resulting progressive dilution. Aportion of the sample was centrifuged at 8,000 X gfor 20 min. The absorbancy at 260 mg and thechlorophyll content of the supernatant fluid weremeasured. The remainder of the sample was usedto determine the optical density at 680 m, andthe concentration of surviving intact cells.

Ballistic disintegration. Cells from a culturegrown at a light intensity of 1 ft-c were suspendedin 0.05 M phosphate buffer (pH 7.0) to a concentra-tion of 5 mg (dry weight) per ml. An amount (5 ml)of this suspension, together with 2 ml of glassbeads (0.2-mm diameter, Minnesota Mining andManufacturing Co., St. Paul, Minn.), was addedto the cup of the Mickle apparatus. The cups wereshaken with a 9-mm peak-to-peak displacementand were cooled to 4 C every 30 see, which main-tained the temperature below 20 C during theentire treatment. Turbidity, count of intact cells,and sedimentable chlorophyll were determinedas for sonic treatment.

Osmotic shock. The technique of osmotic shockdeveloped by Robrish and Marr (1962) was used.A suspension of cells was mixed with an equal vol-ume of 6 M glycerol. After allowing the mixture tostand 5 min, it was drawn into a syringe and rap-idly ejected into 10 times its volume of mechani-cally stirred buffer at 4 C. The buffer was 0.05 Mtris(hydroxymethyl)aminomethane (Tris) HCI(pH 7.5) containing 0.001 M MgCl2 . After disrup-tion, the preparation was treated with 0.5 ,ug ofdeoxyribonuclease per milliliter for 20 min. Thesample was then prefixed in 0.1% 004 and cen-trifuged at 3,000 X g for 5 min.

RESULTS

The kinetics of release of chlorophyll by sonictreatment of cells of R. rubrum was determinedas a test of the hypothesis that the chromato-phores are discrete components of the cell. A- sub-stance contained in small, independent structureswhich are distributed throughout the cytoplasmshould be released at the same rate as the disrup-tion of the cells. The rate of release of a substancelocated in an extensive structure will be less thanthe rate of disruption of cells.The results of this experiment are shown in

Fig. 1. The rate of destruction of cells and initialrate of release of solutes absorbing at 260 m,uare nearly equal and are greater than the rate ofdecrease in turbidity (optical density, 680 m,).The rate of loss of sedimentable chlorophyll ismuch lower. That the rate of decrease in intact

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MINUTES OF SONIC TREATMENTFIG. 1. Sonic disruption of Rhodospirillum

rubrum. The logarithms of the per cent of the initialvalues of turbidity, of count of intact cells, of releaseof material absorbing at 260 m,g, and of loss of sedi-mentable chlorophyll are shown as a function of thetime of sonic treatment.

cells greatly exceeds the rate of decrease in sedi-mentable chlorophyll is not in agreement withthe idea that the photosynthetic pigments are

contained in small discrete structures, but sug-

gests, rather, that an extensive structure is thesite of the photosynthetic pigments.The results obtained by sonic treatment were

confirmed by a second method of disruption,ballistic disintegration in a Mickle apparatus.The results of ballistic disintegration are shownin Fig. 2. The rate of decrease in sedimentablechlorophyll is much lower than the rate of de-crease of turbidity or the release of solutes ab-sorbing at 260 m,u. This result is also at variancewith the hypothesis of independent chromato-phores and further supports the hypothesis thatthe photosynthetic pigments are contained in an

extensive structure of the cell.The possibility that the structure of the cells

of R. rubrum used in this investigation differedfrom the structure of cells observed by Vatterand Wolfe (1958) was eliminated by electronmicroscopy of thin sections of both whole cells

MINUTES OF MICKLE TREATMENT

FIG. 2. Disruption of Rhodospirillum rubrumby ballistic disintegration. Release of material ab-sorbing at 260 m,u, loss of sedimentable chlorophyll,and decrease in turbidity are shown as a function ofthe time of shaking in the Mickle apparatus.

and cells disrupted by osmotic shock. Figure 3 is asection of cells of R. rubrum grown at a light in-tensity of 1 ft-c. The predominant internal struc-ture shows circular profiles 700 to 1,000 A indiameter, similar to those observed by Vatterand Wolfe (1958). The cell wall and peripheralmembrane are well defined, and the nuclearvacuole is filled with fine fibrils.

Cells grown at 1 ft-c and osmotically shockedare shown in Fig. 4. Both the cell wall and thecytoplasmic membrane are 75 A thick and havethe appearance of "unit membranes." The in-ternal membranes appear to be identical in struc-ture and continuous with the cytoplasmic (pe-ripheral) membrane.The appearance of fragments produced by

sonic treatment and of stereo pairs of sections ofosmotically disrupted cells has established thepresence of an extensive system of internal mem-branes. Fig. 5 shows R. rubrum disrupted bysonic treatment and washed by three successivecentrifugations from deionized water. Aboutone-half of the cell wall has been disintegratedby sonic treatment. A complex of membranesextends through the opening in the wall. Sections

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FIG. 3. Section of Rhodospirillum rubrum grown exponentially at a light intensity of 1 ft-c. Membrane-bounded vesicles (M) extend deeply into the cytoplasm. The nucleoplasm (N), cell wall (W), and peripheralmembrane (PM) are evident in this micrograph. At this light intensity the chromatophores (Vatter and Wolfe,1958) are distributed throughout the cytoplasm. Main fixation, 12 hr; poststained with lead hydroxide.X 32,000.

FIG. 4. Transverse section of Rhodospirillum rubrum grown at 12 ft-c and osmotically shocked. The rup-ture of the cell wall is evident in one of the cells. The intracytoplasmic membranes (chromatophores) are stillretained in the opened cell. Possible peripheral connections of the chromatophores to the peripheral membraneare indicated by the arrows. Main fixation, 12 hr; poststained with lead hydroxide. X 85,000.

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HOLT AND MARR

FIG. 5a, b. Cells of Rhodospirillum rubrum disrupted by sonic treatment: (a) 30-sec expos?ure; (b) 60-secexposure. Both micrographs are of air-dried samples shadowed with uranium at an angle of 110. Note theinterconnected tubular appearance of the extended membranes. X 34,000.

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LOCATION OF CHLOROPHYLL, IN R. RUBRUM

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FIG. 6-16. Stereo-electron micrographs of sections of Rhodospirillunm rubrunt grown at 1 ft-c and emoptiedof cytoplasm by osmotic shock. The tubular structure of the membrane is indicated by the arrows. Peripheralttachwnents of the chromatophores are indicated by the double arrows. The dotted areas show interconnectionsof membranes. Main fixation, 12 hr; poststained with lead hydroxide. The marker indicates 0.25 u.

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through this network might be expected to show graphs of relatively thick sections of R. rubrum

circular profiles. grown at low light intensity (Fig. 6 through 16).The three-dimensional appearance of the in- The circular profiles are evident, but the struc-

ternal membranes in envelopes which had been ture responsible for these profiles can be identi-

emptied of cytoplasm by osmotic shock was also fied in stereo as tubules originating from the

investigated by preparing stereo-electron micro- periphery of the cell.

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Fig. 17 is a composite representation based onthe observation of a large number of stereo pails;all of the structures illustrated have been ob-served independently by at least three observers.In this composite section the intracytoplasmicmembrane is a tubular structure originating from

the peripheral membrane. Thin sections of thismembranous continuum give the circular profilesobserved by previous investigators. Without thebenefit of stereo observations, the structural basisof the circular profiles is difficult if not impossibleto determine.

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Figure 18 is an artist's concept of the fullthree-dimensional structure which, in section,would appear as Fig. 17.

DIscussION

The isolation of chromatophores by Schach-man et al. (1952), together with the observationsof apparently independent vesicles by Vatter andWolfe (1958), had suggested that the photosyn-thetic pigments are contained in small independ-ent structures within the cytoplasm of the cell.However, the differential release of chlorophyllduring sonic and ballistic disruption of the cellsis not in agreement with the location of chloro-phyll in such independent vesicles. The rationaleof these techniques is that substances containedin independent structures in the cytoplasm shouldbe released at the same rate as the disruption ofthe cells, provided that the breach in the envelopeis sufficient to allow the structures to escape. Therate of release of chlorophyll was found to bemuch lower than the rate of disruption of cells.Therefore, chlorophyll must be contained in an

extensive structure and not in small, independentstructures distributed throughout the cytoplasm.

l3oth direct electron microscopy of sonicallydisrupted cells and stereo microscopy of sectionsreveal a network of tubules attached to theenvelope. The reconstruction of the three-dimen-sional arrangement (Fig. 17, 18) of the internalmembranes developed from the observation ofmany stereo pairs was made by a scientific il-lustrator who had had no previous experience inbacterial cytology. The representations suggestthat the internal membranes containing thephotosynthetic pigments originate from theperipheral membrane in the form of sphericalv-esicles that later develop into bulged and some-times flattened tubes. It is our contention thatchromatophores are fragments of these tubularinternal membranes.

Several observations support the hypothesisthat the intracytoplasmic membranes originatefrom the peripheral membrane. Flexer, Sistrom,and Chapman (1960) presented evidence that thechromatophore (in the sense of Vatter and Wolfe)

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8LOCATION OF ChILORlOPIlYLL IN l?. RUBI? 1411

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FIG. 17. Artist's reconstrzuction of the three-dimstensional appearance of the previous stereo-electron ,m 'CI'O-graphs (Fig. 6 through 16). This figure is a comtiposite representation of a large number of stereo-electronmicrographs. Only those structurles that hare been observed by several independent observers have been indi-cated.

FIG. 18. Hypothetical three-dimensional representation of the internal membrane systemit of Rhodo-spirillum rubro;m.

originates at the peripheral membrane. Boatmanand Douglas (1962), Cohen-Bazire and Kunisawa(1963), Giesbrecht and Drews (1962), and lBoat-man (1964) also concluded that the chhromato-phores are formed through invaginations of theperipheral membIane.

Tubular intracvtop)lasmic membranes are notconfined to the photosynthetic bacteria. Thereare many p)ublishedl micrographs of gram-negativeorganisms showing a tubular intracytoplasmicmembrane system. Pangborn, MIarr, and Robrish(1962) observed tubules, 15 to 20 m,u in diameter,throughout the cytoplasm of Azotobacter agilis.

In Spirillomnl serpens "simnple intrusives" ex-tending into the cytoplasmic riegion have beeenobserved by MAIurray (1963). Smith (1960) andCota-Robles and Coffman (1964) observed intra-cvtop)lasmnic membranes in sections of Escherichiacoli.

ACKNOWLEDGMENTS

This investigation was stipported by grantt-iin-aid (G-23767 from the National Science Fooinda-tion.

This study could not have been unidertaIkeniwithout the genierous help and advice given to uts

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HOLT AND MARR

by Germaine Cohen-Bazire and Jack Pangborn.We wish to thank Mlrs. F. T. Addicott for thepreparation of Fig. 17 and 18.

LITER.ATURE CITED

AARONSON, S., AND H. A. BAKER. 1959. A com-parative biochemical study of two species ofOchromonas. J. Protozool. 6(3):282-284.

BOATMAN, E. S. 1964. Observations on the finestructure of spheroplasts of Rhodospirillttinrubrum. J. Cell. Biol. 20:297-311.

BOATMAN, E. S., AND H. C. DoUGLAS. 1962. Proto-plast morphology and chromatophore formationin Rhodospirillum rubrumn. Intern. Congr. Elec-tron Microscopy, 5th, p. RR-y.

COHEN-BAZIRE, G., AND R. KUNISAWN A. 1963. Thefine structure of Rhodospirilluai rubrumn. J. CellBiol. 16:410-419.

COHEN-BAZIRE, G., W. R. SISTROM, AND R. Y.STANIER. 1957. Kinetic studies of pigment syn-thesis by non-sulfur purple bacteria. J. CellularComp. Physiol. 49:25-68.

COTA-ROBLES, E. H., AND MI. 1). COFFMIAN. 1964.Electron microscopy of lysis from without ofEscherichia coli B by T2. J. Ultrastruct. Res.10:304-316.

FLEXER, A. S., W. R. SISTROM, AND G. B. Ci.Vp-MAN. 1960. Chromatophore formation in Rhodo-spirillumn rutbrum. Bacteriol. Proc., p. 52.

GIESBRECHT, P., AND G. DREWN-S. 1962. Electronen-mikroskopische Untersuchungen uber dieEntwicklung der "Chromatophoren" VOIlRhodospirillum molischianuins giesbergen. Arch.Mikrobiol. 43:152-161.

HUXLEY, H. E., AND G. ZUBAY. 1960. Electronmicroscope observations on the structure ofmicrosomal particles from Escherichia coli. J.Mol. Biol. 2:10-18.

MARR, A. G. 1960. Localization of enzymes in

bacteria, p. 443-468. In I. C. Gunsalus and R.Y. Stanier [ed.], The bacteria, vol. 1. AcademicPress, Inc., New York.

M1LLONIG, G. 1961. A modified procedure forlead staining of thin-sections. J. Biophys. Bio-chem. Cytol. 11 :736-739.

MURRAY, R. G. E. 1963. The organelles of bac-teria, p. 34-39. In 1). Mazia and A. Tyler [ed.],The general physiology of cell specialization.McGraw-Hill Book Co., Inc., New York.

PANGBORN, J., A. G. MARR, AND S. A. ROBRISH.1962. Localization of respiratory enzymes inintracytoplasmic membranes of Azotobacteragilis. J. Bacteriol. 84:5S69-678.

ROBRISH, S. A., AND A. G. MARR. 1962. Locationof enzymes in Azotobacter agilis. J. Bacteriol.83 :158-168.

RYTER, A., AND E. KELLENBERGER. 1958. Etude aumicroscope electronique de plasmas contenantde l'acide deoxyribonucleique. Z. Naturforsch.136:597-605.

SCHACHMAN, H. U., A. B. PARDEE, AND R. Y.STANIER. 1952. Studies on the macromolecularorganization of microbial cells. Arch. Biochem.Biophys. 38:245-260.

SMITH, K. R. 1960. AIn electron microscopic studyof methionine deficient Escherichia coli. J.Ultrastruct. lies. 4:213-221.

STANIER, R. Y. 1963. The organization of thephotosynthetic apparatus in purple bacteria,p. 242-252. In D. Mazia and A. Tyler [ed.],The general physiology of cell specialization.McGraw-Hill Book Co., Inc., New York.

TUTTLE, A. L., AND H. GEST. 1959. Subeellularparticulate systems and the photochemicalapparatus of Rhodospirillumz rubrum. Proe.Natl. Acad. Sci. U.S. 45:1261-1269.

VATTER, A. E., AND R. S. WOLFE. 1958. The struc-ture of photosynthetic bacteria. J. Bacteriol.75:480-488.

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