JOURNAL OF BACTERIOLOGY, Nov. 1978, P. 730-7410021-9193/78/0136-0730$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 136, No. 2

Printed in U.S.A.

Comparison, by Freeze-Fracture Electron Microscopy, ofChromatophores, Spheroplast-Derived Membrane Vesicles,

and Whole Cells of Rhodopseudomonas sphaeroidesMARY ANN JORDAN LOMMENt AND JON TAKEMOTO*

Department of Biology, ltah State University, Logan, Utah 84322

Received for publication 17 August 1978

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonassphaeroides were compared with cytoplasmic membrane and intracellular vesiclesof whole cells. In whole cells, the extracellular fracture faces of both cytoplasmicmembrane and vesicles contained particles of 11-nm diameter at a density ofabout 5 particles per 104 nm2'. The protoplasmic fracture faces contained particlesof 11 to 12-nm diameter at a density of 14.6 particles per 104 nm2 on thecytoplasmic membrane and a density of 31.3 particles per 104 nm2 on the vesiclemembranes. The spheroplast-derived membrane fraction consisted of large vesi-cles of irregular shape and varied size, often enclosing other vesicles. Sixty-sixpercent of the spheroplast-derived vesicles were oriented in the opposite wayfrom the intracellular vesicle membranes of whole cells. Eighty percent of thetotal vesicle surface area that was exposed to the external medium (unenclosedvesicles) showed this opposite orientation. The chromatophore fractions con-tained spherical vesicles of uniform size approximately equal to the size of thevesicles in whole cells. The majority (79%) of the chromatophores purified onsucrose gradients were oriented in the same way as vesicles in whole cells, whereasafter agarose filtration almost all (97 /) were oriented in this way. Thus, on thebasis of morphological criteria, most spheroplast-derived vesicles were orientedoppositely from most chromatophores.

Several photosynthetic bacteria possess an ex-tensive internal vesicular membrane system,which houses all or part of the components ofthe photosynthetic apparatus (14). The intracel-lular vesicles are believed to be part of a contin-uous membrane svstem which includes the cellsurface cytoplasmic membrane (7, 9). TI'hus, itappears that the intracellular vesicles are formedas a conse(luence of invagination of the cyto-plasmic membrane.

'I'wo principal methods of isolating this mem-brane system are used. The first, involvingFrench-press or sonic disruption of cells, pro-duces discrete, closed vesicles, termed "chro-matophores," which are capable of catalvzingseveral reactions related to photosynthetic me-tabolism (6, 18). Recently, a second methodinvolving osmotic lvsis of spheroplasts has alsobeen uLsed (8, 13).

In Rhodopseudomonas sphaeroides, accu-mulating biochemical evidence indicates thatchromatophores are oriented in the same wav asthe intracellular vesicles and opposite to the

t Permanent address: Department of Biological Sciences,University of California. Santa Barbara, CA 93106.

cvtoplassmic membrane, whereas spheroplast-derived vesicles are oriented in the reverse man-ner, that is, in the same wav as the cvtoplasmicmembrane and opposite to the intracellular ves-icles. For example, Hellingwerf et al. (81 -btainedmembrane vesicles of R. sphaerioidl-s u.er os-motic lysis which were capable of light-eliergizedtransport of amino acids in the same directionas whole cells. Chromatophores, however, dem-onstrate an opposite polaritv to whole cells inthe translocation of protons and ions (11, 19).Matsuura and Nishimura (13) recentlv demon-strated that the shift in the carotenoid absorp-tion band induced by potassium ions in chro-matophores is opposite to that induced in lspher-oplast-derived vesicles; this evidence indicatespossible opposite membrane orientation in thetwo prep)arations, since there is evidence thatthe carotenoid spectral change depends on thepolaritv of the membrane potential (1(). Finally,Prince et al. (16) demonstrated that cytochromec molecules are localized both within the inter-nal space of isolated chromatophores and in theintracellular vesicle comlpartrnents of wholecells, which are (ontinuouI with the periplasmicspace. Thtus, on the basis of cytochrome c., lo-

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FREEZE-FRACTURE OF R. SPHAEROIDES 731

cation, it appears that chromatophores are ori-ented in the same direction as the intracellularvesicles in vivo.The technique of freeze-fracture coupled to

electron microscopy has proven invaluable forevaluating the orientation of membrane vesiclesisolated from erythrocytes (20) and Escherichiacoli (1). The value of the technique lies in thefact that freeze-fracturing cleaves the central,hydrophobic plane of biological membranes, re-sulting in the exposure of inner half-membranefaces (3). The exposed faces are characterized byparticles believed to be protein complexesembedded in the membrane, and the asymmet-ric distribution of the particles observed on thehalf-faces serves as markers for the respectivemembrane halves. By correlating the particledistribution with the convexity or concavity ofthe membrane halves, conclusions about theorientation of the vesicles can be formulated.

In view of the recent biochemical evidence onthe chromatophores and spheroplast-derivedvesicles of R. sphaeroides, we sought to inde-pendently investigate and quantitate the ori-entation of these membrane preparations byusing the technique of freeze-fracture electronmicroscopy. Our results indicate that, on thebasis of morphological criteria, most chromato-phores are oriented similarly to most intracel-lular vesicles and oppositely from the majorityof spheroplast-derived vesicles.

MATERIALS AND METHODS

Organism, growth conditions, and harvestingprocedures. R. sphaeroides (NCIB 8253) was grownphotosynthetically under semi-anaerobic conditions.One-liter-capacity serum bottles were filled to capacitywith MG medium (12) and inoculated with 2 ml of anactive culture. The bottles were capped and incubatedat 30°C with an incident light intensity of approxi-mately 20,000 lx. During midlogarithmic growth, whenthe cell cultures attained an optical densitv of approx-imately 1.0 measured at 680 nm (1-cm path length),the cells were harvested by centrifugation at 3,000 xg for 10 min. These cells contained about 8 nmol ofbacteriochlorophyll per mg (dry weight) of cells. Thecells were suspended in either 10 mM tris(hy-droxvmethyl)aminomethane (Tris) -hvdrochloride(pH 7.5) or 20 mM potassium phosphate (pH 7.5) toan optical density of 40 measured at 680 nm. Harvest-ing procedures were performed between 0 and 5°C.

Preparation of spheroplast-derived mem-brane vesicles. Ten milliliters of fresh cell suspensionwas diluted with 5.5 ml of distilled water. With slowstirring at 370C, the following were added in order: 5ml of 1 M Tris-hydrochloride (pH 8.0), 25 ml of 40%(wt/vol) sucrose, 2.5 ml of lysozyme (10 mg/rnl ofwater, Sigma, grade I), and 2 ml of 0.05 M ethylene-diaminetetraacetic acid. After 30 min of incubation,spheroplasts were evident by examination with a lightmicroscope; then 75 ml of 10 mM Tris-hydrochloride

(pH 8.0) was added with stirring at room temperature.Brij-58 was then added to a final concentration of 0.104(wt/vol), and the mixture was incubated for an addi-tional 30 min at room temperature.Two milliliters of 0.1 M MgCl2 and 1 to 5 mg of

deoxyribonuclease I (Sigma, crude) were added to thecrude extract. The mixture was incubated for 30 minat 37°C with gentle stirring. The crude extract wasthen centrifuged at 3,000 x g for 5 min, and thepigmented supernatant was recovered and centrifugedat 48,000 x g for 30 min. The pellet was suspended in10 mM Tris-hydrochloride (pH 7.5) and gently ho-mogenized with a Dounce homogenizer. The suspen-sion was then layered onto 30 to 55% (wt/vol) linearsucrose gradients made up in the same buffer andcentrifuged for about 10 h in a Beckman SW41 rotorat 150,000 x g. The pigmented material sedimentingat approximately 38% (wt/vol) sucrose was recoveredand washed by suspension in 10 mM Tris-hydrochlo-ride (pH 7.5) and centrifugation at 48,000 x g for 30min.

Preparation of chromatophores. Chromato-phores were prepared by two different methods.Method 1 (sucrose gradient): Suspended cells weredisrupted by one passage through a French press cellat 1,265 kg/cm2. Approximately 200 tig of deoxyribo-nuclease I was added per ml, and the crude extractwas centrifuged at 10,000 x g for 20 min. The super-natant was recovered and centrifuged at 150,000 x gfor 1 h. The crude membrane pellet was suspended in10 mM Tris-hydrochloride (pH 7.5) and gently ho-mogenized in a Dounce homogenizer. The suspensionwas then layered onto 30 to 55% (wt/vol) linear sucrosegradients prepared in 10 mM Tris-hydrochloride (pH7.5) and centrifuged for 10 h at 150,000 x g. Thepigmented chromatophore fraction sedimenting at ap-proximately 38% (wt/vol) sucrose was recovered, sus-pended in buffer, and centrifuged at 150,000 x g for 1h.Method 2 (gel filtration): Chromatophores were also

purified by a modification of the method described byFraker and Kaplan (5). The suspended and homoge-nized crude membrane fraction obtained as describedabove for method 1 of chromatophore preparation waslayered onto a column bed (0.9 by 60 cm) of Bio-GelA-150M (100 to 200-mesh, Bio-Rad) and eluted with10 mM Tris-hydrochloride (pH 7.5). The peak frac-tions of pigmented material were pooled and concen-trated bv centrifugation at 150,000 x g for 1 h.The procedures for preparing spheroplast-derived

vesicles and chromatophores were performed between5 and 10°C. In some experiments, all Tris-hydrochlo-ride buffers used in the procedures for membranepreparations were replaced with potassium phosphatebuffers of equivalent ionic strength and pH.

Freeze-fracture. Cells or vesicles were suspendedin 20 to 100 mM potassium phosphate buffer (pH 7.5),and glutaraldehyde was added to give a concentrationof 4% (vol/vol). After 1.5 to 2.5 h of fixation at about10°C, sufficient glycerol in unbuffered glutaraldehydewas added slowly, over 20 to 30 min, with mixing togive a final concentration of 4% (vol/vol) glutaralde-hyde and 30% (vol/vol) glycerol in 10 mM potassiumphosphate buffer. Fixed material was centrifuged, andsmall samples of the pellet were rapidly frozen in

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732 LOMMEN AND TAKEMOTO

aluminum cups by plunging into liquid Freon 22 at-160°C. Frozen samples were transferred rapidly witha prechilled specimen wrench to the specimen mouInt-ing post, maintained at -140°C, of a Denton freeze-fracture apparatus. During pumping to high vacuum(15 to 20 min), the specimen temperature was raisedto -105°C, and the blade was held firmly against theliquid nitrogen-cooled shroud. From one to five spec-imens, maintained at -105 to -115°C, were then rap-idly fractured with the cold blade and replicated im-mediately after the last fracture. Replicas were cleanedwith commercial sodium hypochlorite (Clorox) anddistilled and filtered water.

Electron microscopy. Specimens were examinedwith either a JEM 1OOB electron microscope at anaccelerating voltage of 80 kV or a Zeiss EM-9S at 50kV. Microscope magnification was calibrated using acarbon grating replica (E. F. Fullam) and did not varymore than 3`%.Measurements. Vesicle dimensions were mreasured

on micrographs enlarged to between x4(,000 andx55,000. Measurements of vesicle dimension on freeze-fractured material produce an underestimate of truedimension but were performed here for comparativepurposes. Particle population densitv was counted onmicrographs enlarged to between x100,000 andx130,000. Particle dimensions were measured perpen-dicular to the direction of shadowing on micrographsenlarged to x200,000; a Camrex x5 magnifier wasused. To compensate partially for different amountsof etching and shadowing, measurements of particlesfrom different membrane halves from am,v one givenpreparatory method were made, as far as possible, onthe same micrographs. However, the varving angles offracture within any one micrograph and the corre-sponding differences in accumulation of shadow intro-duce an error in particle measurement for which it wasnot possible to compensate.

RESULTSWhole cells. The main structural features

seen in freeze-fractured whole cells of R. sphae-roides correspond to those described by previousworkers (4, 21) in thin sections. The outer mem-brane of the bacterium was usually cross-frac-tured (Fig. 1 and 2), and its cleaved surfaceswere rarely seen. However, the cleavage planefrequently exposed the extracellular fracture(EF) face and the protopasmic fracture (PF)face (terminology of Branton et al. [2]) of thecytoplasmic membrane.The EF face of the cytoplasmic membrane is

a concave face with a population of sparsely (4.8particles per 104 nm2) and randomly distributedsmall particles with a mean diameter of 10.7 nm(Fig. 1; Tables 1 and 2). This face is marked byseveral raised areas, which represent indenta-tions of the membrane into the cell cvtoplasni;the raised areas also often exhibit the smallparticles which are characteristic of the otherregions of this membrane half.The PF face of the cytoplasmic membrane is

a convex face with a popl)ation of (lensely (14.6particles per 10( nmr ) andci randomlly distributedsmall particles with a mlean (liamiieter of 11.4 nmri(Fig. 2; Tables I and 2). This face is mlarke(d byseveral depressions of abouLt the samle size as theraise(l areas on the EF face. I'hese (lel)ressionssomletimiies appear to be devoid of l)articles. bttwhen the (lepressions are viewed in a (ross-frac-tured plrofile, t hey ar e seen to be line(d withdlenselv packedi parti(les (Fig. 2). 'I'he (lelpres-.sion.s on the PF face and(i the raiseri areas on theEF face are interlpretedi to 1)e areas where intra-cellular vesicies are forming Or ha-ve fornmed hyintvagination (of the cvtopl)asrlic membrane.

'I'he cytoplasm contains small s(attered singlel)arti(les, occasional srooth-surfaced Sphericalbodies of diameters varying from 5)) to 180) nmnfotind in the central or the l)erip)heral cytoplasm,anrI memrnihane-bouneId vesicles largely confinedto the pleriphel-al cytoplasm ( Fig. 2 to 4). 'I'hevesicles are roughly sp)herical, with arn averagelerngth otf 4 nmii anid anl average withh oif 59 nrm(Table 3).

'I'he nembrarne fracture faces (of these intra-celuIlar vesicles resembled very (cIo.sely the frac-ture faces (of the (ctoplasmic membranes, exceptthat their (onmcavit and cmonvexity were reversed(Fig. :3 ari(I 4). 'I'he convex fracture face, that is,the fractUre face of the interior half of the vesiclememl)rarle or the EF face, UStIually hadI a sparseiopulation of particles (5.2 particies l)er 10' urim'with an average dIiameter of 10.5r-)nm. 'I'he ( on-cave or ITF face uIsuLally hacI a iernse IolItlationof plarticles with an average (liamileter 12.2 nm;these particles were mlore eienselv l)a(kedl (31.3particles 1per 1)' nnvm ) than those on the (Yto-p)lasmic miemibranie (Tables I and(i 2).

From ani analysis off 209 spherical vesiclesin(ludInLIg the smooth-SUrfaced b(oies fronmwhole cell preparations, 72%i were fotinrI to haveeither a (onvrex (HF) face with sparse p)arti(les(conxvex-sparse) Or a oncave (PF) face withdlenselv (list ribut ed p)articles (concave-dense)(Table 4). T'he smnooth-Surfaced h()olies, whichwere oftern larger antI more centrally loc-ate(dthani typ)ical photosynthetic vesi;icles (Fig. 4).may replresent StrtLctural elements in the tell notrelated to the photosynthetic nienmbr-anie sVstem,for example, 1)oly-/3-hytlroxybut vrate granulesor gas vactroles. If these were eliirinat ed fromimthe anialyNis, then 88%' of the remilaininlg vesiclesshowed an orientation of either (Inmvex-slparse mrconcave-dense.Spheroplast-derived vesicles. 'I'he pig-

miiernte(i miemibranie fraction isolate(d after (os-m(otic lysis (if spheroplasts antI )subse(qUent (lern-sity gradient cent rifugation was characterized by!vesicles of irregular shape arl(i varietl size, whichofteni appeared to enc(lose other vesicles (Fig. 5,

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FIG. 1. Freeze-fracture replica of whole cell. Fracturing has exposed the EF face (outer leaflet) of thecytoplasmic membrane (CM). Raised areas of the cytoplasmic membrane are marked by arrows. The outermembrane (OM) is seen in profile. 97,OOOx. The bar in the upper right of all figures equals 0.1 or 0.2 ,im, asdesignated. The arrow in the lower right indicates the shadowing direction.

FIG. 2. Replica of whole cell showing the PF face (inner leaflet) of the cytoplasmic membrane (CM) as wellas fractured cytoplasm. Depressions (arrows) occur in the cytoplasmic membrane, and particles (P) aresometimes seen to line the depressions. Outer membrane (OM). 97,000x.

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lables :3 and 4). 'I'he enclosed vesicles weresonmetillmes neste(l one inside another. Somiie ofthe apparently enclose(l vesicles muitist be thereesuLlt of infoldings of the surface of auLtonomiiouLsve,sicles; suc-h infoldinigs were occasionally seenin l)rofile (Fig. 5). 'I'he average length of all thevesicles, enclosed an(l unenclosed, was 14(0 nm,and their average width was 100 nm (Table 3).

Analysis of about 400 vesicles of this fraction(Table 4) revealed that the majority have thereverse orientation fromn vesicles obser\ved inwhole tells, that is. 66%i were either convex withdensely distributed p)articles (convex-dense) orconcave with sparse particles (concave-sparse).

'T'o estinmate the orientation of only the mnemil-brane surface area which was exposed to theexternal mnediumL1, only Unenclosed vesi(les wereconsidlered next. Estinmation of the nmemibranesurface area of these vesicles combined withscoring of their orientation showed that about80()(' of this exposed memiibrane surface area wasorientecl in the convex-dense or concave-sparsemanrner.Sparse particles were distributed with a den-

sitv equal to that in the whole cell membranes.However, on densely populated surfaces, parti-cles were present at a density of 27.9 p)articlesper 10.1 nm2 (Table 1), a value between thoseobserved for cytoplasmic and vesicle menmbranesin whole cells. As in the whole cells, the sparseparticles were slightly smaller in diameter thanthe denselv distributed particles (Table 2). Ves-icle preparations isolated in Tris-hydrochlorideor in phosphate buffers were not observed todiffer from each other in morphology oI' vesicleorientation.Chromatophores. The chromatophore frac-

tion isolated from sucrose gradients after

TI'ABLE 1. Population density ofparticles onfractured faces of whole cell membranes,

spheroplast-deriued cesicles, and chromatophoresNo. of particles per 10' nrn2of half-membrane sturface

area`Membrane source

Sparsely Densely pop-populated ulated sur-surfaces faces

Whole cellsCytoplasmic membrane 4.8 ± 0.3 14.6 ± 0.7Vesicles 5.2 ± 1.3 31.3 ± 1.2

Spheroplast-derived vesi- 4.8 ± 0.4 27.9 ± 1.4cles

ChromatophoresSucrose gradient 5.5 ± 1.4 26.7 ± 1.0Gel filtration 5.4 ± 0.8 27.2 ± 1.5

" ±Standard error.

TABLE 2. Diameters ofparticles on fractured facesof whole cell membranes, spheroplast-dericed

c,esicles, and chromatophoresSparsely 1)popu- Denisely populatedlated surfaces sUrfaces

Membhrane source --___Diameter' X Diameter IV

(nmi) ' (nniWhole cells

Cvtoplasmic mem- 10.7 ± 0. 1 98 11.4 + 0.2 97brane

\esicle.s 10.() -0. 1S 12.2 + il 7f0

Spheroplast-derived 11.5 ±+(0.2 50 12.4 +0.:3 30vesicles

Chronatop)horesSucrose gr-adienit 11.9 ± 0.2 50 12.3 0.4 56Gel filtrationl 107±( 0 90 50 12.3 + 0.( 50

Meani ± standard etrror.N. NUml)er of particles nmeasuLr ed.

French-press cell disruption contained nearlyspherical individual vesicles of slightly variablesize (Fig. 6; Table 3). Their average dimensionswere 79 by 65 nm, and only 1%e were enclosed inother vesicles (Table 4). Their orientation wassimilar to that found in the whole cells, that is,79% were either convex-sparse or concave-dense(Table 4). Often very large vesicles, fragments ofmembranes, and occasional fragments of cellsenclosing cytoplasm were seen in this fraction.The chromatophore fraction purified by aga-

rose filtration after French-press disruption ofcells was characterized by spherical individualvesicles of more uniform size (Fig. 7; Table 3).Their average length was 67 nm, slightly smallerthan that in whole cells, and their average widthwas 61 nm. No vesicles were enclosed in othervesicles, and 97% were oriented in the convex-sparse or concave-dense manner, that is, simi-larly to vesicles in whole cells (Table 4). Thefraction contained only rare small membranefragments.

In both chromatophore preparations, thesparse particles were slightly smaller than thedensely distributed particles, and the populationdensities of particles were similar to those ofspheroplast-derived vesicles (Tables 1 and 2). Asin the case with spheroplast-derived vesicles,chromatophores prepared with potassium phos-phate buffers did not differ from those preparedin Tris-hydrochloride buffers.

DISCUSSIONWhole cells. The only significant difference

observed between the cytoplasmic membranesand vesicle membranes of whole cells was thatthe PF face particles of the vesicles were twiceas densely packed as those of the cytoplasmic

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FIG. 3 and 4. Replicas of whole cells showing several vesicles (V). Fracturing has exposed the concave (PF)face of some vesicles and the convex (EF) face of others. The fracture faces are usually either densely (d) orsparsely (sp) populated with particles. Several smooth-surfaced bodies (sm) are seen. Figure 4 shows the kindof large and centrally located smooth-surfaced body that is frequently seen in cells and is clearly distinctfrom the photosynthetic vesicles. In Fig. 3, the smooth-surfaced bodies might be considered photosyntheticvesicles, but as concave surfaces they would then usually exhibit a dense particle distribution (see Table 4).PF and EF faces of the cytoplasmic membrane (CM) are shoun. Figure 3, 95,OOOx; Fig. 4, 106,000x.

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736 LOMMEN AND TAKEMOTO

membrane. This suggests that condensation ofthese particles in the vesicle membranes mayoccur during formation of photosynthetic vesi-cles from cytoplasmic membrane. In contrast,the EF particles in both kinds of membraneswere equally sparsely distributed. All the parti-cles averaged 11 to 12 nm in diameter. (Forevery membrane type and preparation exam-ined, the densely packed particles were slightlylarger than the sparse particles; however, giventhe errors in particle measurement discussedunder Materials and Methods, it is difficult toascribe any significance to this difference.)Membrane vesicles. The results presented

indicate that by morphological criteria the ma-jority of membrane vesicles derived from spher-oplasts are oriented oppositely from the majorityof chromatophores. Furthermore, the chromat-ophores are oriented similarly to intracellularvesicles occurring in whole cells. These findingsare consistent with previous biochemical evi-

TABLE 3. Vesicle dimensions in whole cells and inspheroplast-dericed and chromatophore

preparations

1No. of Avg ofLength' Width' vesicles lengthVesicle source (nm) (nm) mea- and

sured width)~~~~nm)

Whole cells 74 + 2 59 ± 1 196 67

Spheroplast-derived 140 ± 6 100 + 3 199 120vesicles

ChromatophoresSucrose gradient 79 ± 2 65 ± 1 201 72Gel filtration 67 ± 1 61 ± 1 200 64

Mean ± standard error.

dence on membrane preparations of R. sphae-roides as cited in the introduction. Virtually allof the chromatophores (97%) purified by agarosefiltration were oriented in the same direction asthe intracellular vesicles, and 66% of the spher-oplast-derived vesicles were oriented in the op-posite direction. Moreover, about 80% of themembrane area exposed to the medium in spher-oplast-derived vesicle preparations was orientedin the opposite direction.

In a previous study, Reed and Raveed (17)observed, by freeze-fracture electron micros-copy, chromatophores obtained from strain R-26 of R. sphaeroides, a mutant incapable ofsynthesizing carotenoids. Fractures of chromat-ophores of this strain produced rough faces,covered with 42 particles per 104 nm2 with di-ameters of about 13 nm, and smooth faces withvery few particles. From their micrographs, therough faces appear to be largely concave and thesmooth faces appear to be largely convex. Theseresults are similar to observations made in thepresent study on chromatophores from a wild-type strain. Similar observations have beenmade with membrane preparations derived fromFrench-press cell extracts of a related organism,Rhodospirillum rubrum (15).The chromatophore preparations isolated on

sucrose density gradients were more heteroge-neous in vesicle size and orientation than thepreparations isolated after agarose filtration.The ability to recover more homogeneous chro-matophores after agarose filtration suggests arelationship between vesicle size and orientationin the chromatophore preparations. For spher-oplast-derived vesicles, however, preliminaryanalysis of the size distribution and orientationdid not reveal a similar relationship (data not

TABLE 4. Orientation of vesicles in uhole cells and in spheroplast-derit,ed and chromatophorepreparations"

Orientation

Convex Concave Convex- En- N ofVesicle source dense + Convex+ close( esicles

Cocv-sparse ± ana-Dense Sparse Smooth IDense Sparse Smooth Concave Concave- vlzed((C7, (c)) (7 (e;) (') () sp(aCrse dense (.(C' (CI spa(C-r((e

Whole cells 1 26 10 46 9 7 10 72 0 209Correctedh 2 32 56 11 13 88 0 174

Spheroplast-derived 46 9 1 21 20 3 66 30 32 399vesicles

ChromatophoresSucrose gradient 12 22 1 57 6 3 18 79 1 200Gel filtration 2 34 0 63 1 1 3 97 0 200

" The number of vesicles of each orientation is expressed as a percentage of the total number of vesiclesanalyzed from each source.

h Results after removing smooth-surfaced bodies from whole cell vesicle counts.

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FREEZE-FRACTURE OF R. SPHAEROIDES 737

FIG. 5. Spheroplast-derivied vesicles. Concave (CC) and convex (CV) fracture faces are denselv (d) andsparsely (sp) populated with particles. Smooth-surfaced bodies are rarely seen. Many vesicles are enclosed (e)in others; sometimes several vesicles are nested (n) inside one another. Small arrouw marks an infoldedautonomous vesicle. 57,OOOx.

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FIG. 6. Chromatophores prepared by French-press disruption followed by sucrose gradient centrifugation.Most of the concave (CC) faces are densely (d) populated u'ith particles, and most of the convex (CV) faces aresparsely (sp) populated, although the reverse orientations are also seen. Smooth-surfaced bodies are rare.Several large vesicles (LV) (100 to 180 nm in diameter) are present, although most vesicles are smaller. Whatappears to be a fragment of a cell (fc) including outer membrane, cytoplasmic membrane, and cytoplasm ispresent. (A) 57,000x; (B) 150,OOOx.

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FIG. 7. Chromatophores prepared by French-press disruption followed by agarose filtration. Almost all theconcave (CC) faces are densely (d) populated with particles, and almost all the convex (CV) faces are sparsely(sp) populated. Only one vesicle larger than 100 nm in diameter is present (LV). There are no large cellfragments. (A) 57,OOOx; (B) 150,000x.

shown). In addition, efforts to recover more ho-mogeneous preparations of spheroplast-derivedvesicles by agarose filtration were not successful.

Speculations on how spheroplast-derived ves-

icles and chromatophores may be formed duringcell disruption can be considered in view of theseresults. The spheroplasts swell in hypotonic so-lution, and the intracellular vesicles that are

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740 LOMMEN ANI) T'AKEMOTO

continuous with the cVtoplasmic membrane maybecome part of the stretched limiting surface ofthe spheroplast. With lysis, the surface menm-branes fragment and reseal in an orientationsimilar to the cytoplasmic membrane in situ.With French-press disruption, the intracellularmembranes apparentlI reseal in the original ori-entation. Fragmentation and resealing in thiscase perhaps occur at points of constriction inthe continuous membrane svstem, since thephysical dimensions of the chromatophores Ie-senmble those of the whole cell vesicles.The light intensity used in growing cells for

membrane isolation may havTe significant bear-ing on the orientation of vesicles obtained. Arelatively high light intensity was chosen inthese experiments (20,000 lx) since undei suchconditions the intracellular membrane system islargely confined to the cell periphery (Fig. 2 to4) (4). 'I'hus, during osmotic lNsis, the resealingof membrane fragments into spheroplast-de-rived vesicles with an orientation of the in situcyrtoplasmic membrane might be facilitatedsince the extent of menmbrane invagination isminimized. A similar approach was used by Hel-lingwerf et al. (8) to obtain a preparation ofmembr-ane vesicles fronm R. sphaero ides capableof actively transporting L-alanine.The three kinds of isolated vesicle prepara-

tions were all obser-ed to have densely packedparticle populations with densities intermediatebetween those of the cvtoplasmic and vesiclemembranes in whole cells (Table 1). This sug-gests that both cytoplasmic and intracellularmembranes contribute to the preparations pro-ducing the observed intermediate populationdensities. However, the density differences aretoo small to allow individual vesicles oIr parts ofvesicles to be assigned to one or the other source.Thus, the extent of contribution by the cvto-plasmic membrane in the formation of thesevesicle p)reparations is difficult to assess. Withthe high light intensity used for growth, theamount of cyrtoplasmic rmembrane relativ e tointracellular membrane is higher than withgrowth under lower light intensities. Therefore,the contribution of cytoplasmic membrane tothe preparations may possibly be higher thanfor preparations obtained from low-light-growncells. Chromatophore preparations from cellsgrown with relatively low light intensities havebeen ireported to be essentially free of cvtoplas-mic miienmbrane material (3). Interestingly, (vto-plasmic membrane vesicles obtained from E.coli after French-press clisruption show inside-out orientations as revealed bv freeze-fractureelectron microscopy, (1), and these vesicles re-semble the chromatophores observed in thepresent study in size and particle distribtution.

J. BACTERIOI.

'I'he abilityv to prepare membrane vesicles ofthe phot osvnthetic apparatus of B. sphacroideswith opposite au(l known orientation shotldl al-low fortiher st udies on thie topographical (listri-bution of (oml)onents whi(h comprise this svs-teml.

ACKNOW LEDI)( MENTS\Ve th;ink Katiiherine Ferrell (it iertoriingmearsirerioenitsn

1 i ;111t Y5 tot hlell)fil ilisc ioteih ill tl ,it ndi.at-ori-xx nixelxii ifTI;ialinittaSnIa tIat,tB t lie lis

of dIr kr innri i(wilities in the fin;dl stages ot this wnrk.TI is et'ir.ll(tcli wNassiiil)Itie(l bY grInIstrfon, the N;t innal

S( ienc( Fonii tioni(1in(M 79-02969) and(i the I tah Agriciil-tilnil 1 xlii linliellt taiti"tl.

ADD)ENDUtM IN PROOF

A recent report (P. A. M. Michels anid W. N. Konirngs.Bliochiox.i3iolohYs Acta 507::35;3-368. 11978t descrihes ohser-xations oni freeze-ftactunredl pireparat ions of spheroplast-cle-rived -esicles and chrornatotphores of IR. xrcheroi(des silmilarto those reiported here.

LITERATURE (ITEI)

1. Altendorf, K. H., and L. A. Staehelin. 1974. Orienittiorrit onittiihrane vesiiles fliit Fsxtcti/hica (oi( aIs detectedt) treifi-iez(iaxte eleit r(un 11riicroscolx. .1. 4licterioliI i7:S88-S99i.

2. 3ranton, 1)., B.Bullivant, N. H. Gilula, M. J. Karnov-sky, M. Moor, K. Muhlethaler, 1). H. Northcote, L.Packer, H. Satir, 1. Satir, V. Speth, L. A. Staehelin,It. I,. Steere, and R. S. Weinstein. 1975. Freeze-t.aitiitre riorrienclatriie. Science 190:54-5f;.

:t. Branton, I)., and I). I)eamer. 1972. Mlerhrarine strurc-tlre r pltsrlat ologiao 1II E 1. Sririrger-Virlag. NewY r k.

4. (ohen-Bazire, G. 1993. Sorne obserxations onl the orga-nization of the photosxnthetic apparatus in purple an(dgreen bacteria, p. 89-10)). In H. C,est, A. -San I'ietro, andLI 1). Vernon (ed.) t Bacterial phctosynthesis. Antiochliress, Yellox Springs, Ohro

5. Fraker, P. J., and S. Kaplan. 141 lxIsoltionr ;ri frac-iiioritution hle phtixt htic nietrbhranots oIrgtlnellefi-onl l¢lsls>svllnler6spherot(idle s. 'J. Bac(ter-iol.10S:4 i5-473

9. Frenkel, A. W., and 1). I). Hickman. 1959. Structureaind photouhem-ical aictivit! of hlirphvll-imitairoiirrpart iles f'ronti Rhdisii.pirillui//n iiirtuuin. J.J. jophyhs. [ii-

cleri. (ti'. 6:285-289..ieshrecht, 1'., and G. I)rews. 1992. Electronrerrnuikris-

kopiische tnt erstichunirgeni ubher (lie Entwicklinig (der4'Chrnitauatoii;horert xonr Khotdopi.r'littui/ ttii/nlh/i(t1ttti?gieshiirgeni Arch9ikriobhl. 43:152-1f1.

. Hellingwerf, K. -J., P'. A. M. Michels, .J. W. IDorpema,and W. N. Konings. 1975. Irasptiort of artnirno acids intrierilir)arne xesicles ofo/Rhodip.seudeomtnon(tls .s)h(oeriide.senergized hY respiratorY antd cYclic electroun flow. Eur.-J. Biocihie-i. 55:.397-406.

9. Holt, S. C., and A. G. Marr. 1965. Location of chlorophyllin Rhodosptri/lunt rtitbrtirn. .J. Bacteriol. 89:140)2-1412.

10. Jackson, J. B., and A. R. Crofts. 1969. 'I'he energy statein chroniatophores froim Rhodopseuidonrona(t.s sphae-rotdes. FEBS; Iett. 4:185-189.

1 1. -Jackson, ,. B., A. R. (rofts, and I-V. von Stedingk.1968. Iotn trarnspiort irnduied hY light arid antibiotits inchrirot itolil)hres frorri iRho/di.spiirillutnt rtui/utint. EKuir. J1.I-io iherni. 6:4 1-54.

12. Lascelles, J. 1959. Adaptation to fornm hacteriochlnro-phYll in Rhodoiosetudornonatcis spheroides: changes inact xiit of enzmies concernmed in pyrrole synthesis. B'i-chern. -J. 72:508-518.

on April 5, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

FREEZE-FRACTURE OF R. SPHAEROIDES 741

13. Matsuura, K., and M. Nishimura. 1977. Sidedness ofmembrane structures in Rhodopseudontonas sphcae-roides. Electrochemical titration of the spectrunochanges of carotenoid in spheroplasts. spheroplastmembrane vesicles, and chromatophores. Biochim. Bio-phys. Acta 459:483-491.

14. Oelze, ,J., and G. D)rews. 1972. Menmbraties (f photosvoy-thetic bacteria. Biochim. Biophys. Acta 265:20)9-239

15. OeIze, J., and J. R. Golecki. 197i-5. P)roperties of reactioo0

ceniter depleted mnembranes of khodospiriluon rubrum.

Arch. M irobl)iol. 102:59-64.

111. Prince, R. C., A. Baccarini-Melandri, G. A. Hauska,B. A. Melandri, and A. R. Crofts. 1975. Asymmetryof an energy transducing membrane. The location ofcvtochrome c, in Rhodopseudomonas sphaeroides andRhodopseudomonas capsulata. Biochim. Biophys.Acta 387:212-227.

17. Reed, 1). W., and 1). Raveed. 197I2. Somiie r)poert es otfthe ATlPase fromii hronmato)phores of U/t(dpseildol00as sp)h loeroideS (1 its sI oral relatiomlship to the

balteriomhli(rophVll lproteins. Ba chimo. Bpiophvs. Al ta283:79-91.

18. Schachman, H. K., A. B. Parde, and R. Y. Stanier.1952. Stt (lies o00 the imacromtolectlar orgaflmiztiotl of

mWirobial cells. Arclh. Biocheml. BiophYs. 38:245-261.

19. Scholes, F., P. Mitchell, and ,J. Moyle. 1969. T'he llIlar-ity of proton traoislo(ati00 i0i somiie photosyntlbetic tlli-

croorgaolisms. Em-. J1. Iiocb(em. 8:450(-454.2(0. Steck, T. L, R. S. Weinstein, .J. H. Strauss, and 1). F.

H. Wallach. 197(o. Irlsi(ie-olit re(i (ell mlemllbratie vesicles: preparation and(i potrificatiom. Scien e 168:255-257.

21. Vatter, A. E., and R. S. Wolfe. 1958. The strllctllre (If

plotosYllt-hetic hbacteria. 1. Bacteriol. 75: 480-488.

VOL. 136, 1978

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