JOURNAL OF BACTERIOLOGY, Jan. 1969, p. 426-440 Vol. 97, No. ICopyright © 1969 American Society for Microbiology Printed in U.S.A.

Fractionation and Characterization of the Plasmaand Mesosome Membrane of Listeria

monocytogenes1B. K. GHOSH2 AND R. G. E. MURRAY

Departmenit of Bacteriology anid Immunology, Untiversity of Westernz O,itario, Londoni, Otitario, Canada

Received for publication 7 October 1968

Protoplasts of Listeria monocytogenies strain 42 were fractionated after controllysis on a Ficoll (a polysucrose) density gradient. Visually, five zones could berecognized in the gradient. The first one was composed of amorphous cytoplasmicsolutes (fraction la) and a mixture of particles (fraction lb). These were: (i) lightparticles that were lipase-sensitive and composed of six subunits and (ii) heavyparticles, sensitive to ribonuclease and devoid of fine structure. The second zoneconsisted of tubules and vesicles still harboring cytoplasmic components (fraction2), whereas the third zone contained only empty vesicles and protoplast ghosts(fraction 3). The material congregating into the fourth zone was morphologicallyidentical to that of the third (fraction 3a). The fifth and heaviest zone contained amixture of (i) particles without any substructure and (ii) partly lysed protoplasts(fraction 4). Fractions lb and 4 were the richest in nucleic acids (ribonucleic acid,11.4 and 9.4%, respectively; deoxyribonucleic acid, 5.1 and 4.8%/, respectively),whereas fraction lb had the highest protein contents (74.6%). Phospholipids weremainly found in fractions 2 and 3. Except for fraction 1, all materials containedsignificant amounts of protein-bound phosphorus. The main concentrations of fourenzymes were: glucose-6-phosphate dehydrogenase (fraction la); adenoXjne tri-phosphatase and reduced nicotinamidV a ine diphosphate oxidase_(fraction 3);nitro blue tetrazolium chloride reductase Tfraction 2). Fractionation of strain 42after addition of 32p during the mid-log phase of growth revealed that the radio-activity was mainly detected in fraction lb, when growth in the presence of themarker was allowed for 10 min, and in fraction 2, when growth was allowed for90 min. The vesicles of fraction 2, often tubular, are probably of mesosomal origin,whereas those of fraction 3, which are always spherical, represent, most likely, thebulk of the cell plasma membrane. Our data showed slight chemical differencesbetween these two fractions, but the differences in enzymatic activities and lipid-phosphorus incorporation during long pulse experiments were most dramatic.

Extensive intracytoplasmic membranous in-vagination has been observed in Listeria mono-cytogenes (7, 18, 23, 38). These invaginationscorrespond to the mesosome of Fitz-James (10)and have been shown to possess the usual mor-phological forms (36).The lack of techniques for isolating pure

mesosomal membrane from disrupted cells hashampered the definition of the physiological

I Part of the result of this investigation was presented in theThird International Symposium of Listeriosis, Bilthoven, Holland,13-16 July 1966.

2 Postdoctoral Fellow of the Medical Research Council ofCanada, 1964-1966. Present address: Institute of Microbiology,Rutgers, The State University, New Bninswick, N.J. 08903.

properties of the structure. Initial attempts bySalton and Chapman were not very successfulbecause the plasma and mesosome membranescofractionated (46). Other difficulties occurduring fractionation of the cell membrane. Theintracytoplasmic membranous organelles undergodisorganization during preparation, and sepa-rated membrane fragments tend to aggregate andfuse or fragments bind to other cell components,notably the cell wall. Differential separation isdifficult.No definite criteria have been established to

distinguish the peripheral plasma membranefrom mesosome membrane, and the function(s) of

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VOL. 97,1969 L. MONOCYTOGENES PLASMA AND MESOSOME MEMBRANE

the mesosome is still a matter for speculation. Theevidence thus far accumulated indicates that themesosome may be related to a variety of physio-logical functions of bacteria; e.g., a site of re-spiratory activity (33, 49, 50); cross wall forma-tion (4, 13, 16); a control center for replicationand separation of deoxyribonucleic acid (DNA)strands (12, 22, 44); a site of new membrane syn-thesis (15).

Protoplast formation (9, 11, 14, 16), tempera-ture shifts (12), or plasmolysis (53) may cause ex-pulsion of mesosomes from the cytoplasm. InL. monocytogenes, large numbers of extrudedmesosomes can be stabilized and these remain asbuds or tubules on carefully stabilized protoplasts(19). The released buds and tubules can be sepa-rated from the bulk of the protoplast fraction on aFicoll density gradient (3; B. K. Ghosh and R. G.E. Murray, Bacteriol. Proc., 1966, p. 108). Anumber of reports have appeared on the isolationand characterization of mesosomes from bacilli(9, 14, 15, 44). In the present paper, we describe amethod for separating the membrane system of L.monocytogenes into mesosomal and peripheralfractions. Chemical and enzymatic differenceswere noted between these mesosomal andperipheral membranous fractions.

MATERIALS AND METHODSMaintenance of the culture and preparation of the

protoplast lysate. L. monocytogenies strain 42 wasgrown as reported previously (19, 35). Protoplastswere formed (19) from logarithmic phase cells grownat 28 C. Washed protoplasts were lysed in a solutioncontaining 0.2 M sucrose, 0.03 M tris(hydroxymethyl)-aminomethane (Tris) buffer, 0.01 M MgCI2, and 0.01 Mglucose, and were incubated at 37 C for 15 to 20 minwith shaking. The lysate was dispersed and its vis-cosity was reduced by homogenization in a Sorvallomnimixer (8,000 rev/min for 30 min). The bucket ofthe omnimixer containing the lysate was immersed inice during homogenization.

Density gradient centrifugation. The protoplastlysate was fractionated on a Ficoll (a polysucrose ofhigh molecular weight and low viscosity; PharmaciaInc., New Market, N.J.) gradient. A 50% solution ofFicoll was dialyzed for 24 hr at 2 to 4 C against dis-tilled water to remove contaminating salts and waslyophilized. Ficoll was dissolved in a solution con-taining 0.25 M sucrose, 0.01 M MgCl2, and 0.03 MTris buffer at pH 8.1. Solutions containing 2, 4, 6, 8,10, 14, 16, and 20% Ficoll were thus obtained. Threemilliliters of each of the above solutions starting from20%, were carefully layered one after another in a34-ml centrifuge tube. After storage for 48 to 60 hr at4 C, 9 ml of protoplast lysate (100 mg, dry weight)was carefully layered on the gradient, and the tubewas centrifuged at 57,000 X g for 30 min (rotor SW25.1) in a Beckman Spinco preparative ultracentrifuge(model L). After centrifugation, distinct zones ofvarying opacity were noted. Each zone was removed

with a Pasteur pipette, its volume was recorded, andit was further treated as described below.The material from the first zone was diluted to twice

its volume with a solution containing 0.03 M Trisbuffer (pH 8.1) and 0.01 M MgCI2 (Tris-MgCl2). Thiswas centrifuged at 105,000 X g for 45 min. The result-ing residue and supernatant fluid were designated asfractions lb and la, respectively. Fraction lb wassuspended in Tris-MgCl2 for analysis and electronmicroscopy.The materials from zones 2 and 3 were diluted to

twice their volume with solutions of 6% Ficoll inTris-MgCl2 and 12% Ficoll in Tris-MgCI2, respec-tively, and were centrifuged at 57,000 X g for 30 min.In some experiments, zone 3 could be separated intotwo bands. In that case, both of the bands weretreated similarly. The supernatant fluids after centrifu-gation were discarded. The residues were designated asfractions 2 and 3, respectively. In some experiments,fraction 3 was divided into fractions 3 and 3a. Electronmicroscopy was done with these fractions before anyfurther washing. For chemical and enzymatic analysis,the fractions were again washed by suspending in thesolution containing 0.03 M Tris buffer (pH 8.1), 0.01 MMgCl2, and 0.05 M NaCl (Tris-MgCl2-NaCl). Theresidues were collected by centrifugation at 105,000 Xg for 45 min.The fifth zone from the original gradient was sus-

pended in a solution of 20% Ficoll in Tris-MgCl2 andwas centrifuged at 57,000 X g for 30 min. The super-natant fluid was discarded; the residue was examinedunder an electron microscope and was further washedfor chemical and enzymatic analysis in Tris-MgCI2-NaCl. This was designated as fraction 4.

Chemical analysis. All of the fractions were ana-lyzed for ribonucleic acid (RNA), DNA, and protein.Acid-soluble, lipid, nucleic acid, and total phosphoruscontents were also measured. Protein was estimated bythe method of Lowry et al. (30) and by the biuretmethod (20). The former tended to give higher valuesthan the latter. Nucleic acid was extracted by Ogur andRosen's method (39) with a slight modification. Por-tions of the protoplast lysate and isolated fractionswere precipitated with 10% cold trichloroacetic acidand were centrifuged at 4,000 X g at 4 C. The phos-phorus content of the supernatant fraction was esti-mated as an acid-soluble fraction. The residue wasthen extracted with 70% alcohol containing 0.1%perchloric acid (PCA) followed by 70% alcohol. Thesupernatant fractions were discarded. To obtain thelipid phosphorus fraction, the residue was extractedwith chloroform-methanol at 60 C; the extract wasevaporated and the residue was suspended in pure dis-tilled chloroform; after decanting the chloroform-soluble material, the undissolved residue was dis-solved in pure methanol. Phosphorus was estimated inchloroform- and methanol-soluble material separately.The residue after chloroform-methanol treatment wasextracted for RNA with 1 N PCA at 4 C for 18 hr.To extract for DNA, the residue was further treatedwith 0.5 N PCA at 70 C for 20 min.

Phosphorus was estimated in a 6 N PCA digest oflipid, RNA, DNA, and protein by Ernster's method(8). RNA and DNA were determined by the orcinol

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(39) and diphenylamine (47) reactions and werefurther checked by ultraviolet measurements. Frac-tions (0.1 to 0.2 ml) were heated on aluminum foil at70 to 80 C for 2 hr, dried over P20, in vacuo for 18 hr,and weighed for dry weight determination.The amount of phospholipid was calculated by

multiplying the lipid phosphorus content by 20. Themultiplying factor was obtained by establishing thatthe average phosphorus content (expressed as dryweight) of phospholipid from L. monocytogenes lipidmaterial is approximately 5%. The method of isola-tion of phospholipid by thin-layer chromatographywas described previously (18). This provides a con-venient way of determining the amount of phospho-lipid (this may not be very accurate) of various- frac-tions. The usual gravimetric procedure of lipid estima-tion could not be followed because of the smalleryield of fractions.

Enzymatic analysis. Adenosine triphosphate (ATP)phosphohydrolase (EC 3.6.1.3) was estimated bymeasuring the release of inorganic phosphorus fromATP (25). Reduction of nitro blue tetrazoliumchloride (NBT) was estimated separately in the pres-ence of reduced nicotinamide adenine dinucleotide(NADH), lactate, and succinate as substrates accord-ing to the method of Lester and Smith (28). NADHoxidase was estimated by measuring the reduction inoptical density (OD) at 340 nm (31). D-Glucose-6-phosphate: nicotinamide adenine dinucleotide phos-phate (NADP) oxidoreductase (EC 1.1.1.49; glucose-6-phosphate dehydrogenase) was estimated by meas-uring the increase in absorbancy at 340 nm withglucose-6-phosphate as the substrate and NADP asthe electron acceptor (26).

Incorporation of p32. The cells were grown in aVirtis fermentor at 28 C. When H3P3204 (40 ,uc/liter)was added during the lag phase at the beginning ofgrowth, the cells were harvested at the early sta-tionary phase after 12 hr of growth; when it wasadded at the mid-logarithmic phase (i.e., 7 hr afterinoculation), the cells either were grown for another90 min, then centrifuged and washed, or they wereharvested 10 min after the addition of phosphate.

Protoplasts, prepared from the 32P-labeled cells,were lysed and fractionated by the method alreadydescribed. Incorporation of 32p into total and lipidphosphorus was measured in a D-47 gas flow counterwith micromil window (Nuclear-Chicago Corp.,Des Plaines, Ill.).

Lipid extraction. A portion of each fraction waslyophilized and was shaken overnight with chloro-form-methanol (2:1). The filtered extract was evapo-rated in a flash evaporator at 37 C, and the residuewas extracted with distilled chloroform and assayedfor phosphorus and radioactivity. The results of in-corporation were expressed as specific activity; i.e.,counts per minute per ;&g of phosphorus.

Electron microscopy. Electron micrographs weremade on Kodak fine grain, positive film with a PhilipsEM 100 at 60 kv. The negatively stained specimenswere prepared with 1% phosphotungstic acid (PTA;adjusted to pH 6.1 with KOH) or 1% uranyl acetate(pH 5.1 in sodium acetate buffer). Sometimes PTAwas dissolved in ammonium acetate buffer, pH 6.0.

Each fraction was diluted before staining, and asmall drop of the suspension was placed on Formvar-(Ladd Research Industries, Inc.,. Burlington, Vt.) andcarbon-coated grids. Excess fluid was then removedwith a piece of filter paper, and a small drop of stainwas immediately placed on the grid. The fluid fromthe grids was removed after about 10 sec with filterpaper. The dried grids were examined immediatelyor kept in an evacuated dessicator. Thin sections ofprotoplasts were made as described previously (16).Parson's surface spreading technique (41) was alsoused for negative staining of protoplasts.

RESULTSStructure of various fractions: whole protoplasts.

Protoplasts suspended in a solution that contained10% Ficoll, 0.5 M sucrose, 0.03 M Tris buffer(pH 8.1), and 0.01 M MgC12 had many extrudedbuds and tubules which are believed to originatefrom mesosome (Fig. 1). These tended to sepa-rate very easily from the body of the protoplastswhen they were not properly stabilized (e.g.,acidic pH, absence of MgCl2). But these struc-tures could not be removed by sonic treatmentwhen the protoplasts were suspended in the abovemedium. Therefore, it seemed that optimal os-motic and ionic conditions are necessary for thestability of these tubules and vesicles attached tothe protoplasts.A section of such a preparation (Fig. 2) showed

a tubule (arrow) attached to the protoplast andbounded by an extension of the peripheral plasmamembrane.Lysed protoplasts. Lysis by osmotic shock of

fully stabilized protoplasts showed the presenceof many vesicular or tubular structures inside ofthe lysing protoplasts. They were irregular in sizeand shape and lacked obvious connection withthe peripheral membrane (Fig. 3, 4, and 5).Peripheral plasma membrane (pm) can be clearlydefined in the partly lysed protoplasts. By stainingaccording to Parson's surface spreading technique(41), we were able to demonstrate that stabilizedprotoplasts contained coiled tubules and vesicles(Fig. 3) and that these structures could be liber-ated intact from the lysing protoplast (Fig. 5).

Fraction lb. Fraction lb was composed ofparticles; there was no contamination withtubular and vesicular material. Differential cen-trifugation revealed two morphologically differentparticles. The heavier fraction (pellet at 50,000 Xg) was composed of particles 2 to 3 pm in size(Fig. 6); these particles were disintegrated byribonuclease treatment or MgCI2 depletion. Thelighter particles (supernatant fraction at 50,000 Xg) were 1 to 1.5 pm in width (Fig. 7) and theywere disintegrated by lipase treatment. This frac-tion also contained some membrane material.Negative staining with PTA showed that this

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FIG. 1. Negatively stained preparation ofprotoplast suspended in Ficoll-sucrose (0.5 M)-Tris-MgCl2 medium.Slender tubules (st) and vesicles (v) can be seen; note the point of separation of a tubule and vesicle from thesurface ofthe protoplast (arrow). All markers indicate 0.2 ,um, unless otherwise stated.

FIG. 2. Thin section ofa protoplast having an attached tubule; note the tubule bounded by an extension ofplasmamembrane (arrow).

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FIG. 3. Negatively stained preparation by Person's surface spreading technique (41). Note the tightly coiledtubular structures and vesicles inside of the partly lysed protoplast (same suspending medium as in Fig. I but0.2 Am sucrose was used). Clear definitioni ofperipheral membrane can be seen (pm).

FIG. 4 and 5. Negatively stained preparation oflysed protoplasts (same suspendinig medium as in Fig. I withoutany sucrose). Note the disruption ofperipheral membrane (arrows) and the escape of tubules and vesicles frominside ofthe protoplast into the medium (Fig. 4). Many vesicles (v), tubules (t), and vesicles having tubules attachedto them (vt) can be seen inside of lysed protoplast ghosts (Fig. 5). Many such released vesicles and tubules canbe seen in the medium (arrow).

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FIG. 6 and 7. Negatively stained preparation offraction 1. The 105,000 X g residue offraction I was suspendedin Tris-MgCl2 and centrifuged at 50,000 X g for I hr; the resulting residue was stained with 0.5% uranyl acetatein ammonium acetate buffer at pH 5.1 (Fig. 6), and supernatant fraction was stained with 1% PTA (Fig. 7).Note that the particles ofFig. 6 do not htave any obvious substructure, whereas the particles of Fig. 7 are composedofcircularly arranged subunits (arrow).

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FIG. 8. Cruide preparationt of fractionz 2 was stained with PTA before washinig. The fraction is composed oftubiules (t), vesicles (v) conztainiing some material (because PTA did nlot penetrate iniside), antd some empty vesicles(ev). Both of the suirfaces of the membrane cant be seen because of tlhe penietrationi of PTA i)ito the empty vesicles.(These vesicles may be contaminiated from fractioni 3.) Note the presenice oJ maniy particles having circularly ar-raniged subunits (Fig. 8B, arrow).

material was composed of a ring of six subunitswith a diameter of 0.3 to 0.4 pm.

Fraction 2. Fraction 2 was composed of amixture of tubules and vesicles and was not sig-nificantly contaminated with particulate materialfrom fraction lb. PTA did not penetrate insidethese vesicles, which may indicate that thesevesicles contain some cytoplasmic material. Incontrast, empty vesicles stained with PTA ex-hibited penetration of stain on both sides of themembrane. The vesicles had a variable morphol-ogy. Figures 8 and 9, respectively, present thecrude and clean preparations of the negatively

stained fraction. These tubules and vesicles werecomparable to the structures present inside thelysing protoplasts (Fig. 3 to 6).

Fraction 3. Fraction 3 was composed of largeprotoplast ghosts and many smaller vesicles (Fig.10). These appeared to be devoid of any cyto-plasmic material. Both the inner and outer sur-faces of the membranes were stained with PTAwhich indicates the lack of cytoplasmic contents.

Fraction 4. Fraction 4 contained partiallylysed protoplast ghosts and a large number ofdense bodies lacking any fine structures.

Morphological characteristics of various frac-

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FIG. 9 and 10. Preparations of washed fractions 2 and 3 negatively stained with PTA. Note the absence (unlikeFig. 8A anid B) ofparticles and empty vesicles in fraction 2 (Fig. 9). Fraction 3 (Fig. 10) is composed of largeempty ghosts and empty vesicles (possibly rolled-up pieces ofperipheral membrane). Note the penetration of PTAinto the empty vesicles and the clear definition ofboth sides of the membrane.

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tions and their correspondence to the differentzones in the original gradient tube are shown inTable 1.

Chemical composition. Table 2 shows the pro-tein and nucleic acid concentrations of variousfractions. Comparison of fractions 2 and 3 showsthat the former had a considerably higher con-centration of DNA (almost double) than thelatter; RNA concentration was also higher infraction 2 than in fraction 3.Though fraction 3 and 3a are morphologically

similar, the latter contained considerably moreprotein and RNA. The highest amounts of RNAand DNA were present in fractions lb and 4. Thepeak protein concentrations were found in frac-tions lb and 3a.The results of phosphorus fractionation studies

are presented in Table 3. The phosphorus solublein 10% trichloroacetic acid represents compara-tively low molecular weight nucleotides, sugarphosphates, teichoic acid, inorganic phosphorus,and various other minor components (detectedby qualitative paper chromatography). The high-est concentration of this 10%c trichloroacetic acid-

TABLE 1. Morphological character of various frac-tionis and their correspondence to the various

zones in the gradienta

Zones

gradient Fraction after Morphological characteristictubeb

1 Soluble cyto- No definite detectableplasm (la) structure

lb Mixture of two kinds oparticles: 2- to 3-pm

heavy particles hav-ing no substructure;1- to 1.5-pm lightparticles composed ofsix subunits

2 2 Tubules and vesicles con-taining material inside

3 3 Empty membranousghosts of varioussizes

4 3a Empty membranousghosts of varioussizes

5 4 Particles of varioussizes lacking sub-structure and somecontamination ofpartly lysed proto-plasts

a Fraction numbers indicated in all other tablescorrespond to the numbers shown in the secondcolumn of this table.

b Zones are from top (1) to bottom (5).

TABLE 2. Protein and niucleic acid composition ofvarious subcellular fraction2s from the gradienta

Fraction Protein RNA DNA

Whole cell lysate 54.8a 5.4 1.7lb 74.6 11.4 5.12 58.4 2.1 1.73 55.5 1.6 0.863a 79.8 2.9 0.954 47.1 9.4 4.8

a Results are expressed asweight. The data represent anidentical sets of experiments.

percentage dryaverage of five

soluble phosphorus was found in the soluble cyto-plasmic fraction. Fractions 1 and 3 contained verylow amounts of 10% trichloroacetic acid-solublephosphorus, but significantly higher concentra-tions of this material were present in fractions 2and 4.

After the extraction of the acid-soluble phos-phorus, the material was washed with 70%7 alco-hol and 70% alcohol containing 0.1 N PCA at 4 C.The supernatant fractions of these washes con-tained significant amounts of phosphorus. How-ever, the results of this estimation are not in-cluded here because of their extreme variability.We were unable to identify the origin of this ma-terial or the cause of the variability.The material washed in alcohol-PCA was then

extracted for lipid. The phosphorus contents ofthe chloroform- and methanol-soluble lipid ma-terial were estimated. The bulk of lipid phos-phorus was found in fraction 3 (25.6%) and frac-tion 2 (17.1 %). Small but significant amount ofmethanol-soluble lipid phosphorus was found infractions 2 and 3.A significant amount of methanol-soluble lipid

phosphorus was present in soluble cytoplasm, butthe chloroform soluble lipid material was verylow in this fraction.

Nucleic acid phosphorus was estimated in thelipid-extracted residue. It represented about 71 %of the total phosphorus content of fraction lb.In all other fractions, the concentrations weremuch lower (26 to 27%): fraction 3 containedonly 11.6%.The recovery of phosphorus (first four columns

of Table 3) indicated that only 50 to 80% of thetotal phosphorus was accounted for. The phos-phorus remaining bound to protein and the phos-phorus soluble in the alcohol washes accountedfor this disparity.

In all fractions (Table 3), fraction 1 excluded,there was a considerable amount of protein-bound phosphorus, which varied widely frombatch to batch. Similar observations were made

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TABLE 3. Distributioni ofphosphorus fractionis in differelnt cell fractionsa

Trichioro Lipid phos- Lipid phos- Nucleic acidFraction Trichloroacetic phorus (chloro- phorus (net- phosphorus Protein-bound Percentage recovery1cdsoul fomslbehnlslbe (05wN PCA- phosphorus PrFraction acidsporubs omslbe)hnlslbe soluble at 70 C)

Whole lysate 24.6 8.0 2.1 17.6 20-3lb 72.3-83.3la 45.4 0.9 2.4 26.0 5.2-11.1 79.9-85.8lb 5.0 4.7 0.5 71.0 0-5.0 81.2-86.22 17.6 17.1 1.9 26.0 25.1-35.0 97.7-97.63 6.9 25.6 2.5 11.6 30.5-41.2 77.1-87.83a -C7.2 - - 21.0-4 9.7 12.4 1.3 27.0 31.2-41.5 81.6-92.0

a Results are expressed as percentage of whole phosphorus content. The figures represent an averageof five different sets of experiments.

b Protein-bound phosphorus shows extreme variability; the ranges of values in five different sets havebeen shown.

c Due to small amount all the estimations could not be done.

by Logan et al. (29). In a previous publication,we reported the presence of phosphoserine in acidhydrolysates of membrane (20).Table 4 shows the ratio of protein to phospho-

lipid in the different fractions. The whole lysatecontained about 12 units of protein per unit ofphospholipid (arbitrary unit), whereas fractions2 and 3 (membrane material by morphologicalcriteria) contained about 3 units of protein perunit of phospholipid. Both whole lysate and frac-tions 2 and 3 had almost the same amount of pro-tein (Table 2), so the decrease in ratio resultedfrom the increase in phospholipid content of thelater fractions.

Fraction lb contained 8 units of protein perunit of phospholipid. But the protein content ofthis fraction was much higher than that of wholelysate or that of fractions 2 and 3 (Table 2). Asubstantially larger increase in the phospholipidcontent can explain the lower ratio of protein tophospholipid in fraction lb. The results furtherimply that, although there was a considerable in-crease in the phospholipid content of fraction lb,the relative proportion of phospholipid is lowerthan that in morphologically identifiable mem-brane material (fractions 2 and 3).The high value of the ratio in fraction la was

due to its high protein and very low phospholipidcontent.

Distribution of some enzymes. The relative dis-tribution of certain enzymes in subcellular frac-tions of lysed protoplasts is illustrated in Table 5.Complete separation of enzyme activities in vari-ous fractions involved exhaustive washing. Thistreatment caused a very substantial loss of en-zyme activity. An increase in specific activity ofan enzyme after fractionation over that of a wholelysate was deemed evidence of the localization ofthe enzyme in that fraction. Due to the loss of

TABLE 4. Relative proportions of protein antdphospholipid in various membrane fractions

Fraction Ratio of protein toPhospholipid

Whole lysate 12.3la 71lb 82 2.83 2.93a 4.64 3.6

material from every fraction during isolation andpurification procedures, the total quantity of en-zyme present in the whole lysate could not be re-covered and this recovery varied from batch tobatch.Adenosine triphosphotase activity was sig-

nificantly concentrated in fraction 3. Some in-crease in specific activity was also noted in frac-tion 2. NADH oxidase activity was found to behighly concentrated in fraction 3. The reduction ofNBT (NBT reductase) was studied separately inthe presence of various substrates, NADH, suc-cinate, and lactate; the results showed relativelyunspecific distribution, because the electron trans-fer may occur through various flavoprotein en-zymes. This may explain the fact that a consider-able reduction was noted in all of the fractions.Of course, the highest activity was always foundin fraction 2. Glucose-6-phosphate dehydrogenase(NADP specific) was localized solely in the frac-tion la.

32p incorporation. Results of the 32p incorpora-tion studies into lipid of various membrane frac-tions (Table 6) can be summarized as follows(comparison of specific activities): when 32p waspresent throughout the growth cycle (Table 6b),

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TABLE 5. Assay of some enzymes in different fractions of protoplast lysate from gradienta

Amt of adenosine NBT reductasebFractions rgiphonsipseum NADH oxidase phosphate

activated) NADH Succinate Lactate dehydrogenase

Whole lysate 6.1 0.83 0.055 0.055 0.5 0.053lb 0 (-) 0.56 (0.6) 0.1 (1.8) 0.1 (1.8) 0.1 (0.2) 02 11.1 (1.8) 0.87 (1.05) 0.12 (2.2) 0.18 (3.3) 0.4 (0.8) 03 18.3 (3.0) 0.31 (0.3) 0.065 (1.2) 0.065 (1.2) 0.73 (1.54) 04 4.1 (0.6) 0.45 (0.5) 0.102 (1.8) 0.088 (1.6) 0.53 (1.06) 0la 1.1 (0.2) 0.63 (0.6) 0.057 (1.0) 0.085 (1.5) 0.006 0.19 (3.7)

a Numbers in parentheses show the ratio of the specific activity of the enzymes of any fraction to thatof the whole protoplast lysate. All of the results are expressed as the specific activity of the enzymes.Adenosine triphosphatase = micrograms of inorganic phosphorus liberated per minute per milli-gram of protein; NBT reductase, optical density at 530 nm per minute per milligram of protein; NADHoxidase, decrease in the optical density at 340 nm per minute per milligram of protein; glucose-6-phos-phate dehydrogenase, increase in the optical density at 340 nm per milligram of protein per minute.

b See reference 28.

TABLE 6. Incorporation of 32p inito the lipid ofdifferent membrane fractions of L.

moniocytogenes-

Fraction Lipidb Lipid LipiddFranphoSphorus phosphorusc phoSphorusd

lb 700 234 5832 457 505 1853 650 251 1893a 700 58 1594 615 245 243

Whole lysate 364 138

a Results are an average of three different setsof experiments and are expressed as the specificactivity (counts per minute per microgram ofphosphorus) of 321p

b 32p was present throughout the growth cycle(12 hr of growth).

C 32p was added 7 hr after inoculation, and thenthe cells were grown for another 90 min beforeharvesting.

d 32p was added 7 hr after inoculation, and thecells were harvested 10 min later.

fraction lb = 3 = 3a = 4 > 2; when there wasa90-min pulse mid-log (Table 6c), fraction2 >> lb = 3 = 4 > 3a; when there was a 10-minpulse mid-log (Table 6d), lb >> 4 > 2 = 3 > 3a.The lipid of the particulate fraction (Ib) showed

a very rapid turnover of 32p, but when an equi-librium was established by prolonged exposure(Table 6b), the specific activity of "P incorpora-tion into lb was similar to that of fraction 3 (pre-sumably peripheral membrane). Fraction 2(presumably mesosome membrane), on the otherhand, showed significantly higher "P uptake inthe mid-log phase of growth only (Table 6c). Thelevel of radioactivity in fraction 2 was much lower

than that of particulate and peripheral membranefractions when equilibrium in the radioactivitywas established by prolonged exposure to 32P(Table 6b).

DISCUSSION

The widely diverse functions of the bacterialmembrane include energy generation and trans-fer, control of the molecular traffic in and out ofthe cell, genome separation, and cell wall biosyn-thesis. The recent demonstration of a membrane-bound long-lived messenger RNA (54) and anRNA of a very rapid turnover (2, 37) has led tospeculation that the membrane takes part in aspecialized aspect of protein and RNA synthesis.Active participation of membrane in secretion ofenzymes has also been suggested (27); however,due to the lack of fractionation techniques, therelationship between the structure and functionof the bacterial membrane has not been clearlydemonstrated, although complex intracytoplas-mic morphology of membrane has been ob-served in recent years (1, 36, 45, 51, 52).This communication contains details of pro-

cedures for gentle fragmentation of protoplastmembranes and fractionation and identificationof components in a suitable density gradient.Early attempts to fractionate lysed protoplastswere unsuccessful, as the methods were too drasticand caused fragmentation of membrane. We ex-ploited the observations that, above a critical glu-cose concentration (>0.01 M), actively respiringprotoplasts lysed, and that this lysis was furtherenhanced when the osmotic support of the proto-plasts was reduced by partial reduction of the os-molarity (0.2 M sucrose) of the stabilizing

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medium. Lysis and release of internal constituentswere gradual with minimal fragmentation, be-cause the protoplasts did not abruptly burst.Ficoll, a polysucrose compound of high molecu-lar weight and low viscosity, was selected as amedium for density gradient membrane fractiona-tion, because it is reputed to enhance the sta-bility of fragile biological structures like yeastvacuoles (32). In early experiments, we found thatsucrose in concentrations in excess of 0.25 M

caused fragmentation of mesosomes released fromlysing protoplasts. However, osmotic support wasnecessary to stabilize these structures; thus, an0.25 M sucrose concentration was always main-tained during fractionation. The final problemwas the lack of techniques available for charac-terizing membrane fragments adequately.A specific enzyme system, which can be used as

a marker, was not found in mesosomes. The mostwidely accepted view at the present time does notsupport the idea of differential distribution of en-zyme chains transferring electrons in peripheralcytoplasmic membrane or intracytoplasmic(mesosome) membrane (17, 33).Though it has been shown by cytochemical

studies in L. monocytogenes (24) that telluritereduction takes place only at the plasma mem-brane, the malic dehydrogenase-linked telluiritereductase (48) is entirely concentrated in thesoluble cytoplasmic fraction (unpublished data).

In view of the failure of this specific approach tofraction characterization, a more general investi-gation was undertaken. This included studies of(i) morphology under the electron microscope,(ii) chemical composition, (iii) distribution ofenzyme activities known to be membrane bound,and (iv) 32p incorporation as an indicator ofmetabolic activity.

Morphological differentiation of membraneswas rarely observed in the bacterial cell. However,with the introduction of negative staining (41),new possibilities became available. The three ma-jor fractions isolated on the gradient varied ingross morphology.

Fraction lb was a mixture of two kinds of par-

ticles. Certain properties of heavier particles, e.g.,ribonuclease sensitivity, sensitivity to the depriva-tion of MgCl2, and an average diameter of 2 to 3pm, suggest that they may be membrane-boundribosome particles. But the RNA concentrationwas much lower than that reported for bacterialribosome. These particles may be membrane-bound RNA particles and have a different com-position than cytoplasmic ribosome.Although the lipase-sensitive lighter particles

are morphologically similar to the adenosinetriphosphatase particles of Bacilluls megaterium(34), we could not detect any adenosine triphos-

phatase in these particles. The particles may beassociated with some oxido-reduction enzymesystem. Because these particles contain a signifi-cant amount ofNBT reductase, the activity of thisenzyme in the particles was much higher thanthat in the peripheral plasma membtane but lowerthan that in the tubular and vesicular (mesosome)fraction. On the other hand, the NADH oxidaseactivity of the particles was much lower than thatof the peripheral plasma membrane. This differen-tial distribution strongly suggests that some of theparticles originated from the mesosome fraction.Similar reports have been made by cytochemicalstudies that deposition of formozan takes placeonly in the mesosomes of L. monocytogenes (23).The particles (ib) contain very large amounts

of RNA, protein, and DNA. Phosphorus frac-tionation can also be used as a distinguishingfeature of these particles. Unlike other fractions,nucleic acid phosphorus constitutes the majorportion of the phosphorus content. The particles(Ib) differ from fraction la in their very low 10%trichloroacetic acid-soluble phosphorus and rela-tively higher chloroform-soluble lipid phos-phorus.The lower ratio of protein to phospholipid in

the particles, in comparison to the whole celllysate, indicates an increase in the phospholipidconcentration with respect to protein. But thisratio is much higher than that found in the mem-brane (fraction 2 and 3). It may be possible thatthe phospholipid in the particles is required forsome enzymatic functions and that the relativeconcentration of phospholipid to protein is muchlower than that required for the formation ofmembrane.A high rate of turnover of 32p into the lipid may

include both de novo synthesis and exchange intothe phospholipid. It may be possible that biosyn-thetically the particles are precursors of mem-brane material or that they have some role in thetransport process of the membrane (21).

Electron micrographs of isolated fractions sug-gest that two main membrane fractions are liber-ated when protoplasts are lysed in a medium ofsuitable osmotic and ionic strength. These frac-tions are characterized by (i) empty ghosts ofvarious sizes (fraction 3) and (ii) tubular struc-tures frequently terminating in vesicles and con-taining some cytoplasmic material inside (fraction2). By studying protoplasts in the process oflysing, it was determined that these fractionscorrespond with the peripheral membrane andthe intracytoplasmic membrane bodies (meso-some), respectively. Further criteria were soughtto prove this contention. These tubules andvesicles could have been artifacts resulting fromthe reaggregation of peripheral membrane of

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lysed cells or a new conformation of the periph-eral membrane. This can be countered by the argu-ment that artifacts of this type would be in greaterevidence after more drastic lysis. Furthermore,observations during lysis would suggest a corre-spondence between the two fractions and thestructure observed in the unlysed and partlylysed protoplasts.The distribution of RNA, protein, and lipid

phosphorus content of the separated fractionsindicate no marked dissimilarities between thetwo membrane fractions (2 and 3). A higher con-centration of DNA in fraction 2 than in fraction3 may indicate that the DNA remains bound tothe mesosome. Attachment of nuclear materialand mesosome has been suggested (45). Therewas about a twofold difference in the DNA con-centration of fractions 2 and 3, but the crude ma-terial from the gradient showed a much larger(fourfold) difference (unpublished data).Certain enzymes are clearly concentrated in

certain fractions. Thus, there are higher adenosinetriphosphatase and NADH oxidase activities inthe peripheral membrane (fraction 3), whereasNBT reductase is more evident in the mesosomemembrane (fraction 2). The low levels of theseenzymes in other fractions are probably indica-tive of mutual contamination of one fraction withthe other and are most probably a reflection of thephysical connection of the fractions and of arbi-trary fragmentation of the connection. Neithercytochrome oxidase nor the absorption band ofcytochrome c could be detected in any fraction.However, there was a strong NADH oxidase ac-tivity, suggesting that the organism uses a cyto-chrome-independent pathway for electron trans-fer like other facultative anaerobes (6).

It is interesting to note the differential distribu-tion of NADP oxidase and NBT reductase ac-tivity in the two membrane fractions. No properphysiological explanation can be offered from thepresent results. However, it suggests that the oxi-doreduction properties of the two membranefractions are different. The very high NADH oxi-dase activity further suggests that physiologically-significant electron transport system may be lo-calized in the peripheral membrane.The incorporation of 32p into the membrane-

lipid of fraction 2 (mesosome) was much higherthan that of fraction 3 (peripheral membrane)during log phase. But radioactivity did not reachan equilibrium in the membrane-lipid of fractions2 and 3 when 32p was present throughout thegrowth cycle. These data suggest the possibilitythat the rate of turnover in the membrane of frac-tion 2 is comparatively lower after its synthesis.The higher uptake by fraction 2 (mesosome)during log phase can be explained by its rapid syn-

thesis during the period of maximal cell division.The possible relationship of mesosome to genomeseparation (12) and cross wall formation (4) hasbeen suggested by many workers.

Similar observations have been made in mam-mals; the myelin sheath of the nerve has a verylow turnover rate of 32p into lipid after the syn-thesis of the membrane (5).

Fitz-James reported that 32p incorporation intothe forespore membrane is unrelated to incorpo-ration into the peripheral membrane. He alsosuggested, from his '4C-acetate incorporationstudies (16), that there is a precursor product re-lationship between mesosome and peripheralmembrane. But our 32p studies suggest thatL. monocytogenes mesosome membrane is syn-thesized independently of peripheral membrane ata specific period of the growth cycle. It is possiblethat the higher incorporation of 32p results from ahigher exchange rate in response to some acceler-ated functional activity. In the future, it will beinteresting to resolve this problem by studying theincorporation of phospholipid biosynthesis intointermediates as well as 'IC incorporation intomembrane proteins.However, although the significance of the 32p

studies cannot be fully assessed until more infor-mation is obtained, we found that 32p incorpora-tion into lipid can be used as one of the criteria todistinguish peripheral plasma membrane frommesosome membrane. In conclusion, it can besaid that L. monocytogenes membrane materialcan be separated, after lysis of protoplasts underspecific conditions, into three major fractionshaving structural, enzymatic, and metabolicdifferences. One of these fractions probablyoriginates from the mesosome of this organism.

ACKNOWLEDGMENT

This investigation was supported by the Medical ResearchCouncil of Canada.We thank K. K. Carroll for his suggestions and for laboratory

facilities and K. P. Strickland for the supply of 32P. We also thankM. G. Sargent and H. Lechevalier for their help in preparingthe manuscript and John Marak and D. J. Groot Obblink forproviding technical assistance in electron microscopy and invarious other ways.

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