CHARACTERIZATION OF THE PROTOPLASMIC CONSTITUENTS OFBACILLUS MEGATERIUM'

CLAES WEIBULL2Department of Bacteriology and Virus Laboratory, University of California, Berkeley, California

Received for publication June 22, 1953

It has been shown (Weibull, 1953) that it ispossible to isolate protoplasts from Bacillusmegaterium by lysozyme treatment of cellssuspended in 0.1 to 0.2 M sucrose. When proto-plasts are resuspended in phosphate buffer(pH 7), rapid lysis occurs, and the protoplasmicconstituents are released. This procedure doesnot include any mechanical disintegration; inaddition, only a small fraction of the lysozymeinitially applied is present in the final lysate sothat enzymatic or nonenzymatic interaction be-tween lysozyme and the protoplasmic constit-uents is largely avoided. This paper describesthe properties of the protoplasmic material soobtained.

MATERIALS AND METHODS

B. megaterium, strain KM, was grown andharvested as has been described earlier (Weibull,1953). The experimental procedure for obtaininglysates was as follows. Washed suspensions in0.15 M sucrose + 0.03 M phosphate (pH 7.0)containing ca 10 mg bacterial dry weight per mlwere treated at room temperature with lysozyme(final concentration 0.5 mg per ml). After theenzymatic reaction had reached completion, anequal amount of sucrose solution was added andthe protoplasts were precipitated by centrifuga-tion at 5 C. The protoplasts were resuspended atroom temperature in an appropriate amount ofbuffer. Complete lysis occurred almost instan-taneously. Direct lysis of cells, i.e., not viaprotoplasts, was conducted similarly in theabsence of sucrose.Mechanical disruption of bacterial cells was

achieved either by the use of a 9 kc Raytheon1 This investigation was supported in part by a

grant-in-aid to Dr. R. Y. Stanier by the AmericanCancer Society on recommendation of the Com-mittee on Growth of the National ResearchCouncil.

2 Rockefeller Research Fellow, 1952-1953. Pres-ent address: Institute of Biochemistry, Uni-versity of Upsala, Upsala, Sweden.

magnetostriction oscillator or by grinding withalumina (McIlwain, 1948).The lysozyme used was the Armour crystalline

product prepared from egg white. Crystallinedesoxyribonuclease and ribonuclease preparedaccording to Kunitz (1940; 1950) were purchasedfrom Worthington Biochemical Sales Co.; crystal-line trypsin was purchased from General Bio-chemicals, Inc. Ribonucleic acid was determinedaccording to Schneider (1945).

Analytical ultracentrifugations were conductedin a Spinco instrument equipped with a Philpot-Svensson optical system at an average centrifugalfield of 130,000 G. The total amount of bacterialmaterial in the solutions investigated was 10 to15 mg per ml. Preparative ultracentrifugationswere performed in the Spinco Model L centrifugeusing the no. 40 rotor (diameter of tubes 16 mm,tubes inclined 260 to rotor axis, average centrif-ugal field 105,000 G at 40,000 rpm). The averagetemperature of the solutions during the runs was13 C. In order to perform reproducible centrifuga-tions, the viscosity of the lysates had to belowered by treatment with desoxyribonuclease(final concentration 0.0005 per cent).Specimens for electron microscopy were

prepared as described earlier (Weibull, 1953).RESULTS

When suspensions of B. megaterium, even veryconcentrated ones, are treated with lysozymein the presence of sucrose, no marked increasein the viscosity of the suspension occurs. How-ever, when centrifuged protoplasts are resus-pended in buffer, the resulting lysate is highlyviscous. This viscosity increase may result ingel formation if the concentration of bacterialmaterial exceeds 20 mg dry weight per ml.The high viscosity disappears in a few minutes ontreatment with desoxyribonuclease but is un-changed by treatment with ribonuclease ortrypsin. These findings show that the viscosityis caused by a protoplasmic substance containing

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PROTOPLASMIC CONSTITUENTS OF B. MEGATERIUM

desoxyribonucleic acid. Direct lysis of B. nwga-terium with lysozyme also yields a highly viscouslysate, a fact noted earlier in similar studies onother bacteria (Utter et al., 1946; Militzer et al.,1950).

Analyticat ultracentrifugations. Schachman et al.(1952) have shown that components withsedimentation constants of 40, 29, and 5 S arereleased from the cells of a number of bacteriaby mechanical disruption. In addition, a "spike"composed of a substance containing desoxy-ribonucleic acid was observed frequently. B.megaterium was not studied by these workers;hence analytical ultracentrifugations have beenperformed on lysates obtained from B. mega-

of a gel, sedimenting faster than the othermacromolecular components. Apparently somebonds in this substance, proved above to containdesoxyribonucleic acid, are destroyed in 0.03 Mphosphate but not in 0.15 M sodium chloride.The sedimentation constants are not markedly

influenced by the viscosity of the preparations(see table 1). This is in accordance with the ob-servations of Schachman and Harrington (1952)on the sedimentation patterns of mixed systemscontaining free desoxyribonucleic acid.

Investigations based on centrifugations in thepreparative ultracentrifuge. The aim of theseexperiments has been to characterize further thematerials found in the protoplasmic lysates,

TABLE 1Ultracentrifugal analyses of macromolecular components of Bacillus megaterium

PEPTIVEP lTENIQUE MEDIUM SEDIMENTATION CONSTANTS

I. Direct lysis 0.03 M phosphate 41.6 26.2 14.9* 2.8II. Direct lysis 0. 15 mNaCl + 0.01 41.0 26.4 3.0

phosphateIII. Lysed protoplaats 0.03 M phosphate 41.3 26.3 16.2* 3.9IV. Lysed protoplasts + desoxyribonuclease 0.03 M phosphate 43.6 27.7 3.8V. Sonic extract 0.03 M phosphate 42.7 26.8 4.4VI. Alumina grinding 0.03 M phosphate 42.2 31.3 3.9

In all preparations (except preparation IV) citrate was added to a final concentration of 0.01 M inorder to inhibit bacterial desoxyribonuclease. Preparations I to III were highly viscous, preparationsIV to VI showed no appreciable viscosity. The sedimentation constants are given in Svedbergs andrefer to pure water at 20 C as solvent.

* Spike.

terium cells by mechanical disintegration and bylysozyme treatment. Table 1 shows the results.The distribution pattern of macromolecularcomponents is very similar to that reported forother species by Schachman et al. (1952).Some comments on the behavior of the sub-

stance containing desoxyribonucleic acid arerequired. Mechanically disrupted B. megateriumcells give extracts of low viscosity, and no spikeis found in the ultracentrifugal diagrams (table 1,preparations V and VI). The spike appears,however, following direct lysis with lysozyme inphosphate buffer or lysis of protoplasts inphosphate buffer (preparations I and III) butnot after direct lysis with lysozyme in thepresence of 0.15 M sodium chloride (preparationII). In the latter case centrifugation at lowerspeed shows that the material causing the highviscosity of the preparation has the properties

especially components which are too large tobe studied in the analytical ultracentrifuge.Table 2 shows the results of two independentexperiments in which lysates of protoplasts,prepared as described under Methods, havebeen centrifuged at different centrifugal fields.Aliquots of the original lysates and of eachsupernatant were analyzed for dry weight andribonucleic acid content; from these data valuesfor the successive precipitates were calculated bydifference. Between centrifugations, the solutionswere kept at 5 C.

Centrifugation for 15 minutes at 590 G gives awhite precipitate, comprising ca 10 per cent ofthe bacterial dry weight (table 2). Light micro-scopic observations show that most of the lipidgranules (Weibull, 1953) are precipitated at590 G, no other structures being visible in thisfraction. Figure 1 shows an electron micrograph

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of such washed granules. Qualitative tests on

the washed granules (kindly performed by Dr.M. R. J. Salton) reveal a low content of carbo-hydrate and protein. The granules are almostcompletely soluble in warm alkali or in warm

chloroform. The solution in alkali shows no

specific absorption between 250 and 300 m,i.Thus, these tests confirm the report of Lemoigneet al. (1944) that the granules in the cells ofB. megaterium consist of lipid material, found bythem to be polymerized 13-hydroxy-butyric acid.

TABLE 2Amounts of bacterial substances (dry weight) and

ribonucleic acid precipitated by means ofpreparative centrifugations

CEN'TR.TIME

7lIinutes

1515151515151545120150

CENTRIF-UGALFIELD,

IMULTI-PLESOFG

5901,0509,49014,800

!29,10059,3001105,400105,400105,400105,400

PER CENTBACTERIALSUBSTANCE

PREC.*

PE ETPER CENT

RIBONUCLEIC PERTICLENT

ACID PRECIPI- PARTI;CALES

TATED

I II I 42 S 27 S

11.4 9.7;13.7 10.7l 1.5i20.2 19.01 3.9; 5.5,22.5? 19.01 7.71 5.524.9 20.7j 16.2'! 9.1126.6 20.4 17.8' 12.7 5.3 3.431.0 22.71 28.71 19.1 8.4 5.943.6' 31.7 59.7 47.3 25.31 16.2

47.3 79.1 67.51 43.352.2T 88.41 84.3 54.2

* These values are calculated in terms of wholecells.

Differential centrifugation for 15 minutesbetween 590 and 14,800 G precipitates a darkyellow layer, comprising about 10 per cent of thebacterial dry weight. Phase contrast microscopyreveals "ghosts" (Weibull, 1953) and some

granules, free or attached to the "ghosts". Figure 2is an electron micrograph of this fraction, washedthree times in distilled water, and fixed withosmium tetroxide. Membrane-like bodies are

predominant; a variable number of granules,of the same shape as the granules illustrated infigure 1, can be seen included within the mem-

branes. Electron micrographs of unfixed or

formaldehyde fixed specimens reveal only amor-

phous agglomerations of material; evidently themembranes are destroyed by drying under theseconditions. A brief treatment of the yellowfraction in the Raytheon sonic oscillator destroys

the "ghost" structure. By subsequent centrifuga-tion at 105,000 G, most of the yellow materialcan be resedimented, forming a uniformlycolored layer overlying a small amount of lipidgranules.

Differential centrifugation between 14,800 and29,100 G gives a faintly yellow sediment, whichcontains only a negligible proportion of thebacterial dry weight. At still higher gravitationalfields the sediments are almost completelycolorless, and the supernatants from suchcentrifugations are also colorless, even in deeplayers. Thus, nearly all the pigmented material ofthe cell is associated with the "ghost" fraction.

Isolated cell walls from B. megaterium giveno "ghosts", detectable by phase contrast mi-croscopy, on treatment with lysozyme. Electronmicroscopical observations also give evidencethat the walls are dissolved completely bylysozyme (Salton, unpublished data). Hence,unless the lysozyme acts in a* different fashionon the cell walls in situ, the "ghosts" cannot belysozyme resistant cell wall material. Therefore,the "ghosts" must be assumed to consist ofcytoplasmic material. They may represent thecytoplasmic membrane; however, denaturationprocesses during the lysis of the protoplasts arenot improbable. The fact that isolated cell wallsform white precipitates when centrifuged, incontrast to the yellow precipitates of the "ghost"fraction, is additional evidence of their non-identity. Whether the yellow pigment is aconstituent of the membranes themselves or ofparticles inside them cannot be settled con-clusively on the basis of the present experimentalevidence. Adsorption phenomena must also betaken into account. The fact that, after sonictreatment of the "ghost" fraction, most of thecolored material can be sedimented in thepreparative centrifuge as a uniformly coloredpellet suggests that the pigment is bound firmlyto the membranes.

Stanier et al. (1953) have found that thecomplete cytochrome system of Pseudomonasfluorescens is located in a "particulate" fraction,largely sedimentable from sonic extracts bycentrifugation for one hour at 22,000 G. Sincemost of the pigmented material of B. megateriumis in the yellow fraction described above, whichis also "particulate", this material was examinedfor the presence of cytochromes. The absorptionspectra of a concentrated suspension of the

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Figure 1. Electron micrograph of lipid granules isolated from Bacillus megaterium.

Figure 2. Electron micrograph of the "ghost" fraction of lysed Bacillus megaterium cells.

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yellow fraction in 70 per cent glycerol (Stanieret al., 1953) before and after reduction withsodium hydrosulfite were measured with theBeckman spectrophotometer. Figure 3 shows theresulting curves, which have been corrected fornonspecific light scattering (Weibull, 1948).The absorption maxima at 530, 558, and 600 m,ucorrespond well to the absorption bands of thecytochrome components in whole cells of B.megaterium (recorded without reference as 531,560, and 603 m,u by Stephenson, 1949). Thissuggests that the entire cytochrome system islocated in the yellow fraction.

.300

z

0

00

400 500 600 400 500 600

WAVELENGTH, mp

Figure S. Absorption spectra of yellow fractionof lysed Bacillus megaterium cells, suspended in70 per cent glycerol. To the right, suspensionbefore reduction; to the left, suspension afterreduction with sodium hydrosulfite.

Schachman et al. (1952) have shown that thebulk of the ribonucleic acid in bacterial cells isassociated with particles having sedimentationconstants of 20 to 40 S. Columns 7 and 8 oftable 2 give the calculated percentages of particleswith the sedimentation constants of 42 and27 S3 which would be sedimented by the gravi-tational fields applied. (These calculations were

performed according to the formula given in theSpinco technical manual.) When a comparisonis made with the actual amounts of precipitatedribonucleic acid (table 2, columns 5 and 6), theresults of Schachman et al. (1952) are confirmed;the ribonucleic acid seems to be associatedmainly with the 42 S particles. However, ap-

proximately 15 per cent of it is precipitated with

3The values for the major component in B.mnegaterium found by analytical ultracentrifuga-tions, table 1.

the "ghosts" and in the fraction sedimentingbetween 14,800 and 29,100 G, perhaps as ag-gregated 42 S particles. In similar experimentsperformed with extracts prepared by directlysis of B. megaterium cells with lysozyme, aconsiderably higher percentage of ribonucleicacid was precipitated between centrifugal fieldsof 30,000 and 60,000 G. In this case, nonenzy-matic aggregation of part of the ribonucleicacid by the highly basic lysozyme would explainthe observations, an explanation further sup-ported by the fact that a slight turbidity appearswhen lysozyme is added to a suspension of lysedprotoplasts.

DISCUSSION

The experiments reported above show thattreatment of bacterial cells with lysozyme hascertain advantages as a method for the liberationof protoplasmic constituents over the previouslyused techniques (Schachman et al., 1952; Weibulland Hedvall, 1953) of cellular disintegrationby mechanical means. Specifically, two com-ponents not observed in extracts prepared bymechanical disintegration have been detected:"ghosts", and a gel containing desoxyribonucleicacid.The "ghosts" have been isolated reasonably

free from other constituents of lysates by differ-ential centrifugation between 590 and 14,800 Gfor fifteen minutes. Microscopically, they appearto consist of membranes associated with somegranular material. The membranous structure iseasily destroyed; for example, by drying withoutproper fixation or by sonic oscillation. Similarstructures have been briefly described by Herbertand Pinsent (1948) and Murphy and Bang(1952); the bodies found by Georgi et al. (1951),further characterized by Burns and Militzer(1953), in lysozyme treated preparations ofBacillus stearothermophilus also could be inter-preted as "ghosts" containing other cytoplasmicconstituents. Almost all the pigmented materialof the B. megaterium cells is associated with the"ghost" fraction, whose absorption spectrumshows that it contains the bacterial cytochromesystem. At first sight, the size and form of the"ghosts" suggest that they might consist ofresidual cell walls. However, no "ghosts" can bedetected by phase contrast microscopy inpreparations of pure cell walls after treatmentwith lysozyme. Electron microscopy also indicates

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(Salton, unpublished data) that the cell walls ofB. megaterium are completely lysed by lysozyme;furthermore, the cell walls of this organism arecolorless, whereas the "ghost" fraction is stronglypigmented. In view of these facts, it may besuggested that the "ghosts" represent cytoplas-mic membranes, either intact or denatured.This interpretation is strengthened by theobservations made by Tomcsik and Guex-Holzer(1952) and by Robinow and Murray (1953).The almost exclusive association of the bacterialcytochrome system with this fraction of extractsis undoubtedly of great interest, suggesting thatthis system may be localized in the cytoplasmicmembrane; however, definite conclusions regard-ing the site of the cytochrome system in theliving cells cannot be made on the basis of thepresent experimental evidence.

Earlier attempts (Schachman et al., 1952;Weibull and Hedvall, 1953) to isolate the bac-terial nuclear equivalents (i.e., discrete structurescontaining desoxyribonucleic acid) from mechani-cally prepared bacterial extracts have not beensuccessful. Under such conditions, the desoxy-ribonucleic acid is sometimes liberated in highlyviscous form as a material which gives a sharpspike with a sedimentation constant of approxi-mately 5 to 10 S in the ultracentrifuge, andsometimes (notably in sonic extracts) evenfurther degraded so that it is not detectable as anultracentrifugal component. The present worklikewise indicates that the nuclear equivalentsmust be extremely sensitive to changes in theenvironment, since lysis with lysozyme undermost conditions yields desoxyribonucleic acid inthe form which gives a spike with a sedimentationconstant of approximately 15 S. However, whenlysis with lysozyme is conducted in the presenceof 0.15 M sodium chloride, a desoxyribonucleicacid containing gel with different physicalproperties results, which encourages the hopethat an isolation of the nuclear equivalentseventually may prove possible by the use of theproper medium.

ACKNOWLEDGMENTS

The author wishes to thank Dr. H. K. Schach-man for collaboration in the analytical ultra-centrifugation experiments, Drs. R. Y. Stanierand M. R. J. Salton for stimulating suggestionsand advice.The electron micrographs were taken by

Dr. R. C. Williams; the preparative ultra-centrifugations were performed in the laboratoriesof Dr. D. Mazia. Their help is gratefullyacknowledged.

SUMMARY

The protoplasmic constituents of Bacillusmegaterium have been investigated by removalof the cell walls with lysozyme and subsequentlysis of the protoplasts, as well as by mechanicaldisintegration of the cells. Earlier investigationsof the macromolecular organization of themicrobial cell have been confirmed. Two largestructures constituting part of the protoplastshave been characterized: "ghosts", which mayrepresent the cytoplasmic membrane, and lipidgranules. The cytochrome system of B. mega-terium sediments with the "ghost" fraction indifferential centrifugations. The bacterial lysatesdo not contain any discrete nuclear equivalents.

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