JOURNAL OP BACTERIOLOGY, Jan., 1967, p. 427-437Copyright © 1967 American Society for Microbiology

Vol. 93, No. 1Printed In U.S.A.

Production and Ultrastructure of Lysozyme andEthylenediaminetetraacetate-Lysozyme Spheroplasts

of Escherichia colilD. C. BIRDSELL AND E. H. COTA-ROBLES

Department ofLife Sciences, University of California, Riverside, California

Received for publication 19 August 1966

ABsTRAcrSpheroplast production by lysozyme and ethylenediaminetetraacetate (EDTA)

was examined as a means of obtaining osmotically sensitive cells for studies ofenzyme localization. Physiologically young cells plasmolyzed with 0.5 M sucrose in0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7, 8, or 9) were quanti-tatively converted to plasmolyzed osmotically sensitive rods after lysozyme treat-ment. Although such cells were osmotically sensitive, a 1:1 dilution in Tris bufferwas necessary for conversion of rods into spheroplasts. Addition of EDTA resultedin a rapid conversion of the plasmolyzed spheroplasts into spherical structuresdevoid of a plasmolysis vacuole. These structures, which we call EDTA-lysozymespheroplasts, contained a number of attached membranes. We believe that this con-version results from a weakening of the outer trilaminar component of the cell wallby EDTA, resulting in the collapse of the plasmolysis vacuole. Dilution of sucrosebelow 0.15 M also resulted in the collapse of the plasmolysis vacuole. Both the lyso-zyme spheroplasts and the EDTA-lysozyme spheroplasts were osmotically sensitive.Thin sections of the EDTA-lysozyme spheroplasts demonstrated that the outertrilaminar component of the cell wall was broken, exposing large areas of thecytoplasmic membrane to the environment.

Spheroplasts of gram-negative bacteria havebeen and are being used in a variety of differentinvestigations, for example, release of enzymes(5, 15, 19-21), accumulation of metabolites (25),isolation of polyribosomes (7), purification ofenzymes (16), and assays of the biological activityof viral nucleic acids (9, 17). The standard tech-nique which has evolved for production of sphero-plasts involves treatment of exponential- orstationary-phase cultures with ethylenediamine-tetraacetate (EDTA) and lysozyme at an alkalinepH in the presence of a stabilizing solute, usuallysucrose. The resulting cells are spherical andsensitive to lysis by osmotic shock.Our interest in spheroplasts has resulted from a

desire to control their production prior to under-taking comprehensive studies of enzyme localiza-tion in cells of Escherichia coli. The presentpaper describes some parameters for the produc-tion of two types of spheroplasts from "physio-logically young" E. coli B. The first type is pro-

1Portions of this report were presented at the 66thAnnual Meeting of the American Society for Micro-biology, Los Angeles, Calif., 1966.

duced by the addition of lysozyme alone. Thesecells are referred to in the text as "lysozymespheroplasts." The second type are spheroplastsproduced by the addition of EDTA to lysozymespheroplasts. These are referred to as "EDTA-lysozyme spheroplasts." A further aspect of thisreport details the pattern of spheroplast produc-tion as determined by phase and electron micros-copy. In this report, the cell wall of E. coli isconsidered to consist of an outer trilaminar com-ponent (outer membrane) plus a rigid mucocom-plex. Our micrographs, as well as those of manyothers, seldom resolve the rigid mucocomplex.Murray, Steed, and Elson (18) found a structure,between the outer membrane and the cytoplasmicmembrane in cells of E. coli, which they identifiedas the rigid layer. These workers feel that othersdo not resolve this structure primarily as a resultof insufficient staining with a suitable metal salt.

MATERIALS AND METHODSSpheroplast formation. E. coli cultured aerobically

for 12 hr at 37 C in Fraser and Jerrel's glycerol me-dium (8) on a rotary shaker (500 rev/min) was inocu-lated into 500 ml of fresh medium in 2-liter flasks

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(1%, v/v) and further incubated with shaking. Atgiven times of incubation, cultures were harvested bycentrifugation at room temperature, washed once withan equal volume of 0.01 M tris(hydroxymethyl)amino-methane (Tris)-chloride (pH 8.0), and suspended toa final cell density of approximately 5 X 108 cells permilliliter in the same buffer supplemented to contain0.5 M sucrose. To prepare lysozyme spheroplasts,lysozyme (Sigma Chemical Co., St. Louis, Mo.) wasadded to a concentration of 20 /Ag/ml; the cells wereincubated at room temperature for 5 to 10 min, anddiluted 1:1 with Tris buffer. For preparation ofEDTA-lysozyme spheroplasts, EDTA to a final con-centration of 10-3 M was added to lysozyme sphero-plast suspensions. Formation of spheroplasts wascomplete within 10 to 15 min as determined byosmotic sensitivity and phase-microscopic examina-tion.

Electron microscopy. Cell suspensions were fixed1 hr in 10% formalin followed by the standard Kellen-berger and Ryter osmium fixation procedure (11) andwere stained for 1 hr in uranyl acetate prior to dehy-dration through a graded acetone series and embed-ding in Vestopal W. Sections were cut with either glassor diamond knives on an LKB Ultrotome, stainedwith lead citrate (26), and examined in either anHitachi HUll or a Zeiss EM9 electron microscope.

Phase microscopy. Unfixed cell suspensions wereexamined and photographed in a Zeiss StandardGFL phase microscope. At least 500 cells were ob-served and counted to determine the percentage ofspheroplast conversion after lysozyme treatment orthe percentage of ghost formation after osmotic shock.

r W2244

.4 |- lysozyme EDTA

.3 no additions

(08 to,X I

Osmotic sensitivity. Initially, osmotic sensitivitywas measured by determining the decrease in absorb-ancy at 600 ma after diluting the suspensions 1:5 inglass-distilled water and correcting for the dilutionfactor. In all later work, the cells were centrifugedfrom suspension and resuspended in the same volumeof glass-distilled water.

Phage adsorption. After equilibration for 10 minin a water bath (37 C), 0.5 ml of cell suspension (ap-proximately 108 cells) and 0.5 ml of coliphage T4(5.7 X 108 per milliliter) were mixed and allowed toreact for 10 min at 37 C. Portions (0.1 ml) werediluted into 9.9 ml of precooled buffer of the follow-ing composition (3.0 g of KH2PO4, 7.0 g of Na2HPO4,4.0 g of NaCl, 0.2 g of MgS04.7H20, 10 ml of 1%solution of gelatin, and 1 liter of distilled water) andwere immediately centrifuged at 10,000 X g for 5 min.Appropriate dilutions were made of the supernatantfraction and were assayed by the agar overlay tech-nique (1) for enumeration of nonadsorbed phage.

RESULTS

Osmotic sensitivity. Figure 1 demonstrates theosmotic sensitivity of physiologically young E.coli B and K-12 W2244 after treatment withlysozyme alone, EDTA alone, or EDTA followinglysozyme. Both strains became sensitive to lysisby osmotic shock upon lysozyme treatment andprior to the addition of EDTA, with little furtherincrease in osmotic sensitivity after EDTA wasadded. EDTA treatment alone also rendered thecells osmotically sensitive; however, the resulting

r B

lysozyme EDTAI I

.4I no additions

.300

80

Minutes MinutesFIG. 1. Osmotic sensitivity of Escherichia coli B and K-12 W2244. Symbols: O, no additions; 0, 20 ug/ml

of lysozyme; 0, 10-3M EDTA; A, EDTA after lysozyme treatment.

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"ghosts" retained the shape of the cell beforetreatment. Such "ghosts" of rods can also be ob- 100_tained upon osmotic shock of plasmolyzed cellsin the absence of EDTA. This phenomenon willbe discussed in a future paper.

Lysozyme spheroplast production. The concen-tration of sucrose used regulated the extent of 80 _plasmolysis. Figure 2 shows that, within limits,spheroplast production and osmotic sensitivityare dependent upon the concentration of sucroseused in the suspending medium. Thus, sphero- enplast production and osmotic sensitivity are , 60 -

dependent upon the degree of plasmolysis of cells XLa.at the time of lysozyme treatment. This suggests ° /that cells must be plasmolyzed before lysozyme -can degrade the rigid layer. The relationship be- Ltween final sucrose concentration and spheroplast 40 -production is shown in Fig. 3. As the suspensionwas diluted, an increasing proportion of the pop-ulation became spherical until virtually all of thesusceptible cells were typical lysozyme sphero-plasts. The 1:1 dilution step routinely used was 20

----0~~~~~~+ +~~~~~~~- 0 IIII.III,I

-r,.5 A .3 .2FINAL SUCROSE CONCENTRATION (M/L)

80 / FIG. 3. Effect of dilution on formation of spherical/ // * cells. Samples (5-ml) of lysozyme-treated cells were

/ / / diluted with 1.0,2.0,3.0, 4.0, and 5.0 ml of Tris buffer.I / / Formation of spherical cells was determined by phase-

w I / microscopic counts.0-

60 / /t 1 necessary only for formation of spherical cells,SI / / not for osmotic sensitivity. Removing the sucrose

I /, from lysozyme-treated cells before or afterI / sphering led to complete lysis and the formation

40 / 1l of spherical ghosts. Quantitative conversion toI // lysozyme spheroplasts occurred at pH 7, 8, and 9.

/,'/ Microscopic observations of normal cells. Nor-| // mal cells of E. coli B fixed in the growth mediumI /11 ____ sphero Iost - are shown in Fig. 4a and 4b. The fibrillar nucleoid,20 --- Spheroplosts particulate cytoplasm, and triple-layered profiles/I ~~~~Ghosts

/// Turbidity of the outer unit membrane component of the cell/i,;// wall and of the cytoplasmic membrane are well

.2I, defined. Also present are membranous prolifera-C' ___________________________________ tions varying from simple vesicles (Fig. 4a) to

SUCROSE CONCENTRATION 4 .5 .6 7 more complex configurations (Fig. 4b). InternalSUN(M/L BEFORE DILUTION membranes in cells of E. coli have been reportedFIG. 2. Effect of sucrose concentration on sphero- by Ryter and Jacob (24) and by Cota-Robles

plast production. After washing in Tris buffer, equal (J. Ultrastruct. Res., in press).amounts of cells were suspended in Tris buffer supple- Microscopic observations of plasmolyzed cells.mented to contain the sucrose concentrations given. . .Osmotic sensitivity is reported as per cent decrease in Upon plasmolysis in 0.5 M sucrose, the cells as-turbidity and per cent "ghost" formation. "Ghost" sumed the structural pattern shown in Fig. 5a.formation and spheroplast production were measured by The cytoplasmic membrane limits the more con-phase-microscopic counts. densed cytoplasm, and the convoluted outer

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430 BIRDSELL AND COTA-ROBLES J. BACrERIOL.

IM~~~~~~

ji~ ~M,i..r.OXS

RI :; ..........7 &

.... {. .. ..... > - a s

*~~ ~ ~~Si F .tiVX;... 'a ..............stu .'se'k.' 'St; f.' ' s'W'>', ........................................ 2..^s.~~~~~~~~~~~At *z._ . ._ fi,, B, .

tM* . .. s..... . : . ..: :. ._

.: .__

.. ............. t* , s ; ...... Y4. b ... . r :S., .............. .* ;; . ¢ .b jlj_j;FIG. 4a and 4b. Thin sections ofnormal Escherichia coli B. N, nucleoid; OM, outer unit membrane of the cell

wall; CM, cytoplasmic membrane; IM, internal membranes; R, ribosomal particles; and M, membranous invagina-tions. The bar in all electron micrographs represents 0.5 I. X 49,400.

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SPHEROPLASTS OF E. COLI

..

-(

FIG. 5a. Thin section ofEscherichia coli B plasmolyzed in 0.5 M sucrose. E, extensions of the cytoplasm; CMcytoplasmic membrane. X 67,500.

FIG. 5b. Phase micrograph ofplasmolyzed E. coli B. X 1,250.

FIG. 6. Phase micrograph oflysozyme spheroplasts of Escherichia coli B. X 1,500.FIG. 7. Phase micrograph ofEDTA-lysozyme spheroplasts ofEscherichia coli B. X 1,500.

membrane retains the shape of the cell prior toplasmolysis. The nucleoid has become quitecompact, and ribosomes are no longer discernibleas defined particles. In all of our sections ofplasmolyzed cells, there appear to be areas wherethe cytoplasmic membrane adheres more firmlyto the outer membrane-rigid layer complex. Thesewere frequently observed as extensions of thecytoplasm, terminating at the cell wall. Both

longitudinal and cross-sections through such ex-tensions are shown in this micrograph. Figure 5bis a phase micrograph of unfixed plasmolyzedcells in which the plasmolysis vacuoles are clearlyvisible.

Microscopic observations of lysozyme sphero-plasts. The appearance of lysozyme spheroplastsas observed by phase-contrast microscopy isshown in Fig. 6. The cytoplasm is crescent-shaped

VOL. 93, 1967

:%~ >

F.P\ ..E~~~~~~~~~~~~~~~~~~~~~~51

431

..:...... ;. ...; . ........ . ....-

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ft

... : v

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...... ...* ........ WJ;

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BIRDSELL AND COTA-ROBLES

...1>s;.t-

-,. 1.1.1 ,.

.. 4..- f,f' 'r.41.0- ik,i

:', t.... -1,

10

1:.9.

1-4. .7. ,. d!7-..s

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_: . 7 ...OM

FIG. 8. Thin sections of lysozyme spheroplasts ofEscherichia coli B. X 51,000.

432 J. BACrERIOL.

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SPHEROPLASTS 'OF E.COLI4

with the concavity facing the large plasmolysisvacuole. Within the vacuole are seen dense bodieswhich appear to be attached to the outer limitingstructure and do not appear to be free within thevacuole. In some instances, the dense bodies canbe seen to be extensions of the cytoplasm.Lysozyme spheroplasts (Fig. 6) differ fromEDTA-lysozyme spheroplasts in that the latterare smaller and lack a plasmolysis vacuole (Fig.7). Thin sections of lysozyme spheroplasts areshown in Fig. 8. The spheroplasts are bounded byboth the outer and cytoplasmic membrane. Thecytoplasmic membrane also limits extensions ofthe cytoplasm. Membranous proliferations areobserved within the cytoplasm and may representan internal membrane system. The nucleoid re-mains somewhat condensed although ribosomalparticles can be resolved.

Microscopic observations of EDTA-lysozymespheroplasts. Treatment of lysozyme spheroplastswith EDTA results in a rapid, dynamic conversionto EDTA-lysozyme spheroplasts. Phase-micro-scopic examination of this process reveals that itoccurs instantaneously. It is possible to observethis conversion directly by viewing the diffusionboundary of EDTA as it travels through a popu-lation of lysozyme spheroplasts. As the EDTAstrikes the spheroplast, there is a disappearanceof the plasmolysis vacuole, and the cytoplasmassumes a spherical shape. EDTA-lysozymespheroplasts can be seen to contain attached blebs(Fig. 7). The rapid conversion of lysozymespheroplasts into EDTA-lysozyme spheroplastsresults in a decrease in optical density, as shownin Fig. 9. It can be seen that this decrease inoptical density is complete within 30 sec after theaddition of EDTA. The spontaneous decrease inoptical density in the absence of EDTA is slow,and would require more than 30 min to approachthe same level observed after the addition ofEDTA. We have designated the spherical struc-tures formed after EDTA treatment of lysozymespheroplasts as EDTA-lysozyme spheroplasts.However, EDTA is not an absolute requirementfor this conversion. A similar change is observedupon mild dilution oflysozyme spheroplasts; how-ever, the effect is not as uniform as with EDTAtreatment. We believe that the outer membraneis weakened by EDTA and then ruptured by theosmotic pressure differential. A similar effectcould be induced by mild dilution.The cell shown in Fig. 10 was apparently fixed

immediately after rupture of the outer mem-brane. The cytoplasm has assumed the sphericalshape characteristic of EDTA-lysozyme sphero-plasts. The ruptured outer membrane has begunto assume a curious configuration. The com-

00(<00

M n u t e s

FIG. 9. Effect of EDTA on lysozyme spheroplasts.EDTA to a final concentration of 1J0 M was added toa suspension oflysozyme spheroplasts at 2 min as indi-cated. The decrease in optical density was measured ina Zeiss PMQ IH spectrophotometer at 600 mu. Thenumbers above the curves represent minutes after thestart of the experiment and demonstrate that a slowdecrease in optical density occurs with time of incuba-tion.

plexity of these configurations is emphasizedin Fig. 11. The complex coils of the rupturedouter membrane would correspond to the blebsobservable by phase microscopy (Fig. 7). It isabundantly clear that a single membrane, whichmust be the cytoplasmic membrane, is the outer-most boundary between the cytoplasm and theenvironment over large areas of the spheroplastsurface. The ruptured outer membrane remainsattached to the cell, albeit to a small area of thesurface.

Phage adsorption studies. The pronouncedeffect which the various treatments related tospheroplast production have on the adsorption ofcoliphage T4 to E. coli B is shown in Table 1.Lysozyme spheroplasts adsorb significantly lessT4 than washed cells. However, the capacity ofEDTA-lysozyme spheroplasts to adsorb T4 isvirtually one-fifth of that of the untreated celland only one-third of the capacity of lysozymespheroplasts. We have shown that EDTA inducesthe rupture and subsequent coiling of the outerunit membrane component of the cell wall. Thereceptor sites for coliphage T4 are known toreside in this structure (28). Less effective adsorp-tion of T4 to EDTA-lysozyme spheroplasts can-not be attributed to the presence of EDTA in the

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BIRDSELL AND COTA-ROBLES

FIG. 10. Thin section ofEDTA-treated lysozyme spheroplast. Note the break in the outer membrane. X 68,400.

system, since EDTA alone does not influenceadsorption of T4 (Table 1). Protass and Korn(23) recently reported that EDTA treatment ofE. coli does not remove the receptor sites of T4while removing the receptor sites of X vir andphage 434. Thus, we must conclude that T4 can-not adsorb as effectively to EDTA-lysozymespheroplasts as it does to whole cells, because thereceptor sites are not available even though theyare present. Coiling of the outer membrane mustmask the receptor sites. The decreased ability oflysozyme spheroplasts to adsorb T4 is significant,but may merely be a reflection of the lysis ofsome spheroplasts.

DIscussIoNA variety of techniques [enumerated by Noller

and Hartsell (22)] have been utilized to rendergram-negative bacteria susceptible to lysis bylysozyme. All of these alter either structurally orchemically the outer membrane exposing therigid layer to enzymatic degradation. We findthat plasmolysis of E. coli, harvested duringexponential phase but particularly when "physio-logically young" (late lag or early exponentialphase), enables lysozyme to degrade the rigid

layer at pH 7, 8, or 9. The resulting spheroplastsresemble the crescent-shaped cells produced bypenicillin treatment (12), diaminopimelic aciddeprivation (14), or D-amino acid treatment (6),without the increase in size observable in sphero-plasts produced by metabolic manipulation.

Figure 12 is a diagrammatic representation ofspheroplast formation and osmotic sensitivity ofphysiologically young cells of E. coli B. Cellsharvested in early exponential phase, washed inTris buffer, plasmolyzed in 0.5 M sucrose, andtreated with lysozyme retain their rod shape,although they are susceptible to lysis by osmoticshock. The 1 :1 dilution step appears necessary todecrease the external osmotic pressure belowthat within the cell to allow uptake of sufficientwater to produce the spherical form. The in-creased pressure may be required to overcomeresidual binding of the rigid layer to adjacentpolysaccharide chains, or to the membraneswithin which it is apparently sandwiched (18),or perhaps to overcome rigidity not attributableto the mucocomplex.

Evidence is accumulating which indicates thatdivalent cations have an important role in main-tenance of the lipopolysaccharide of the outer

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VOL. 93, 1967 SPHEROPLASTS OF E. COLI 435;~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~W_~~~~~~~~~~h

4...

'.. .. ....*' '' , *~~~~~~~~~~~~~~~~~~~~~~..s":.._i..... ....:

S' ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ..... ..-- :] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.*: .'

Mb-|Ls ^k; 0000 00 0 0 _ X; i i . jff it,;0000 ' .R ', 0, 'ip i ,,FIG. 11. Thin section ofEDTA-lysozyme spheroplasts. Note the coils of outer membrane and the presence ofa

single (cytoplasmic) membrane bounding the cytoplasm. X 95,000.

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BIRDSELL AND COTA-ROBLES

membrane of gram-negative bacteria (2, 13).Leive (13) in particular has clearly shown thatEDTA treatment of E. coli results in a release ofcell wall lipopolysaccharide. Carson and Eagon(4) reported that lysozyme-treated Pseudomonasaeruginosa retained their rod shape. Similar ob-servations were reported by Voss (27) and byAsbell and Eagon (2). Asbell and Eagon proposedthe term osmoplast to describe osmoticallyfragile rods such as those mentioned in thispaper. It is conceivable that formation of sphero-plasts after lysozyme treatment has been over-looked in some investigations as a result of themaintenance of rod morphology after lysozymedegradation. The conversion of osmoticaly sensi-tive rods into osmotically sensitive spheres afterlysozyme treatment requires dilution of the stabi-lizing solute. Regardless of the sucrose concentra-tion in which we have prepared lysozyme sphero-

TABLE 1. Adsorption of coliphage T4 to spheroplastsof Escherichia coli

Expt ia Expt 26

Phage PhageSample re- Sample re-

moved moved

Washed once 93.2 Washed once 98.9Washed once + 96.9EDTA

Lysozyme sphero- 68.2 Lysozyme 84.4plasts spheroplasts

EDTA-lysozyme 18.0 EDTA-lyso- 34.2spheroplasts zyme sphero-

plasts

Reaction carried out for 3 min.b Reaction carried out for 10 min.

plasts, dilution is necessary to obtain sphericalforms. Thus, it seems likely that an abrupt osmoticimbalance must be introduced before the rod canbe converted into a sphere.Treatment of lysozyme spheroplasts with

EDTA weakens the outer membrane, permittingrupture of this membrane by the differential be-tween internal and external pressure. The brokenmembrane coils upon itself and exposes thecytoplasmic membrane to the environment.Dilution of lysozyme spheroplast suspensionsbelow a critical level could permit the pressuredifferential to induce the rupture of the outermembrane in the absence of EDTA. The resultingcells after either treatment have a spherical profilein which the outer membrane remains attachedin a highly coiled configuration. Hofschneider(10) observed a single membrane limiting someof the cells in a suspension of E. coli treated withEDTA and lysozyme. He suggested that this wasthe cytoplasmic membrane. Our results sub-stantiate his findings. Brenner et al. (3) suggestedthe restriction of the term "protoplast" to thosecells in which there is good reason to believe thataU cell wal components are absent. It must berecognized that our knowledge of what comprisesthe ceUl wall is limited. If the waU includes boththe outer membrane and the rigid layer, then theEDTA-lysozyme treated cells described in thiswork are not protoplasts. On the other hand, ifthe wall refers only to the rigid layer, such cellsare indeed protoplasts. In any case, our resultsindicate that the rupture of the outer membraneexposes large areas of the cytoplasmic membraneto the environment, and thus EDTA-lysozymetreated cells are truly "functional" protoplasts.The fact that the cytoplasmic membrane ofEDTA-lysozyme spheroplasts is extensively ex-

EDTA

sucrose lysozyme dilute 1:1

washed cells

osmotic osmoticX ShOC k oso c shoc

0 shock~~~soc

GHOSTS" "GHOSTS"FIG. 12. Schematic representation of spheroplast formation and osmotic sensitivity of Escherichia coli B.

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SPHEROPLASTS OF E. COLI

posed makes it less difficult to understand theutility of such spheroplasts as acceptors of freeviral nucleic acids.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService fellowship 5-F1-GM-25, 917-02 from theNational Institute of General Medical Sciences andby Public Health Service grant A104829-03 from theNational Institute of Allergy and Infectious Diseases.

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3. BRENNER, S., F. A. DARK, P. GERHARDT, M. H.JEYNES, 0. KANDLER, E. KELLENBERGER, E.KLINEBERGER-NOBEL, K. MCQUILLEN, M.RUBIO-HUERTOS, M. R. J. SALTON, R. E.STRANGE, J. ToMcsIK, AND C. WEIBULL. 1958.Bacterial protoplasts. Nature 181:1713-1714.

4. CARSON, K. J., AND R. G. EAGON. 1966. Lysozymesensitivity of the cell wall of Pseudomonasaeruginosa: further evidence for the role of thenonpeptidoglycan components in cell wallrigidity. Can. J. Microbiol. 12:105-108.

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7. DRESDEN, M., AND M. B. HOAGLAND. 1965. Poly-ribosomes from Escherichia coli: enzymaticmethod for isolation. Science 149:647-649.

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17. MEYER, F., R. P. MACKAL, M. TAO, AND E. A.EVANS, JR. 1961. Infectious deoxyribonucleicacid from bacteriophage. J. Biol. Chem. 236:1141-1143.

18. MuRRAY, R. G. E., P. STEED, AND H. E. ELSON.1965. The location of the mucopeptide in sec-tions of cell wall of Escherichia coli and othergram-negative bacteria. Can. J. Microbiol. 11:547-560.

19. NEU, H. C., AND L. A. HEPPEL. 1964. On the sur-face location of enzymes in E. coli. Biochem.Biophys. Res. Commun. 17:215-219.

20. NEU, H. C., AND L. A. HEPPEL. 1964. The releaseof ribonuclease into the medium when Esche-richia coli cells are converted to spheroplasts.J. Biol. Chem. 239:3893-3900.

21. NEU, H. C., AND L. A. HEPPEL. 1965. The releaseof enzymes from Escherichia coli by osmoticshock and during the formation of sphero-plasts. J. Biol. Chem. 240:3685-3692.

22. NOLLER, E. C., AND S. E. HARTSELL. 1961. Bac-teriolysis of Enterobacteriaceae. I. Lysis by fourlytic systems utilizing lysozyme. J. Bacteriol.81:482-491.

23. PROTASS, J. J., AND D. KORN. 1966. Impairmentof temperate bacteriophage adsorption by brieftreatment of Escherichia coli with dilute solu-tions of ethylenediaminetetraacetate. J. Bac-teriol. 91:143-147.

24. RYTER, A., AND F. JACOB. 1966. E1tude morpho-logique de la liaison du noyau 'a la membranechez E. coli et chez les protoplasts de B. subtilis.Ann. Inst. Pasteur 110:801-812.

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