JOURNAL OF BACTERIOLOGY, OCt 19711, p. 553-563Copyright 0 1971 American Society for Microbiology

Vol. 108, No. IPrinted in U.S.A.

Effect of Ethylenediaminetetraacetic Acid, TritonX-100, and Lysozyme on the Morphology andChemical Composition of Isolated Cell Walls of

Escherichia coliCARL A. SCHNAITMAN

Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22901

Received for publication 29 June 1971

Extraction of a partially purified preparation of cell walls from Escherichia coliwith the nonionic detergent Triton X-l00 removed all cytoplasmic membrane con-

tamination but did not affect the normal morphology of the cell wall. This Triton-treated preparation, termed the "Triton-insoluble cell wall," contained all of theprotein of the cell wall but only about half of the lipopolysaccharide and one-thirdof the phospholipid of the cell wall. This Triton-insoluble cell wall preparation was

used as a starting material in an investigation of several further treatments. Reex-traction of the Triton-insoluble cell wall with either Triton X-100 or ethylenedi-aminetetraacetic acid (EDTA) caused no further solubilization of protein. How-ever, when the Triton-insoluble cell wall was extracted with a combination ofTriton X-100 and EDTA, about half of the protein and all of the remaining lipopoly-saccharide and phospholipid were solubilized. The material which remained insol-uble after this combined Triton and EDTA extraction still retained some of themorphological features of the intact cell wall. Treatment of the Triton-insolublecell wall with lysozyme resulted in a destruction of the peptidoglycan layer as seenin the electron microscope and in a release of diaminopimelic acid from the cellwall but did not solubilize any cell wall protein. Extraction of this lysozyme-treatedpreparation with a combination of Triton X-100 and EDTA again solubilizedabout half of the cell wall protein but resulted in a drastic change in the mor-phology of the Triton-EDTA-insoluble material. After this treatment, the insolublematerial formed lamellar structures. These results are interpreted in terms of thetypes of noncovalent bonds involved in maintaining the organized structure of thecell wall and suggest that the main forces involved are hydrophobic protein-proteininteractions between the cell wall proteins and to a lesser degree a stabilization ofprotein-protein and protein-lipopolysaccharide interactions by divalent cations. Amodel for the structure of the E. coli cell wall is presented.

Cell walls of Escherichia coli and other gram-negative bacteria are complex both in mor-phology and in chemical composition. When sec-tioned preparations of E. coli cells or cell wallsare examined by electron microscopy, three dis-tinct regions or structures can be identified. Theoutermost region, that of the lipopolysaccharide(LPS) carbohydrate chains, cannot be revealedby normal staining techniques. Staining with fer-ritin antibody has shown the LPS to be on theouter surface, and that 0 antigen-specific carbo-hydrate extends quite far out from the surface ofthe cell (19). Beneath this LPS coat is a mem-brane bilayer, identical in appearance to the cy-toplasmic membrane (16). This structure is re-

ferred to as the outer membrane, or, because ofits involvement with LPS, as the LPS membraneor L membrane (4). A granular layer identifiedas peptidoglycan (murein) is tightly attached tothe inner surface of the outer membrane (16).The cell wall may be separated from the cyto-plasmic membrane by centrifugation of brokenenvelope preparations in sucrose gradients, andthe isolated cell wall has the same structure asobserved in intact cells (16).The peptidoglycan layer is the best understood

of these three regions, largely because it can bereadily isolated and studied. Weidel and Peltzer(20) isolated this structure by extracting cellwalls with hot sodium dodecyl sulfate and

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SCHNAITMAN

showed that it was a continuous "bag" studdedwith granules of protein and formed in the shapeof the intact cell. The protein which is attachedto the peptidoglycan has now been extensivelystudied by Braun and his associates (1, 2). Thisprotein has a molecular weight of 7,000 daltons,has an unusual amino acid composition, containscovalently bound lipid, and is itself covalentlylinked to diaminopimelic acid residues on thepeptidoglycan. Gross morphological changes areobserved in intact envelopes when this protein iscleaved from the peptidoglycan by trypsin, indi-cating that this protein plays an important rolein the maintenance of the organized structure ofthe cell wall.The organization of the outer membrane and

the attached LPS is less obvious. Recent studieson the chemical composition of the cell wall sug-gest that the outer membrane is a true mem-brane in that it contains phospholipid and pro-tein in a ratio similar to that observed in otherbiological membranes (16). The protein compo-sition is somewhat unique in that a protein witha molecular weight of about 44,000 daltons ac-counts for up to 70% of the total protein of thecell wall (16, 18). Both the cell wall from normalcells (18) and the outer membrane isolated fromspheroplasts (4) are quite resistant to dissolutionby the nonionic detergent Triton X-100. Part ofthis resistance to detergent lysis must be due to astabilizing effect of divalent cations, since De-Pamphilis (3) has recently shown that treatmentof the outer membrane isolated from sphero-plasts with a combination of Triton X-100 andethylenediaminetetraacetic acid (EDTA) leads tocomplete dissolution of this membrane, and thatit can be reconstituted by dialysis against Mg2+-containing buffer. EDTA has previously beenshown to have a number of effects on E. coli cellwalls. Treatment of intact cells with EDTA al-lows lysozyme to attack the peptidoglycan layer(11), results in a partial release of LPS from thecell (9), and promotes the release of periplasmicproteins by osmotic shock (7). All of these find-ings suggest a role for divalent cations in main-taining the integrity of the outer membrane andin the attachment of LPS to the outer mem-brane.

In this study, the effects of several mild treat-ments on isolated cell walls are examined. Thisstudy was undertaken for three reasons: first, toattempt to provide more information about thelocalization and organization of proteins in thecell wall; second, to obtain some informationabout the types of noncovalent bonds involved inmaintaining the organized structure of the cellwall; and third, to provide some means for frac-tionating cell wall proteins.

MATERIALS AND METHODSBacteria and culture conditions. E. coli J-5 was used

for all experiments except as noted. This organism wasgrown on minimal medium with glucose as a carbonsource as previously described (15). The E. coli K-12derivative AT1047 was obtained from A. L. Taylor,The University of Colorado Medical Center, Denver.This strain requires proline, thiamine, lysine, and dia-minopimelic acid, and was grown on minimal mediumas above, with 50 gg of proline, 20 gg of lysine, 50 ugof DL-diaminopimelic acid, and I ALg of thiamine per mlof medium. When labeling of the diaminopimelic acidwas required, 0.1 ,Ci of 3H-labeled 1-diaminopimelicacid was added per ml of medium. Late log-phase cul-tures were used in all experiments.

Preparation and extraction of envelope. Envelope wasobtained by breakage with a French pressure cell, andthe cell wall-enriched fraction was obtained by centrifu-gation on discontinuous sucrose gradients (16). Thecell wall-enriched fraction from the gradient was ex-tracted with 2% Triton X-100 for 10 min at 23 C fol-lowed by ultracentrifugation (18). The pellet after thisextraction is referred to as "Triton-insoluble wall."This was further extracted for 10 min at 23 C with 2%Triton X-100 in 10 mm N-2-hydroxyethylpiperazine-N'-2'-ethane sulfonic acid (HEPES) buffer, pH 7.4,containing 5 mM EDTA adjusted to pH 7.4 withNaOH. After this extraction, the suspension waschilled and centrifuged for I hr in a Spinco 5OTi rotorat 50,000 rev/min. The pellet from this suspension isdesignated as the "Triton-EDTA insoluble wall" andthe supernatant fraction is designated as the "'Triton-EDTA supernatant."

Other methods. Electron microscopy was carried outas previously described on samples fixed in microcen-trifuge tubes first with glutaraldehyde and then withOsO4 (16). Preparations were block-stained after theosmium fixation with uranyl acetate in Veronal acetatebuffer as described by Palade and Bruns (10). Sectionswere then post-stained with uranyl acetate and lead cit-rate (16).

Gel electrophoresis was carried out as previouslydescribed (14, 15) after removal of lipid and Triton X-100 by gel filtration on Sephadex LH-20 in acidifieddimethyl formamide (18).

Reagents. Lysozyme and Triton X-100 were ob-tained from Calbiochem, Los Angeles, Calif. Uni-formly 3H-labeled 1-diaminopimelic acid (specific ac-tivity 0.2 Ci/mmole) was obtained from Amersham-Searle, Inc., Des Plains, Ill.

RESULTSEffect of lysozyme on the Triton-insoluble wall.

It is likely that the protein associated with thecell wall is primarily localized in the outer mem-brane rather than in association with the peptido-glycan layer (16). This would be true even forthe peptidoglycan-bound protein described byBraun et al. (1, 2) if this protein is in fact a "ce-ment" holding the peptidoglycan to the outermembrane. To test this, the Triton-insoluble wallfraction was treated with lysozyme to see

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whether protein was released when the peptido-glycan layer was broken down. Table I showsthe effect of lysozyme on Triton-insoluble wallisolated from strain J-5. Neither lysozyme nor

EDTA, nor a combination of these, resulted inany substantial release of protein. To demon-strate that lysozyme was active under these con-

ditions, the experiment was repeated with strainAT1047 in which the peptidoglycan was labeledwith 3H-diaminopimelic acid. There was little or

no release of the 3H label in either the firstTriton extraction or in the incubated control(Table 2), showing that autolysis of the peptido-glycan during the incubation periods at 23 C was

minimal. When the Triton-insoluble wall was

incubated with lysozyme, more than 60% of the3H label was released. Figure 1 shows the effectof lysozyme and EDTA treatment on the mor-

phology of the Triton-insoluble wall. Lysozymetreatment resulted in the disappearance of thepeptidoglycan as a discrete layer, and the innersurface of the outer membrane took on a raggedor "sawtooth" appearance (arrow, Fig. ID).The most likely interpretation is that the"teeth" on the inner surface of the outer mem-

brane represent fragments of peptidoglycanwhich are still attached to the inner membraneby the peptidoglycan-bound protein. This appear-

ance is probably accentuated by the presence oflysozyme bound to these fragments, as examina-tion of these preparations by gel electrophoresishas indicated that they contain a substantialamount of tightly bound lysozyme. EDTA hadlittle effect on the morphology, other than to in-crease the tendency of fragments of outer mem-

brane to roll up to form multilayered structuresas seen in Fig. I D.

In a previous study (15) it was noted that 10to 15% of the protein of the envelope failed todissolve in acidified dimethyl formamide. Be-cause high-molecular-weight carbohydrates such

555

as the peptidoglycan are insoluble in this organicsolvent, it is probable that some of this solvent-insoluble protein represented the lipoproteinwhich is covalently bound to the peptidoglycan.Braun et al. (1, 2) have indicated that this pro-tein accounts for about 10% of the total envelopeprotein. Breakdown of the peptidoglycan by lyso-zyme should permit this protein to dissolve in thesolvent. To test this, the solubility of the Triton-insoluble cell wall protein in acidified dimethylformamide was examined before and after lyso-zyme treatment. Before lysozyme treatment,about 15% of the cell wall protein was insolublein acidified dimethyl formamide (Table 3), butthis solvent-insoluble protein was reduced almostto zero by lysozyme treatment.The size of the peptidoglycan-bound protein

TABLE 1. Release of 3H-labeled protein from Triton-insoluble wall of strain J5a

Total Super- Re-Treatment broteI natant leasedpoen fluid" (%

Incubated control 6,840 92 1.3Plus 5 mm EDTA 7,420 24 0.3Plus lysozyme (0.2 mg/ml) 7,184 64 0.9Lysozyme plus EDTA 7,212 48 0.7

a Strain J-5 was grown on glucose minimal mediumcontaining 3H-leucine and 3H-tyrosine as previouslydescribed (15), and the Triton-insoluble cell wall wasobtained by extraction with 2% Triton X-100 for 10min at 23 C (18). The Triton-insoluble wall was incu-bated at a protein concentration of approximately 2mg/ml for 20 min at 23 C in 10 mm HEPES buffer(pH 7.4) containing lysozyme and EDTA as indicated.The suspension was then chilled and centrifuged at144,000 x g for I hr. Portions were removed forcounting before centrifugation and from the superna-tant after centrifugation to calculate the percentage oflabel released.

b Expressed as 3H counts per minute per milliliter.

TABLE 2. Release of "4C-labeled protein and 3H-labeled DAP from the wall-enriched fraction and Triton-insoluble wall of strain A T104 7a

Protein (14C counts per min per ml) DAP (3H counts per min per ml)

Treatment Super- Re- Super- Re-Total natant leased Total natant leased

fluid (%) fluid (%)

Triton extraction of wall-enriched 13,660 2,194 16.1 14,892 764 5.1fraction

Triton-insoluble wallIncubated control 5,120 94 1.8 6,254 152 2.5Plus lysozyme (0.2 mg/ml) 5,034 40 0.8 6,996 4,312 61.9

a Strain AT1047 was grown on glucose minimal medium containing 3H-labeled diaminopimelic acid (DAP) and' 4C-tyrosine. Triton extraction of the wall-enriched fraction was carried out as previously described (18). TheTriton-insoluble wall was incubated, centrifuged, and sampled as above.

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Mi~~~~~~~Y

4 KA:

A7

FIG. 1. Effect of lysozyme and EDTA on Triton-insoluble cell wall. Samples of Triton-insoluble wall weretreated as in Tables I and 2. A, Incubated control. B, Treated with EDTA only. Note that morphology is essen-tially identical to the incubated control. C, Treated with lysozyme only. D, Treated with lysozyme plus EDTA.Arrow indicates the characteristic "sawtooth" appearance ofpeptidoglycan layer after lysozyme treatment. Barindicates 0.1 gm.

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TABLE 3. Solubility of cell wall protein in DMFplus HCI after lysozyme treatmenta

Total DMF- In-Treatment pro- HCI in- soluble

teinb soluble" (%)

Incubated control 35.3 5.6 15.9Plus 5 mm EDTA 30.8 5.7 18.5Plus lysozyme (0.2 mg/ml) 35.5 0.3 0.8

aTriton-insoluble wall was isolated from a culturegrown on 3H-leucine and 3H-tyrosine, and identicalportions were incubated with buffer alone, lysozyme,and EDTA as described in Table 1. After incubation,the suspensions were centrifuged at 144,000 x g for 1hr, and the pellets were dissolved in a solvent con-taining nine parts of dimethyl formamide (DMF) andone part of 0.12 N HCI. The final protein content ofthe solvent was approximately 2 mg/ml. This solutionwas allowed to stand for 30 min at 0 C and was thencentrifuged at 20,000 x g for 15 min to sediment anyinsoluble protein. The insoluble pellet was suspended inwater and counted.

b Expressed as counts per minute x 103.

after lysozyme treatment was examined by com-paring the protein profiles of Triton-insolublecell wall from control and lysozyme-treatedsamples on gel electrophoresis (Fig. 2). Thisexperiment was done by preparing two identicalpreparations of Triton-insoluble wall, one labeledwith "4C-amino acids and the other with 3H-amino acids. The 3H-labeled cell wall was incu-bated with lysozyme, and the "4C-labeled cellwall was incubated without lysozyme. The twopreparations were dissolved in acidified dimethylformamide, mixed, and prepared for gel electro-phoresis. Any peaks labeled with 3H but not with14C should represent peptidoglycan-bound pro-tein which is now soluble in the solvent as a con-sequence of lysozyme treatment. A number ofnew small peaks appeared (Fig. 2), indicatingthat lysozyme had produced irregular breaks inthe peptidoglycan resulting in a mixture of var-ious sized fragments to which one or more of the7,000-molecular-weight polypeptides was at-tached. This is in accord with the results ofBraun and Sieglin (2) who found with isolatedpeptidoglycan preparations that lysozymecleavage was not complete. The fastest movingpeak seen in Fig. 2 probably represents the mon-omeric polypeptide attached to a minimum-sizedpeptidoglycan fragment. Peaks with this high amobility are not normally observed in prepara-tions of cell wall which have not been treatedwith lysozyme, indicating that little of this pro-tein is free in the cell wall.

Reextraction of the cell wall with Triton X-100 plus EDTA. The results shown in Table Iindicated that EDTA by itself did not release any

protein from the cell wall. Reextraction withTriton X-100 under the conditions used to solu-bilize the cytoplasmic membrane resulted in nofurther release of protein. However, reextractionwith Triton X-100 in the presence of EDTA re-sulted in a substantial solubilization of cell wallprotein (Table 4). This is similar to the observa-tion of DePamphilis (3), who observed that theouter membrane of spheroplasts was disaggre-gated by a combination of Triton X-100 plusEDTA. The amount of protein solubilized wassomewhat variable, ranging from 35 to 50% ofthe total cell wall protein. The solubilization ismuch less effective at lower temperatures. Re-peating the extraction with Triton X-100 or withEDTA, or with a combination of these, resultedin no further solubilization of protein.Triton extraction of cell wall-enriched frac-

tions in the absence of EDTA was previouslyshown to solubilize only proteins of the cyto-plasmic membrane (18). The electropherogramsshown in Fig. 3 show that reextraction withTriton plus EDTA solubilized proteins which arecharacteristic of the cell wall. The major struc-tural protein of the cell wall (peak 22) appearsto be present in both the Triton-EDTA-solubleand the Triton-EDTA-insoluble fractions. How-ever, there is some specificity in solubilization ofother proteins. Proteins 11 and 16 are enrichedin the Triton-EDTA-soluble fraction, whereas

400 -

2 320 -Cl)-

, 240 -0

0 08160 -

so -

0-

X 0.~~~11.

_ ,_,:1** 560

- 320

240 ,_z

0160 °

80

0

0 10 20 30 40 50 60FRACTION NUMBER

FIG. 2. Effect of lysozyme on the electrophoreticprofile of Triton-insoluble cell wall protein. Solid lineindicates the 3H-protein profile of a sample of Triton-insoluble wall treated with lysozyme as described inTable 3. Dashed line is the 14C-labeled protein profileofan identical preparation incubated without lysozyme.The arrows indicate protein peaks observed only afterlysozyme treatment. The total amount of radioactiveprotein in the areas under the peaks represented about12% of the total 3H-labeled protein. The samples weremixed after dissolving in acidified dimethylformamide.

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558 SCHNAITMAN J. BACTERIOL.

TABLE 4. Solubilization ofprotein and lipopolysaccharide (LPS) by Triton and Triton plus EDTAa

Protein (3H-amino acids) LPS ('4C-galactose)

Extraction ~~~Supematant Solubilized Supematant SolubilizedExtraction Toa uelaat| (o otal fluid | (%S)Total fluid (%)al fui

First Triton extraction 1.36 0.29 21.3 6.02 2.51 41.7Buffer wash 0.780 0.017 2.2 1.95 0.041 0.7

Second extractionIncubated control 0.947 0.015 1.6 2.28 0.009 0.4EDTA only 0.829 0.016 1.9 1.97 0.018 0.9Triton only 0.892 0.044 4.9 2.11 0.281 18.1Triton + EDTA 1.014 0.325 32.1 2.37 2.05 86.5

a Results are expressed as counts per min per ml x 103. Strain J-5 was grown on succinate-minimal mediumcontaining '4C-galactose and 3H-tyrosine (16). The inoculum was induced for galactose incorporation by growthovernight on minimal medium containing 2 mM L-fucose. The wall-enriched fraction (16) was incubated at a pro-tein concentration of approximately 2 mg/ml for 10 min at 23 C in a solution containing 2% Triton X-100 and 10mM HEPES buffer (pH 7.4) (18). The Triton-insoluble wall was then washed once with the same buffer. The solu-bilization of protein in these steps is given in the upper portion of the table. The Triton-insoluble wall was sus-pended at a concentration of 2 mg of protein/ml and extracted again for 10 min at 23 C in the same buffer con-taining 5 mM EDTA and 2% Triton X-100 as indicated. Centrifugation and sampling are the same as in Tables 1and 2.

1200 proteins 5 and 37 are found almost entirely inA. the Triton-EDTA-insoluble fraction. Further

evidence of the specificity of Triton-EDTA ex-900 traction is indicated by the recent observation

that the receptors for colicins E3 and K arefound primarily in the Triton-EDTA-soluble

600 5 37 fraction obtained from E. coli C-600.The morphology of the Triton-EDTA-insol-

uble fraction is shown in Fig. 4. This fraction300 3' still retains some of the morphological characters

27 of the cell wall. Some of the outer membrane isstill visible, although there are gaps appearing in

a' oJ:the outer membrane as if blocks of material were

900 22 removed from it (arrows, Fig. 4B and 4C). In0

A B. other cases, the fragments have a "twisted rib-z bon" morphology, as seen between the arrows ino 1 Fig. 4A. The intact peptidoglycan layer appears600 to be holding the fragments together. When the

Triton-insoluble wall was incubated with lyso-zyme and then extracted with Triton plus EDTA,

300 a totally different morphology was observed8-11 16 27 31 7 (Fig. 5). Instead of long strips, the material is

- , ,. 1 present in lamellar stacks, suggesting that some0 ,_,__,_,_,_,_,_,_,_, form of disruption and reaggregation has oc-

0 20 40 60 80 100 curred. The amount of protein which was insol-FRACTION NUMBER uble in Triton-EDTA was roughly the same

FIG. 3. Comparison of the Triton-EDTA superna- whether lysozyme pretreatment was included ortant fluid and Triton-EDTA-insoluble fractions ob- not. With the preparation shown in Fig. 5, 50%tained from the Triton-insoluble cell wall. Fractiona- of the cell wall protein was found in the Triton-tion was carried out as indicated in Table 4. Dashedline indicates the 'IC-labeled protein profile of unfrac-tionated Triton-insoluble wall. Solid line in A indicates fled dimethyl formamide. The gels are slightly over-the 3H-protein profile of the Triton-EDTA-insoluble loaded to improve the resolution of minor peaks, re-fraction; solid line in B indicates the 3H-protein profile sulting in a broadening and splitting of peak 22 (15).of the Triton-EDTA supernatant. The 3H- and '4C-la- The protein peaks are numbered as previously de-beled samples were mixed and then dissolved in acidi- scribed (18).

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. Iv.

46~~~FIG. 4. Triton-EDTA -insoluble cell wall fraction. This preparation was isolated as described in Materials and

Methods and Table 4. The regions between the arrows in B and C show apparent gaps in the outer membrane.The bar indicates 0.1 gim.

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.mNoW1 - _ 4wa

I k.

FIG. 5. Triton-EDTA insoluble fraction prepared from a sample of Triton-insoluble wall which was treatedwith lysozyme as described in Tables I and 2 prior to Triton-EDTA extraction. The arrow indicates one of thelamellar regions. Bar indicates 0.1 Am.

EDTA-insoluble pellet after lysozyme treatment.This indicated that the insolubility of cell wallprotein in Triton plus EDTA was not due pri-marily to the presence of an intact peptidoglycanlayer per se but to some form of interaction be-tween the proteins themselves.

Extraction of lipid and LPS by Triton andTriton-EDTA. The cell wall contains lipid bothin the form of phospholipid (16) and the lipid Acomponent of LPS. Both of these componentsare extracted by Triton X-100 and Triton plusEDTA. Table 5 shows the phospholipid contentof the cell wall-enriched fraction of the envelopeand the Triton-insoluble and Triton-EDTA-in-soluble preparations obtained from this fraction.About 80% of the lipid was removed by the firstTriton extraction. Some of this lipid is accountedfor by cytoplasmic membrane contamination ofthe cell wall-enriched fraction. In the experimentshown in Table 5, this contamination was unu-sually high, as indicated by the amount of pro-tein removed by the Triton extraction. Neverthe-less, the amount of phospholipid present in thiscytoplasmic membrane contamination can be es-timated based on the phospholipid content of themembrane-enriched fraction reported previously(16), and this would account for less than half ofthe phospholipid removed by Triton extraction ofthe cell wall fraction. Thus, about two-thirds ofthe phospholipid of the cell wall was also re-

moved by Triton extraction. This had little effecton the morphology of the outer membrane,which is identical in the intact cell wall and inthe Triton-insoluble wall (Fig. 1). This is analo-gous to the observations of Fleischer et al. on theinner mitochondrial membrane (5), where themorphology of the membrane was preservedafter removal of much of the lipid.The remaining lipid present in the Triton-in-

soluble wall was removed by Triton-EDTA ex-traction. Hence the remnants of outer membraneobserved in the Triton-EDTA-insoluble fraction(Fig. 4) and the lamellar appearance shown inFig. 5 cannot be due to the presence of phospho-lipid-protein bilayers. It is possible, however,that Triton has replaced the phospholipid inthese structures.A similar situation is seen with LPS. As indi-

cated in Table 4, about 50% of the LPS is re-moved by the first Triton extraction with no al-teration in morphology. This is similar to theobservations of Levy and Leive (9), who re-ported that removal of up to half of the LPSfrom intact cells with EDTA had no effect on themorphology of the cell wall. The remainder ofthe LPS is also removed by the Triton-EDTAextraction. Thus, LPS is not replacing phospho-lipid in the bilayer or lameller structures seenafter Triton-EDTA extraction. It is tempting tospeculate that the gaps visible in the outer mem-

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TABLE 5. Removal ofphospholipids by Tritona

Amt of protein Amt of phospholipid Phospholipid/proteinSample Phosmolesl/mg)el

mg % jmoles %(mo1es/mg)

Crude wall 11.5 100 5.52 100 0.48Triton-insoluble wall 7.35 63.9 1.15 20.8 0.156Triton-EDTA-insoluble wall 3.72 32.3 0.134 2.4 0.036

a Samples were prepared as described in Matefials and Methods and Table 4. Protein and lipid phosphate weredetermined as previously described (16).

brane as seen in Fig. 4B and 4C are due to re-

moval of LPS-lipid-protein complexes from theouter membrane. Soluble LPS-lipid-proteincomplexes are released from intact cells of E.coli and Salmonella under some conditions ofinhibited growth (8, 12).

DISCUSSIONA combination of the results described above

with the recent results of DePamphilis (3, 4) andBraun et al. (1, 2) allows some general conclu-sions to be drawn about the organization of theE. coli cell wall and the types of interactionswhich serve to maintain the integrity of thisstructure. These are outlined below.

Role of the peptidoglycan in cell wall organiza-tion. The outer membrane appears to be attachedquite firmly to the peptidoglycan layer. In no

case was any separation of the peptidoglycanfrom the outer membrane (or the remains of theouter membrane after Triton-EDTA extraction)observed in this study. Digestion of the backboneof the peptidoglycan with lysozyme does not re-

sult in the release of any protein from the re-

mainder of the cell wall. Complete separation ofnoncovalently linked cell wall protein from thepeptidoglycan layer is accomplished only byrather severe treatment, for example the extrac-tion with hot sodium dodecyl sulfate solutionemployed by Braun et al. (1) or the solubiliza-tion in acidified dimethyl formamide which wehave employed in preparing cell wall proteins forgel electrophoresis. At the same time, cleavageof the covalently bound lipoprotein from the pep-tidoglycan with trypsin resulted in gross altera-tion in the properties of the cell envelope (1).This indicates very strongly that the lipoproteindescribed by Braun et al. (1, 2) is responsible forthe attachment of the peptidoglycan to the outermembrane, presumably by very strong hydro-phobic protein-protein interactions. Thus thepeptidoglycan layer appears to be the skeleton on

which the remainder of the cell wall is con-

structed.Cell wall phospholipid. DePamphilis and Adler

reported that the outer membrane isolated fromspheroplasts by Triton extraction contained pro-

tein and LPS but little phospholipid (4). De-Pamphilis suggested that the Triton extractionmay have removed phospholipid (3). The data inTable 5 indicate that this is probably correct.Recent studies in this laboratory on cell wallpreparations purified without detergents indicatethat the cell wall is the site of about half of thephospholipid of the cell. The limited effect of theremoval of phospholipid on the morphology andprotein composition indicates that the phospho-lipid plays only a minor role in the structuralstability of the cell wall. However, this lipid mayhave an important role in determining thepermeability of the cell wall, or it may provide anecessary lipid phase for cell wall biosyntheticreactions of the type described by Rothfield andRomeo (13). It may also provide the anchoringpoint for lipid A.

Attachment of LPS and the role of divalent cat-ions. The requirement for EDTA in the forma-tion of spheroplasts by lysozyme (11) and in therelease of periplasmic proteins by osmotic shock(7) has indicated that divalent cations play a rolein maintaining the impermeability of the cell wallto large molecules. The partial release of LPSfrom intact cells by treatment with EDTA (9)points to an involvement of divalent cations instabilizing the attachment of LPS to the cellwall. In conjunction with Triton, EDTA pro-moted the solubilization of a considerableamount of protein, as well as the LPS and phos-pholipid of the Triton-insoluble wall fraction.Using somewhat different conditions and a dif-ferent starting material, DePamphilis (3) wasable to achieve complete dissolution of the outermembrane isolated from spheroplasts. This couldbe reversed by dialysis against Mg2+-containingbuffer. All of these phenomena indicate t'iat di-valent cations stabilize the cell wall, perh2aps byforming ionic bridges between the phosphategroups in the LPS core and charged groups onadjacent proteins or phospholipids. However,EDTA does not cause any release of proteinfrom the cell wall even after disruption of thepeptidoglycan layer (Table 1), nor does it . -leaseLPS from the Triton-insoluble wall (Table 5). Itis best to consider the effect of EDTA as a weak-

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ening of the cell wall to a point where it is sus-

ceptible to mechanical damage or disaggregationof the LPS and protein components by deter-gents.

Hydrophobic protein-protein interaction. Sev-eral lines of evidence suggest that the majorforce holding the cell wall together is a hydro-phobic interaction between the various proteincomponents. This type of interaction is best illus-trated by the appearance of the Triton-EDTA-insoluble protein shown in Fig. 5. Even in theabsence of native lipid and LPS and in the pres-ence of Triton X-100, the cell wall protein showsa tendency to aggregate into regular lamellarstructures. Protein isolated from E. coli cell wallsshows a very strong tendency towards aggrega-tion. Dissolution of the cell wall in an organicsolvent followed by removal of lipid and dialysisinto sodium dodecyl sulfate-urea solutions hasbeen the only method which in our hands hasprovided satisfactory resolution on gel electro-phoresis. High concentrations of urea or guani-dine hydrochloride or extremes of pH were noteffective in preventing aggregation of either na-

tive or lipid-free preparations of cell wall pro-teins. The failure of these techniques attests tothe strongly hydrophobic nature of the proteininteractions.

Overall organization of the E. coli envelope.The model shown in Fig. 6 is an attempt to sum

up the present knowledge concerning the cellwall of E. coli. The lipoprotein cytoplasmicmembrane contains about half of the phospho-lipid of the envelope and is readily dissociable byTriton X-100 (18). It has a complex proteincomposition typical of other biological mem-

branes (14). This is separated from the inner-most layer of the cell wall by an electron-trans-parent region with a mininum dimension ofabout 10 nm, referred to in Fig. 6 as the peri-plasmic gap. The innermost layer of the cell wallis the peptidoglycan layer, which is visible byelectron microscopy as a finely granular layerand which is presumably a continuous mesh (20),one molecule thick (6). This is shown attachedto the outer membrane by the covalently boundlipoprotein described by Braun et al. (1, 2). Thebond between the subterminal arginine and theterminal lysine residue which is linked to diami-nopimelic acid residues on the peptidoglycan isvery sensitive to trypsin (2). This lipoprotein can

be visualized as extending into the phospholipidlayer of the outer membrane. The outer mem-

brane has a much simpler protein compositionand is characterized by the presence of a singlemajor structural protein with a molecular weightof about 44,000 daltons (16, 18). Similar majorprotein species are observed in envelopes of other

Trypsin cxProtein

..-PeptidoglycanLysozyme ) Periplasmic

Gap"

Triton10

-Phospholipid..-- ~ ----Protein

Cytoplasm

FIG. 6. Simplified model of the cell envelope of E.coli. Various components of the envelope are listed on

the right; probable sites of attack of various disruptiveagents are indicated on the left.

gram-negative species (17). The LPS is indicatedto be on the outer surface of the outer membranewith the lipid A component inserted into thephospholipid layer and the 0-specific side chainsextending out into the external medium. This is

consistent with the observation that LPS is ca-

pable of penetrating synthetic phospholipid mon-

olayers (13). The primary site of action ofEDTA (hence the site of divalent cation stabili-zation) is envisioned to be at the site of insertionof the LPS into the outer membrane. In thissimplified model, flagella and pili have beenomitted. DePamphilis and Adler (4) presentedevidence that the "L" ring of the flagellar basalbody is inserted into the outer membrane of thecell wall. In this model, both the cytoplasmicmembrane and the outer membrane are illus-trated as simple lipoprotein bilayers, without any

attempt to show penetration of the various pro-

teins into the phospholipid layers. Such penetra-tion of protein into the lipid bilayer may welloccur, but the actual organization of lipid andprotein in these membranes is one of many prob-lems which remain to be solved.

ACKNOWLEDGMENTS

This investigation was supported by Public Health ServiceResearch Career Development Award GM22053 from the Na-tional Institute of General Medical Sciences and bv researchgrant GB-8142 from the National Science Foundation.

I acknowledge the skilled assistance of Judy Colby andDiane Gambardella.

LITERATURE CITED

1. Braun, V., and K. Rehn. 1969. Chemical characterization.spatial distribution and function of a lipoprotein (mu-rein-lipoprotein) and the E. coli cell wall. The specificeffect of trypsin on the membrane structure. Eur. J.Biochem. 10:426-438.

2. Braun, V., and U. Sieglin. 1970. The covalent murein-lip-oprotein structure of the Escherichia coli cell wall. Theattachment site of the lipoprotein on the murein. Eur. J.Biochem. 14:387-391.

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PARTIAL SOLUBILIZATION OF E. COLI CELL WALL

3. DePamphilis, M. L. 1971. Dissociation and reassembly ofEscherichia coli outer membrane and of lipopolysac-charide, and their reassembly onto flagellar basal bodies.J. Bacteriol. 105:1184-1199.

4. DePamphilis, M. L., and J. Adler. 1971. Attachment offlagellar basal bodies to the cell envelope: specific at-tachment to the outer lipopolysaccharide membrane andcytoplasmic membrane. J. Bacteriol. 105:396-407.

5. Fleischer, S., B. Fleischer, and W. Stoeckenius. 1967. Finestructure of lipid depleted mitochondria. J. Cell Biol. 32:193-208.

6. Ghuysen, J. M. 1968. Use of bacteriolytic enzymes in de-termination of wall structure and their role in cell me-

tabolism. Bacteriol. Rev. 32:425-464.7. Hepple, L. A. 1967. Selective release of enzymes from bac-

teria. Science 156:1451-1455.8. Knox, K. W., M. Vesk, and E. Work. 1966. Relation be-

tween excreted lipopolysaccharide complexes and surfacestructures of a lysine-limited culture of Escherichia coli.J. Bacteriol. 92,1206-1217.

9. Levy, S. B., and L. Leive. 1968. An equilibrium betweentwo fractions of lipopolysaccharide in Escherichia coli.Proc. Nat. Acad. Sci. U.S.A. 61:1435-1439.

10. Palade, G. E., and R. R. Bruns. 1968. Structural modula-tion of plasmalemmal vesicles. J. Cell Biol. 37:633-649.

11. Repaske, R. 1958. Lysis of gram-negative organisms andthe role of Versene. Biochim. Biophys. Acta 30:255-231.

12. Rothfield, L., and M. Perlman-Kothencz. 1969. Synthesis

563

and assembly of bacterial membrane components. A li-popolysaccharide-phospholipid-protein complex excretedby living bacteri,a. J. Mol. Biol. 44:477-492.

13. Rothfield, L., and D. Romeo. 1971. Role of lipids in thebiosynthesis of the bacterial cell envelQpe. Bacteriol.Rev. 35:14-38.

14. Schnaitman, C. 1969. Comparison of rat liver mito-chondrial and microsomal membrane proteins. Proc.Nat. Acad. Sci. U.S.A. 63:412-419.

15. Schnaitman, C. 1970. Examination of the protein composi-tion of the cell envelope of Escherichia coli by polyacryl-amide gel electrophoresis. J. Bacteriol. 104:882-889.

16. Schnaitman, C. 1970. Protein composition of the cell walland cytoplasmic membrane of Escherichia coli. J. Bac-teriol. 104:890-901.

17. Schnaitman, C. 1970. Comparison of the envelope proteincompositions of several gram-negative bacteria. J. Bac-teriol. 104:1404-1405.

18. Schnaitman, C. 1971. Solubilization of the cytoplasmicmembrane of Escherichia coli by Triton X-l00. J. Bac-teriol. 108:545-552.

19. Shands, J. W. 1965. Localization of the somatic antigen ofgram-negative bacteria by electron microscopy. J. Bac-teriol. 90:266-270.

20. Weidel, W., and H. Peltzer. 1964. Bagshaped macromole-cules-a new outlook on bacterial cell walls. Advan.Enzymol. 26:193-232

VOL. 108, 1971

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