Vol. 169, No. 6JOURNAL OF BACTERIOLOGY, June 1987, p. 2482-24870021-9193/87/062482-06$02.00/0Copyright © 1987, American Society for Microbiology

Structure of the Staphylococcus aureus Cell Wall Determined by theFreeze-Substitution Method

AKIKO UMEDA,* YUJI UEKI, AND KAZUNOBU AMAKODepartment ofBacteriology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan

Received 25 November 1986/Accepted 6 March 1987

The fine structure of the Staphylococcus aureus cell wall was determined by electron microscopy with the newtechnique of rapid freezing and substitution fixation. The surface of the cell wall was covered with a fuzzy coatwhich consisted of fine fibers or an electron-dense mass. Morphological examination of the cell wall, which wastreated sequentially with sodium dodecyl sulfate, trypsin, and trichloroacetic acid, revealed that this coat waspartially removed by trypsin digestion and was completely removed by trichloroacetic acid extraction but wasnot affected by sodium dodecyl sulfate treatment, suggesting that the fuzzy coat consists mostly of a complexof teichoic acids and proteins. This was confirmed by the application of the concanavalin A-ferritin techniquefor teichoic acid and antiferritin immunoglobulin G technique for protein A.

Full knowledge of cell wall structure is important inunderstanding bacterial physiology, the interaction with ahost in infection, and the mechanisms of antibiotic action. Inthis sense we still do not have sufficient knowledge of the cellwall of Staphylococcus spp. The cell wall of Staphylococcusaureus is composed of peptidoglycan, teichoic acids, andproteins (21). Many reports have been published on the finestructure of the cell wall of this bacterium, but little infor-mation on the arrangement of the polymers in the cell wall isavailable (3, 5, 10, 11, 19, 22). Thin-sectioned profiles simplyshow a thick electron-dense layer of the cell wall, as hasbeen seen in many gram-positive bacteria. Chemical analysisof the cell wall indicates that more than 70% of the weight ofthe cell wall is peptidoglycan and that the teichoic acid iscovalently bound to the peptidoglycan through aphosphodiester bond (9). However, how proteins interactwith these polymers is still not known. In a previous report(22) we showed that the arrangement of the polymers on thecell wall is circular and evidence that the distribution ofbacteriophage receptors on the cell wall surfaces is local-ized. These data are not sufficient, however, in helping us tounderstand the three-dimensional distribution of the poly-mers in the cell wall.

Recently, a mild fixation method, the rapid freezing andsubstitution fixation method, has been developed and ap-plied to the study of bacterial morphology (2, 12). By thismethod several new profiles of various bacterial structuressuch as the capsule (6), the nucleoid (4, 13), and the outermembrane (2, 12) have been demonstrated. In this study wedetermined the fine structure of the cell wall of S. aureus bythis technique, and a new feature of the structure of the cellwall is reported.

MATERIALS AND METHODS

Bacterial strains and culture conditions. S. aureus Cowan Iwas mostly used in this study. A clinically isolated strain,PSA-4, was used for the study of the interaction of teichoicacids and concanavalin A (ConA) because of its high reac-tivity with ConA. These strains were maintained on ordinary

* Corresponding author.

nutrient agar slants. For examination of the cell wall thestrain was cultured aerobically in PYK broth (5 g ofpolypeptone, 5 g of yeast extract, 3 g of K2HPO4, 2 g ofglucose, and 1,000 ml of distilled water [pH 7.2]) at 37°C andharvested at its log phase when the optical density (at 660nm) of the culture reached 120 Klett units.

Isolation and chemical extraction of the cell wall. Theisolation of the cell wall was carried out as describedpreviously (7). From 1 liter of a PYK broth culture of strainCowan I the bacteria were harvested by centrifugation (5,000x g, 30 min) and disintegrated with small glass beads with acell disintegrator (Dyno-Mill; Willy A. Bachofen, Maschin-enfabrik, Basel, Switzerland). After the glass beads and thenondisrupted cells were removed by centrifugation (2,000 xg, 10 min), the cell wall fraction was pelleted by centrifuga-tion at 15,000 x g for 20 min, suspended in distilled water,and kept in ice until use. This cell wall fraction was called thecrude wall.The crude wall was then treated with 2% (wt/vol) sodium

dodecyl suflate (SDS) at 37°C for 30 min to remove thecytoplasmic membrane and most of the cell wall proteins.This cell wall was termed the SDS wall. The small amountsof protein that remained associated with the SDS wall couldbe removed by treating it with 200 ,ug of trypsin per ml(bovine pancreas; P-L Biochemicals, Inc., Milwaukee, Wis.)for 18 h at 37°C. This fraction was termed the trypsin wall. Insome experiments the crude wall was treated directly withtrypsin. This process also removed cell wall proteins almostcompletely (7). To remove wall teichoic acid from the SDSor the trypsin wall, they were further extracted with 5%(wt/vol) trichloroacetic acid (TCA) at 4°C for 45 h and thenwith 10% (wt/vol) TCA at 90°C for 10 min. This fraction wastermed the TCA wall.

Chemical analysis. The protein content was determined bythe method described by Lowry et al. (15), with bovineserum albumin used as the standard, The amounts ofteichoic acid were estimated from the phosphorus content bythe method described by Allen (1). The amounts of peptido-glycan were estimated from the amounts of N-acetylglu-cosamine measured by a modification of the Morgan-Elson procedure (18). The amounts of these wall

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FIG. 1. Thin sections of the S. aureus Cowan I crude wall by the conventional fixation method (a) and the freeze-substitution method (b).Bars, 500 nm.

components were expressed as micrograms per milligram(dry weight) of each lyophilized cell wall fraction.

Electrophoresis of the cell wall fraction. Protein profiles inthe cell wall fractions were determined by SDS-polyacryl-amide gel electrophoresis- by the method described byLaemmli (14) with somne modifications. The concentrationsof acrylamide for stacking and separating gels were 4 and12.5%, respectively. Before application to the gel the cellwall fractions were treated with 100 ,ug of lysostaphin (SigmaChemical Co., St. Louis, Mo.) at 37°C for 4 h to solubilizethe cell wall and then boiled in the buffer. Electrophoresiswas carried out on a slab gel at 10 mA per gel under a

constant current until the tracking dye reached the separat-ing gel, and then the current was increased to 20 mA per gel.Electrophoresis was completed when the tracking dyereached the end of the gel. The gel was then stained withCoomassie brilliant blue. For molecular weight markers a

molecular weight kit (Pharmacia, Uppsala, Sweden) was

used.Electron microscopy. The specimens for thin sectioning

were prepared by two methods: conventional chemical fix-ation add rapid freezing and substitution fixation. Chemicalfixation was performed by the currently used double fixationmethod with 1% glutaraldehyde in cacodylate buffer (pH 7.2)for 30 mim at room temperature and 1% OS04 in Veronal(Winthrop Laboratories, Div. Sterling Drug Co., New York,N.Y.) acetate buffer overnight at 4°C (20). The rapid freezingmethod was performed by a previously described procedure(2). The outline of the method is as follows. The cell wallfraction was pelleted by centrifugation and quickly mixedwith 2% (wt/vol) melted agar. The agar was immediatelyspread onto a glass slide and cut into small agar blocks(5-mm square). A block of this agar-cell wall mixture was

applied onto the plunger of a rapid-freezing device (typeRF-2; Eiko Engineering Co., Ltd, Tokyo, Japan) and rapidlyfrozen by pressing it onto a copper block that was precooledin liquid nitrogen. Freezing was then completed in liquidnitrogen. Substitution fixation was carried out in 4% (wt/vol)OS04 in acetone in a mixture of dry ice-acetone for about 40h. After the specimen was allowed to stand at room temper-ature for about 3 h, it was dehydrated with acetone andembedded inl Epoxy resin. Thin sections were cut with adiamond knife, stained with uranyl acetate and lead citrate,and examined with a JEM 100C electron microscope (JEOLLtd., Tokyo, Japan) at 80 kV.

Conjugation of ferritin and ConA. ConA (10 mg; ICNPharmaceuticals Inc., Cleveland, Ohio) and ferritin (80 mg;horse spleen; Sigma) were mixed in 6 ml of 0.1 M phosphatebuffer (pH 7.2) with 0.05% glutaraldehyde for 120 min at10°C. After nonreacted glutaraldehyde was inactivated withglycine, the mixture was dialyzed against 0.1 M Tris hydro-chloride buffer (pH 7.5) overnight. The ConA-ferritin conju-gate was then collected by centrifugation at 40,000 x g for 3h and suspended in a small volume of phosphate-bufferedsaline and kept at 4°C until use. For labeling the cell wallswith ConA-ferritin, the cell walls isolated from strain PSA-4were treated with 200 ,ug of trypsin per ml for 18 h and thenmixed with ConA-ferritin for 30 min at room temperature.Probe for the labeling of protein A. Protein A on the cell

wall was labeled as follows. The SDS wall of strain Cowan Iwas treated with rabbit anti-ferritin immunoglobulin. Afterrepeated washing by centrifugation, this cell wall fractionwas treated with ferritin, washed several times to removeexcess ferritin, and then processed for electron microscopy.Anti-ferritin serum was produced by injecting a rabbit withferritin. The immunoglobulin fraction was collected by salt-

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2484 UMEDA ET AL.

ing it out with ammonium sulfate. This fraction, at a con-centration of an 80-fold dilution, formed a clear precipitationline between ferritin (10 mg/ml) and antiferritin immunoglob-ulin in the agar gel diffusion method.

RESULTS

Thin-sectioned profiles of the cell wail examined by thefreeze-substitution method. Thin-sectioned profiles of thecrude wall of strain Cowan I prepared by the conventionalfixation and the substitution fixation methods are presentedin Fig. 1. By the conventional fixation method the cell wallwas seen as a thick electron-dense layer with a rather flat andsmooth outer surface (Fig. la). The density of the wall washigh at both edges of the wall. On the exactly tangentiallysectioned wall a middle, less-dense layer was seen to besandwiched between the dense inner and outer edges, Theexternal half of the wall often was seen to be denser than theinternal half.By the freeze-substitution method the surface of the wall

was seen to be covered with a fuzzy coat (Fig. lb). Theaverage thickness of the coat was about 36 nm. A similarstructure was also found on the inner face, but it was thinnerthan that of the outer surface. On some parts of the cell wallthis fuzzy coat was seen as an arrangement of fine fibers, buton most parts it was seen as the accumulation of a densemass. The overall density of the stained cell wall was lesswhen determined by the freezing method than by the con-ventional fixation method. The difference in the density atthe inner and the outer half of the wall was also seen in thissection (Fig. lb). In the following experiment we examinedthe morphological relationships of the cell wall teichoic acidor proteins to the fuzzy coat.

Chemical composition of the cell wall fractions. The chem-ical composition of the cell wall after the extractions ispresented in Table 1. Although most of the cell wall-associated proteins were removed by treatment with SDS, asmall amount of protein still remained associated with thecell wall. These proteins were removed completely bytreatment with trypsin. Analysis of the cell wall-boundproteins by SDS-polyacrylamide gel electrophoresis re-vealed that there were only a few peptides on the gel of theSDS wall and that none of the peptides appeared on thetrypsin wall (Fig. 2). The most extensively stained band,appearing at 64,000 daltons in the SDS wall, seemed to bethe band of protein A, 'as estimated from its molecular size.Teichoic acids were removed by treatment with TCA.

Structure of the cell wall fractions. The structure of the cellwall fractions is presented in Fig. 3. The cell wall profile ofthe SDS wall was very much like that of the crude wall,except that the fibrous structures of the fuzzy coat of theouter surface were more delicate and fine and those of theinner surface became less visible (Fig. 3a). Treatment of theSDS wall with trypsin greatly reduced the thickness of thefuzzy coat (Fig. 3b). The overall density of the cell wall was

TABLE 1. Chemical composition of the S. aureus Cowan Icell wall fraction

Amt of the following components (,Lg/mg of cell wall):Cell wallfraction N-Aceco-samine Phosphorus Protein

Crude 160 12 123SDS 190 10.5 44Trypsin 195 10.5 6TCA 230 0 6

94-...

43 _'.

30 dUbIn

l

FIG. 2. SDS-PAGE analysis of cell wall-associated proteins ofthe S. aureus Cowan I SDS wall. Lane a, molecular weight markers(in thousands); lane b, cell wall proteins extracted by treatment withSDS; lane c', SDS wall digested with Iysostaphin; lane d, trypsin walldigested with lysostaphin. The peptide band indicated by the arrow-head is the band of lysostaphin.

not changed by the treatments. Drastic changes in thestructure were found on the TCA wall. This wall seemed tohave lost the rigidity that is characteristic of the cell wall,and the thickness of the wall appeared irregular (Fig. 3c).The density of the wall was also reduced, and it becamedifficult to take a picture with good contrast. A moreimportant finding was that the fuzzy coat was no longerfound on this wall.

Localization of protein A. To study the relation of the fuzzycoat to the wall-associated proteins, we determined thelocation of protein A. Results of SDS-polyacrylamide gelelectrophoresis analysis of the SDS wall showed that theprotein that remained associated with the cell wall after SDSextraction was mostly protein A (Fig. 2) in strain Cowan I.We determined the location of protein A with anti-ferritinimmunoglobulinp G and ferritin.- Man'y ferritin particles wereassociated with fibrous materials on both the inner and outersurfaces (Fig. 4).

Localization of teichoic acid. ConA interacts with theteichoic acid of at-glycosylated teichoic acids (17). Thedistribution of the a form of teichoic acid on the cell wall ofS. aureus is said to vary, depending on the strain. Becausestrain Cowan I did not react with ConA, we employed theclinically isolated strain PSA-4, which showed a strongreaction with ConA. The trypsin-digested cell wall of PSA-4treated with a ConA-ferritin probe is shown in Fig. 5. Manyferritin particles were seen on the fibrous layer of the outersurface, but they were only occasionally seen in associationwith the inner surface layer. The ConA-ferritin probe did notreact with the SDS wall of PSA-4, althoug the teichoic acidstill remained associated with the cell wall. It can be as-sumed that treatment with SDS alters the configuration ofteichoic acid.

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......... ...._..............:.'

FIG. 3. Thin sections of the cell wall fractions. (a) SDS wall; (b)trypsin wall; (c) TCA wall. Bars, 500 nm.

DISCUSSION

By using a new fixation method for electron microscopywe demonstrated a new feature of the S. aureus cell wallsurface which was basically different from that obtained bythe conventional fixation method. By this method the cellwall surface was seen to be covered with a fuzzy coat or a

layer consisting of the accumulation of a dense mass. Thelayer was not affected significantly by extraction with SDS,but its density was reduced by treatment with trypsin and thelayer was completely removed by extraction with TCA.These results suggest that the layer consists of a complex ofteichoic acids and proteins. This possibility was confirmedby labeling protein A with anti-ferritin and ferritin and

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aPI,

FIG. 4. Location of protein A on the SDS wall of strain Cowan I.The SDS wall was treated with anti-ferritin antibody and then withferritin. The cell wall was fixed by the freeze-substitution method.Bars, 100 nm. (a) Section was stained with uranyl acetate and leadcitrate. Ferritin particles are seen as small particles (arrows) on bothsides of the cell wall. (b) Section was not stained. Ferritins are more

clearly recognized in panel b.

labeling teichoic acid with ConA-ferritin conjugate. Thesemarkers were specifically detected on the fuzzy coat. Theparticipation of peptidoglycan in this layer is still unknown,because markers specific for peptidoglycan are not yetavailable. In a previous report (22) we demonstrated that thebacteriophage receptor is located on the old surface of thecell wall. Because the receptors of most staphylococcalphages are said to consist of a complex of peptidoglycan and

FIG. 5. Location of teichoic acids on the trypsin wall of strainPSA-4. The crude wall of the strain was treated with 100 ,ug oftrypsin per ml for 18 h at 37°C and then incubated with a ConA-ferritin probe. Ferritins are seen on the outer surface of the cell wall(arrowheads). Bar, 100 nm.

FIG. 6. Structure model of the S. aureus cell wall. The thick walllayer is composed mostly of the peptidoglycan layer (solid lines).Teichoic acids are bound covalently to peptidoglycan, and most ofthem extend outward to the external space and some extend into theinner space. Proteins (open circles) are probably bound to theteichoic acid chains (wavy lines). These extended teichoic acidchains and the associated proteins form the fuzzy coat.

teichoic acids, it is conceivable that on some parts of the cellwall peptidoglycan is a component of the fuzzy coat.From the data obtained in this study we made a model for

the S. aureus cell wall (Fig. 6). Peptidoglycan forms a fullthick layer of the cell wall. Teichoic acids are bound topeptidoglycan and extend their chains to the outer surface ofthe wall, forming the main components of the fuzzy coat.Proteins are presumed to bind to teichoic acid chains cova-lently or ionically, depending on their nature. Some teichoicacid chains could be extended to the inner surface of thewall. Because the cell wall components generally undergoturnover, on some parts of the wall, especially the old wall,this regular arrangement of the wall polymers might bedisturbed to some extent.The presence of such a fuzzy coat has been reported in

some other bacteria. In Streptococcus pyogenes the Mprotein layer forms a fuzzy coat (16). Bacillus subtilis has asimilar layer which is presumed to be the layer of teichoicacid (4, 8). The flat and even surface of most gram-positivebacterial cell walls obtained by the coventional fixationmethod could be an artefactual structure induced by chem-ical fixation. The surface of the bacterial cell wall must be amore complicated structure than is currently believed.

ACKNOWLEDGMENTS

We thank A. Takade for excellent technical assistance with theelectron micrographs and M. R. Robbins for preparing this manu-script.

This research was supported in part by grant 60480165 from theMinistry of Education and Science of the Japanese government anda grant from the Kazato Science Foundation.

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