JOURNAL OF BACTERIOLOGY, Oct. 1973, p. 483-487Copyright © 1973 American Society for Microbiology

Vol. 116, No. 1Printed in U.S.A.

Effect of Lysozyme Treatment on Cell WallUltrastructure in Sarcina [lava

M. I. S. HUNTER, D. D. M. MUIR,' AND D. THIRKELL

Department of Biochemistry, University of St. Andrews, North Street, St. Andrews, Fife, Scotland

Received for publication 17 July 1973

Treatment of Sarcina flava NCTC 7503 cells with lysozyme resulted inincomplete removal of the cell wall and imparted a layered appearance to it.

Although the fine structure of Sarcina flavahas so far been reported only briefly (D. Thirkelland M. I. S. Hunter, Biochem. J. 120:13P,1970), that of the closely related species Sarcinalutea (4, 5) and Micrococcus lysodeikticus (19,10, 15, 22) has been described in more detail. Inboth bacteria, the cell wall appears to be a thickamorphous structure typical of gram-positivebacteria.The removal of the gram-positive bacterial

cell wall by digestion with lysozyme (N-acetylmuramide glycanohydrolase, EC 3.2.1.17) toform protoplasts or protoplast membrane prep-arations is particularly well documented forBacillus megaterium (27) and M. lysodeikticus(12, 23, 24), although for neither species is thecell wall composed entirely of the peptidoglycansubstrate (7, 18). Nevertheless, the peptidogly-can must be sufficiently accessible to the lyso-zyme to account for the considerable and rapidhydrolysis of the ,B-1, 4-glycosidic bonds, andthis peptidoglycan component must be respon-sible for the structural integrity of the wall sinceits hydrolysis results in the disintegration of theentire cell wall substance.Although many gram-positive bacteria, in-

cluding S. lutea, are lysozyme sensitive, severalare resistant to the enzyme. This may be due tothe composition of the wall substance itself, astypified by the extreme halophile Sarcinamorrhuae, whose walls contain no muramic acid(3), or to the occlusion of the substrate by acomplex arrangement of overlying cell wallcomponents as found in Bacillus polymyxa (19).Variation in lysozyme sensitivity is not onlyinterspecific, as seen in the genus Bacillus, butcan also be intraspecific as found for differentstrains of Listeria monocytogenes (11). Resist-ance to lysis by lysozyme is also observed inspecies of the genera Nocardia, Mycobacteria(21, 25), and Corynebacteria (6), whose cellwalls contain little amino sugar, the major

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carbohydrate constituents being galactose andarabinose. Lysozyme treatment of Mycobacte-rium phlei disintegrates only the "basal layer"of the cell wall, destroying the shape and rigidityof the cell but leaving intact the bulk of theoverlying cell wall (26).

In this work, the sensitivity of S. flava NCTC7503 to lysozyme was investigated under condi-tions employed for the preparation of proto-plasts from M. lysodeikticus and B.megaterium. Cells were grown in nutrient broth(Oxoid Ltd.) enriched with 1% glucose at 34 C,harvested at 48 h (early stationary phase), andtreated with egg white lysozyme (Sigma Ltd.)by the method of Baird-Parker and Woodroffe(1). Cells were suspended to give a concentra-tion of 20 mg (wet wt) per ml in a mediumcontaining 0.01 M Sorensen double phosphatebuffer (pH 7.0), 0.005 M MgCl2, 0.01 M NaCl,and 0.75 M sucrose. Lysozyme (100 Ag/ml) wasadded, and digestion was allowed to proceed at30 C for 30 min.

After lysozyme treatment, the cells were pre-fixed and fixed according to the modified Ryter-Kellenberger method of Highton (16), fixationbeing carried out overnight at room tempera-ture (22 C). After fixation the cells were dehy-drated and embedded in Araldite (CIBA Ltd.).Thin sections, prepared using an LKB Ul-trotome I, were collected on uncoated coppergrids, poststained with uranyl acetate and leadcitrate (20), and examined in an AEI EM 6Belectron microscope.The cell wall of S. flava is relatively resistant

to attack by lysozyme, since only in a few cells(Fig. 1) was rupture seen to be complete.Usually, the wall was seen to be largely intact(Figs. 2 and 3), in that the rigidity and originalpacket formation were not destroyed. Neverthe-less, some digestion of peptidoglycan occurred,since the wall material of all treated cells wasfrayed and less compact than that of untreatedcells (Fig. 4).

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FIG. 1. S. flava cell in which fission of the cell wall has occurred. Layering and splitting between the layers ofthe wall are also apparent. Characteristic "unit" cytoplasmic membrane surrounds nascent protoplast. Markerbar in this and all subsequent figures represents 0.2 usm.

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FIG. 2. Packet of cells showing incomplete digestion of wall material. Area at center ofgroup shows greatestdegree of attack.

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FIG. 3. Tetrad of cells in which layering of lysozyme-resistant wall substance islysozyme-resistant material envelopes all four cells.

r4. V..I-rapparent. A strand of

FIG. 4. Control S. flava cells untreated with lysozyme but fixed in the presence of 0.75 M sucrose and underthe conditions described in the text.

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FIG. 5. Partially lysed cell with wall material in densely staining strata, separated by light areas. Fourdensely staining bands discernible in places (arrowed).

FIG. 6. Plasmolyzed cell in which wall substance can be seen as three electron-dense layers. MAembrane isattached to the innermost of these layers at intervals. Nuclear material not associated with attachment pointsin this plane of section. A2a

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VOL. 116, 1973

In groups or pairs of cells (Fig. 2 and 3),preferential digestion of cell wall material canbe observed in the region of common cross walls,suggesting that these regions are, in fact, themost sensitive to the action of the enzyme.Others (17) have also shown that the mostrecently synthesized wall substance is the mostsusceptible to attack by lytic enzymes.

Figures 1 to 3, 5, and 6 show that lysozymetreatment imparted a stratified appearance tothe wall. Two or three layers are frequentlvdiscernible (Fig. 1, 2, 3, and 6), and occasion-ally (Fig. 5) as many as four could be distin-guished. These observations suggest that thewall may be layed down as alternate layers oflysozyme-resistant and lysozyme-sensitive com-ponents.

Figure 6 shows a plasmolyzed cell in whichregions of the membrane have remained incontact with the innermost layer of the wall.Areas of membrane-wall adhesion have alreadybeen reported for Streptomyces coelicolor (14),Bacillus subtilis (13), L. monocytogenes (8),and Escherichia coli (2). Bayer (2) suggests thatthese points of attachment are regions of activecell wall synthesis, and showed that nuclearmaterial was often associated with these mem-brane-wall junctions. No such association wasobserved in cells of S. flava.The resistance of the cell wall of S. flava to

attack by lysozyme, when compared with theextreme sensitivity of most strains of the closelyrelated species S. lutea and M. lysodeikticus, isperhaps not surprising in the light of the obser-vations of Ghosh et al. (11) on the vastlydiffering lysozyme sensitivities of strains of L.monocytogenes.

D.T. is grateful to the Science Research Council forfinancial support.

LITERATURE CITED

1. Baird-Parker, A. C., and R. C. S. Woodroffe. 1967.Analysis of the bacterial cell, p. 85-117. In C. H. Col-lins (ed,), Progress in microbiological techniques.Butterworth, London.

2. Bayer, M. E. 1968. Areas of adhesion between wall andmembrane of Escherichia coli. J. Gen. Microbiol.53:395-404.

3. Brown, A. D., and K. Y. Cho. 1970. The walls of theextremely halophilic cocci: gram-positive bacterialacking muramic acid. J. Gen. Microbiol. 62:267-270.

4. Chapman, G. B. 1960. Electron microscopy of cellulardivision in Sarcina lutea. J. Bacteriol. 79:132-474.

5. Cherny, N. E. 1967. Ultrastructure of the intracytoplas-mic membrane systems in Sarcina lutea cells. Izv.Akad. Nauk Kirg. SSSR Ser. Biol. 2:302-305.

6. Cummins, C. S., and H. Harris. 1956. The chemicalcomposition of the cell wall in some Gram-positivebacteria and its possible value as a taxonomic charac-

ter. J. Gen. Microbiol. 14:583-600.7. Czerkawski, J. W., H. R. Perkins, and H. J. Rogers. 1963.

A study of the composition and structure of the cellwall mucopeptide of Micrococcus lysodeikticus. Bio-chem. J. 86:468-474.

8. Edwards, M., and R. W. Stevens. 1963. Fine structure ofListeria monocytogenes. J. Bacteriol. 86:414-428.

9. Friedberg, I., and G. Avigad. 1968. Structures containingpolyphosphate in Micrococcus lysodeikticus. J. Bacte-riol. 96:544-553.

10. Friedberg, I., and G. Avigad. 1970. Fine structure ofpolyphosphate granules in Micrococcus lysodeikticus.Isr. J. Med. Sci. 6:511-518.

11. Ghosh, B. K., and R. G. E. Murray. 1967. The finestructure of Listeria monocytogenes in relation toprotoplast formation. J. Bacteriol. 93:411-426.

12. Gilby, A. R., A. V. Few, and K. McQuillen. 1958. Thechemical composition of the protoplast membrane ofMicrococcus lysodeikticus. Biochim. Biophys. Acta29:21-29.

13. Glauert, A. M., E. M. Brieger, and J. M. Allen. 1961. Thefine structure of vegetative cells of Bacillus subtilis.Exp. Cell Res. 22:73-85.

14. Glauert, A. M., and D. A. Hopwood. 1960. The finestructure of Streptomyces coelicolor. I. The cytoplas-mic membrane system. J. Biophys. Biochem. Cytol.7:479-486.

15. Grula, E. A., and R. D. King. 1970. Inhibition of celldivision in Micrococcus lysodeikticus dis-II. Can. J.Microbiol. 16:317-324.

16. Highton, P. J. 1969. An electron microscope study of cellgrowth and mesosomal structure of Bacilluslicheniformis. J. Ultrastruct. Res. 26:130-147.

17. Mitchell, P., and J. Moyle. 1957. Autolytic release andosmotic properties of protoplasts from Staphylococcusaureus. J. Gen. Microbiol. 16:184-194.

18. Nermut, M. V. 1967. The ultrastructure of the cell wall ofBacillus megaterium. J. Gen. Microbiol. 49:503-512.

19. Nermut, M. V., and R. G. E. Murray. 1967. Theultrastructure of the cell wall of Bacillus polymyxa. J.Bacteriol. 93:1949-1965.

20. Reynolds, E. S. 1963. The use of lead citrate at high pH asan electron-opaque stain in electron microscopy. J. CellBiol. 17:208-212.

21. Romano, A. H., and A. Sohler. 1956. Biochemistry of theactinomycetales. II. A comparison of the cell wallcomposition of species of the genera Streptomyces andNocardia. J. Bacteriol. 72:865-868.

22. Salton, M. R. J., and J. A. Chapman. 1962. Isolation ofthe membrane-mesosome structures from Micrococcuslysodeikticus. J. Ultrastruct. Res. 6:489-498.

23. Salton, M. R. J., and J. H. Freer. 1965. The compositionof the membranes isolated from several Gram-positivebacteria. Biochim. Biophys. Acta 107:531-538.

24. Salton, M. R. J., and J. M. Ghuysen. 1960. Acetylhexosa-mine compounds enzymatically released from Mi-crococcus lysodeikticus cell walls. III. The structure ofdi- and tetrasaccharides released from cell walls bylysozyme and Streptomyces F, enzyme. Biochim. Bio-phys. Acta 45:355-363.

25. Sohler, A., A. H. Romano, and W. J. Nickerson. 1958.Biochemistry of the actinomycetales. III. Cell wallcomposition and the action of lysozyme upon cells andcell walls of the actinomycetales. J. Bacteriol.75:283-290.

26. Takeya, K., K. Hisatsune, and Y. Inoue. 1963. Mycobac-terial cell walls II. Chemical composition of the "basallayer." J. Bacteriol. 85:24-30.

27. Weibull, C. 1953. The isolation of protoplasts fromBacillus megaterium by controlled treatment withlysozyme. J. Bacteriol. 66:688-698.

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