Using a freeze-etching method, the ultrastructure of cell surface of gram-positive cocci was studied by digesting cell wall with lytic enzyme. In M. lysodeikticus, the cell surface revealed a very simplified ultrastructure, i. e. a single cell wall layer and a single
plasma membrane layer. On the contrary, the cell surface of S. aureus exhibited a unique structure composed of two cell wall layers and a single ploasma membrane layer. The wall layers were constituted of 160 -180 A particle layer (CWl) which was unsusceptible to the L-ll enzyme and amorphous layer (CW2) which was susceptible. These results suggested that 160-180 A particles in CWl consisted mainly of the teichoic acid.</p

Kanemasa, Yasuhiro

Tomochika, Ken-ichi

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 28, Issue 6 1974 Article 1DECEMBER 1974Freeze-etch studies on the bacterial cellsurfaces: action of the cell wall lytic enzymeson the gram-positive cocciKen-ichi Tomochika∗ Yasuhiro Kanemasa†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Freeze-etch studies on the bacterial cellsurfaces: action of the cell wall lytic enzymeson the gram-positive cocci∗Ken-ichi Tomochika and Yasuhiro KanemasaAbstractUsing a freeze-etching method, the ultrastructure of cell surface of gram-positive cocci wasstudied by digesting cell wall with lytic enzyme. In M. lysodeikticus, the cell surface revealed avery simplified ultrastructure, i. e. a single cell wall layer and a single plasma membrane layer.On the contrary, the cell surface of S. aureus exhibited a unique structure composed of two cellwall layers and a single ploasma membrane layer. The wall layers were constituted of 160 -180A particle layer (CWl) which was unsusceptible to the L-ll enzyme and amorphous layer (CW2)which was susceptible. These results suggested that 160-180 A particles in CWl consisted mainlyof the teichoic acid.∗PMID: 4282000 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Merl. Okayama 28,381-389 (1974)FREEZE-ETCH STUDIES ON THE BACTERIAL CELLSURFACESACTION OF THE CELL WALL LYTIC ENZYMESON THE GRAM-POSITIVE COCCI -Ken-ichi TOMOCHIKA and Yasuhiro KANEMASADepartment of Microbiology, Okayama University Medical SchoolOkayama, Japan (Director: Prof. J. Tawara)Receivd for publication, May 8, 1974Abstract: Using a freeze-etching method, the ultrastructure of cellsurface of gram-positive cocci was studied by digesting cell wall withlytic enzyme. In M. lysodeikticus, the cell surface revealed a verysimplified ultrastructure, i. e. a single cell wall layer and a singleplasma membrane layer. On the contrary, the cell surface of S. aureusexhibited a unique structure composed of two cell wall layers and asingle ploasma membrane layer. The wall layers were constituted of160 -180 A particle layer (CWl) which was unsusceptible to the L-llenzyme and amorphous layer (CW2) which was susceptible. Theseresults suggested that 160-180 A particles in CWl consisted mainly ofthe teichoic acid.Fine structures of bacterial cell surface, especially the outer membraneor cell wall, are well known in gram-negative bacteria which are generallycomplicated in their fine structures. On the other hand, almost all gram-positive bacreria possess very simplified structures of the cell wall as clarifiedby chemical analyses and electron microscopic observations.To study fine structure, bacterial cell surfaces of gram-negative andgram-positive bacilli were treated with chemicals and lytic enzymes (1, 2, 3).These results demonstrated that cell walls of gram-negative bacilli wereconstituted with several layers as demonstrated in gram-positive bacillus (2).FUTAI et al. state that the cell wall of Staphylococcus aureus is composed oftwo morphologically distinct layers both of which are susceptible to the L-llenzyme (4). This observation is very interesting in contrast to chemicalcompositions of the cell wall reported by ARMSTRONG et al. (5).Recently, freeze-etching or freeze-fracture methods are employed in theanalysis of the cell surface structure (6, 7, 8). These methods provide moreeffective ways for the elucidation of surface structures. For instance, the cellwall structure of Escherichia coli has been extensively clarified by thismethod (6).The present freeze-etching studies were undertaken to establish the fine3811Tomochika and Kanemasa: Freeze-etch studies on the bacterial cell surfaces: action of theProduced by The Berkeley Electronic Press, 1974382 K. TOMocHIKA and Y. KANEMASAstructures of the cell surface of gram-positive cocci, i. e. Micrococcus lysodeik-ticus and S. aureus.MATERIALS AND METHODSCultures and Media: Micrococcus lysodeikticus lAM 1056 and Staphylococcusaureus 209P were cultured in a phosphate nutrient broth with gentle shaking at37°C, and the cells which reached the late logarithmic phase were harvested bycentrifugation at 6, ()()() xg for 10 min.Procedures for the Enzyme Treatment: The harvested cells of M. lysodeikticuswere washed twice with physiological saline solution, and then rewashed with0.1 M phosphate buffer (pH 6.0). Cells were resuspended in the same buffercontaining 0.5 M sucrose for stabilizing protoplast. Egg lysozyme (Boehringerand Soehne Gmbh Mannheim), a bacteriolytic enzyme, was added onto the cellsuspensions at the rate of 20 pg per 1 mg wet cells.Staphylococcal cells were washed twice with physiological saline solutionand rewashed twice with 0.01 M tris (hydroxymethyl) aminomethane (Tris)-HClbuffer (pH 7.2). The washed cells were suspended in the same buffer with0.8M sucrose. Crude L-ll enzymes, that were kindly supplied by Prof. S. KOTANI,Osaka Univ., were purified by the method of KATO et al. (9, 10), and purifiedL-ll enzymes were added at the rate of O. 2 unit per mg of washed cell. Thetreatments with both enzymes were performed under the condition of gentleshaking at 37°C and were checked for a decrease in the optical density whichwas measured at 650nm in a Coleman spectrophotometer. The treatments werestopped by chilling in an ice bath at an appropriate time. The treated cellswere collected by centrifugation at 12,000 xg for 30 min.Freeze-Etching: M. lysodeikticus was washed and immersed with 20% glyce-rol containing 0.5 M sucrose and S. aureus with 20% glycerol containing 0.8 Msucrose, respectively. After the penetration of glycerol into the cells at 4°C for5 hr, cell pellet was placed on a copper block and immediately frozen in Freon-12 cooled by liquid nitrogen. The freeze-etching was carried out in an EE-FEDfreeze-etch apparatus (Japan Electron Optics Laboratory Co. Ltd, Tokyo). Thespecimens were freeze-cleaved at -120°C, etched 2 min at -100°C, shadow.casted with platinum-carbon and replicated with carbon in the high vacuumat 4 to 10 X 10-') Torr. The replicas were cleaned in 70% H 2S04 , rinsed in dis-tilled water and mounted on a bare copper grid. Specimens were examinedwith an electron microscope, model ]EM-7 (JEOL Ltd). In all micrographsshadows appear white, and shadow directions are given by the encircled arrows.The scales indicate 1 pm except for insert in Fig. 6 that has 100 nm scale.RESULTSEnzyme Treatments on M. lysodeikticus: M. lysodeikticus was used for thecomparative studies because the cell wall of this organism is, for the mostpart, composed of peptidoglycan. The cell wall lysis with enzyme treatmentwas shown as an abrupt decrease in optical density, which reached a plateau2Acta Medica Okayama, Vol. 28 [1974], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss6/1Freeze-Etch Studies on the Bacterial Cell Surfacesby 20 min (Fig. 1).a.383100>....9ID_a: E~ c: 50w ~(Ofo)>(0-Q~o-.1-wa:b.o 1.----'1.---'------::-l3o::-----''----'--6~0:-­INCUBATION TIME (min)Fig. 1. Action of lysozyme on M. lysodeikticlIs.a. Enzymatically treated. Condition: incubatedin O. 1M phosphate buffer (pH 6.0) with 0.5 Msucrose.b. Enzymatically treated. Condition: incubatedin the above described buffer containing lyso-zyme 120 pg/mg wet weight cells).Freeze-etch appearances of untreated M. lysodeikticus are shown in Fig.2. In cross fracturing faces the cell wall is seen to be an amorphous anddiffusible single layer which is about 500 A in thickness. There can observedno distinct differences in the surface pattern between the convex face (PM)and the concave face (PM) of the plasma membrane. The cave-in figures areobserved on the fractured face that appeared inside the plasma membrane.These figures seem to be of scooped mesosomal vesicles (ME).Lysozyme-treated cells on incubation for three min. reveal a figure inwhich the cell wall materials have been removed completely (Fig. 3). Inaddition, globular arrangements are observed on the plasma membrane whichis constituted of the particles (arrow) ranging 150-170 A in diameter on crossfracture face and of a convex face of plasma membrane.When the cell treatment was prolonged more than 20 min, osmoticallyfragile cells (Fig. 4a) and membrane fragments (MF) (Fig. 4b) derived fromthe osmotically fragile cells were observed.En<.yme Treatment on S. aureus: A bacteriolytic enzyme called the L-llenzyme prepared from Flavobacterium sp. was used throughout this experi-ments. When S. aureus was exposed to L-ll enzyme, there occurred a gradual3Tomochika and Kanemasa: Freeze-etch studies on the bacterial cell surfaces: action of theProduced by The Berkeley Electronic Press, 1974384 K. TOMOCHIKA and Y. KANEMASAFig. 2. Replica of freeze-etched intact M. lysodeikticus. CW: cell wall, PM:convex face of plasma membrane, PM: concave face of plasma membrane, andME: mesosomal vesicles.Fig.3. Replica of freeze-etched AI. !.ysodeikticus treated with lysozyme for 3 min.Cell wall layer was removed entirely. An arrow indicates the cross fracture faceof the plasma membrane.decrease in turbidity without a lag and the turbidity reached plateau(60~o in relative turbidity) within 30 min.4Acta Medica Okayama, Vol. 28 [1974], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss6/1Freeze-Etch Studies on the Bacterial Cell Surfaces 385The freeze-etch appearances of non-treated S. aUl'eus are shown in Fig. 6.It is demonstrated that the surface layer seems to be composed of plasmaFig. 4. Replica of freeze-etched protoplast (al and membrane fragments. (b)derived from M.l.vsodeikticus treated with lysozyme for 20 min.Fig. 6. Replica of freeze-etched non-treated S. aureUJ. Insert: cross fractureface of surface layer at a high magnification.5Tomochika and Kanemasa: Freeze-etch studies on the bacterial cell surfaces: action of theProduced by The Berkeley Electronic Press, 1974386 1(. TOMOCHIKA and Y. KANEMASAmembrane and cell wall layers, 360-410 A in total thickness. More precisely,the inner-most layer is a plasma membrane formed by an array of 80-130 Aparticles. The cell wall layer is seen to consist of two layers; the outerlayer (CW1) contains 160-180 A particles in proper intervals and the innerlayer is amorphous, existing between the plasma membrane layer and OWl(Insert in Fig. 6).a.b.OL--'''"""'-----''---:3="'::O~-----''---;:60~INCUBATION TlME(min)Fig. 5. Action of L-ll enzyme on S. aureus.a. Enzymatically treated. Condition: incubated in0.01 M Tris-HCl buffer (pH 7.2) with 0.8M suc-rose.b. Enzymatically treated. Condition: incubated inthe above described buffer containing L-ll enzyme10.2 unit/mg wet weight cells).On treating with L-ll enzyme for 5 min (Fig. 7), there appeared remark-able differences in the contour of the cell wall layers from the wall layers ofnon-treated cell. CWI remained to be observed separately from the plasmamembrane, because the cell wall layeI was partially digested and CW2 be-came wider. This phenomenon may indicate that the particles constitutingOWl are not affected by enzyme treatment. Convex face of plasma membrane(PM) reveals the particles which range from 80 to 130 A, and in cross fractur-ing face of plasma membrane its particles appear to be uniform particlesmeasuring 150 A (Fig. 7).The prolonged treatment (30 min) gave the protoplast from which cellwall materials were entirely removed (Fig. 8a, and 8b). Convex face of theplasma membrane (PM) appeared to be smooth, whereas concave face (PM)was densely covered with 80-130 A particles. --6Acta Medica Okayama, Vol. 28 [1974], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss6/1Freeze-Etch Studies on the Bacterial Cell Surfaces 387~.Fig. 7. Replica of freeze-etchedS. aureus treated with L-Il enzyme for 5min. Anarrow indicates the particle layer (CWI) of cell wall.Fig. 8. Replica of freeze-etched S. aurew treated with L-II enzyme for 30 min.Cell wall materials entirely disappeared.DISCUSSIONIn the present investigation, we studied the fine structure of cell surfacein the gram-positive cocci, i. e. M. (l'sodeikticus and S. aureus, using a freeze-7Tomochika and Kanemasa: Freeze-etch studies on the bacterial cell surfaces: action of theProduced by The Berkeley Electronic Press, 1974388 K. TOMOCHIKA and Y. KANEMASACWPMCW1CW2PMa)M. lysodeikticusb) S. aureusetching method.Chemically, the cell wall of gram-positive cocci consists of the peptido-glycan only or both the peptidoglycan and teichoic acid (5, 11). The cellwall of !l1. lysodeikticus contains about90-93% peptidoglycan by dry weight andlacks teichoic acid (11). Consequently,these gram-positive cocci were treatedwith lysozyme having the activity of N-acetyl-muramylhydrolase that forms pro-toplasts easily, and the freeze-etchingfigures of enzymatically non-treated ortreated cells agree reasonably well withthe simplified structure of this organism(Fig.9a).As compared with lvf. lysodeikticus,the cell wall of Staphylococcus consistingFig. 9. Diagrams of the surface approximately of peptidoglycan and tei-layers of M.lysodeiktieus (a) and S.aureus choic acid in an equal amount (12), has(b). b ba unique structure (Figs. 6 and 9) Yshowing distinguishable particle and amorphous layers. Fine structures ofcell walls on S. aureus were previously reported by SUGANUMA (13) and FUTAIet al. (4) by thin sectioning methods and MORIOKA et al. (14) by freeze-etching.SUGANUMA (13) suggested that cell walls of S. aureus were composed of threelayers, of which the two outer layers are relatively rigid and the innermostlayer is fused to plasma membrane. FUTAI et al. (4) drew a conclusion that acell wall of S. aureus was composed of two layers by thin sectioning methodusing L-ll enzyme. However, their conclusion differs slightly from SUGA-NUMA'S report (13). On the freeze-etching studies of S. aureus, MORIOKA et al.(14) have described that the cell wall exhibits multiple layers on the fractureplane. However, the fine structure of cell wall has not been revealed clearly.From the evidence obtained after the treatment with L-ll which is knownto hydrolyze amide and peptide linkages, it is appropriate to consider thatCWI is composed of teichoic acid and peptidoglycan, and CW2 is composedof peptidoglycan only or peptidoglycan-rich materials which are digested byL-ll enzyme.From these results, it seems most reasonable to conclude that the finestructures of cell surface are evidently of stratiform as shown in Fig. 9b.Acknowledgement: The authors wish to thank Mr. Nobuo HAYASHI for his helpful tech-nical advice.8Acta Medica Okayama, Vol. 28 [1974], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss6/1Freeze-Etch Studies on the Bacterial Cell SurfacesREFERENCES3891. SCHNAITMAN, C. A.: Effect of ethy1endiamine-tetraacetic acid, triton X-100, and lysozymeon the morphology and chemical composition of isolated cell walls of Escherichia coli. J.Bact. 108, 553-563, 19712. NERMUT, M. V. and MURRAY, R. G. E.: Ultrastructure of the cell wall of Bacillus polymyxa.J. Bact. 93, 1949-1965, 19673. MARTIN, H. H.: Bacterial protop1asts. A review. J. Theor. Biol. 5, 1-34, 19634. FUTAI, M., OKABAYASHI, K. and MIZUNO, D.: Electron microscopic studies on the outerlayers of Staphylococcus aureus using a lytic enzyme from Flavobacterium. Japan. J. Microbiol.16, 341-350, 19725. ARMSTRONG, J. J., BADDILEY, J., BUCHANAN, J. G., DAVISON, A. L., KELEMEN, M. V. andNEUHAUS, F. C.: Composition of teichoic acids from a number of bacterial walls. Nature184, 247-248, 19596. V;N GOOL, A. P. and NANNINGA, N.: Fracture faces in the cell envelope of Escherichiacoli. J. Bact. 108, 474-481, 19717. FILL, A. and BRANTON, D.: Changes in the plasma membrane of Escherichia coli duringmagnesium starvation. J. Bact. 98, 1320-1327, 19698. WATSON, S. W. and REMSEN, C.C.: Cell envelope of Nitrosocystis oceanus. J. UltrastructureRes. 33, 148-160, 19709. KATO, K., KOTANI, S., MATSUBARA, T., KOGANI, J., HASHIMOTO, S., CHIMORI, M. andKAZEKAWA, 1.: Lysis of Staphylococcus aureus cell walls by a lytic enzyme purified fromculture supernatants of Flavobacterium species. Biken J. 5, 155-179, 196210. KATO, K. and STROMONGER, J. L.: Structure of the cell wall of Staphylococcus aureus. IX.Mechanism of hydrolysis by the L ll enzyme. Biochemistry 7, 2754-2761, 196811. PERKINS, H. R. and ROGERS, H. J.: The products of the partial acid hydrolysis of themucopeptide from cell walls of Micrococcus lysodeikticus. Biochim. J. 72, 647-654, 195912. ACHIBALD, A.R., BADDILEY, J. and HECKELS, J.E.: Molecular arrangement of teichoicacid in the cell wall of Staphylococcus lactis. Nature New Biol. 241, 29-31, 197313. SUGANUMA, A.: Fine structure of Staphylococcus aureus. Ann. N. Y. Acad. Sci. 128, 26-44,196514. MORIOKA, H., SUGANUMA, A., YOKOTA, Y. and TAWARA, K.: Ultrastructure of Staphylo-cocci after freeze-etching. J. Electronmicroscopy 22, 255-266, 19739Tomochika and Kanemasa: Freeze-etch studies on the bacterial cell surfaces: action of theProduced by The Berkeley Electronic Press, 1974

Freeze-etch studies on the bacterial cell surfaces: action of the cell wall lytic enzymes on the gram-positive cocci

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