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Isolation of the membrane-mesosome structures from micrococcus lysodeikticus
J. ULTRASTRUCTURERESEARCH 6, 489-498 (1962)
489
Isolation of the Membrane-mesosome Structures from i~/icrococcus
lysodeikticus
M. R. J. SALTON1 and J. A. CHAPMAN
Department of Bacteriology and Rheumatism Research Centre, University of Manchester, England Received August 23, 1961 Micrococcus lysodeikticus cells possess a membranous organdie of about 2500 ~ diameter. This structure is similar to those found in other bacteria and the term "mesosome", which was proposed by Fitz-James to describe such organelles, has been adopted here. The mesosomes of Micrococcus lysodeiktieus have been isolated by differential centrifugation of lysed protoplasts and from lysates prepared by dissolution of the wall with lysozyme. The isolated mesosomes are composed of a series of concentric shells, each membrane being double and having an over-all thickness of about 75 ~. There are probably four concentric membranes forming the mesosome particle which on isolation is frequently surrounded by the plasma membrane forming a fifth shell. The maximum number of concentric membranes observed in the isolated membranemesosome fractious was 5. The present procedure does not permit a differential separation of plasma membrane from mesosome and it is concluded that previous protoplast membrane preparations of this organism contained the mesosome structures. With the isolation of the major structural components and organelles of the bacterial cell, our knowledge of the chemical and biochemical anatomy of bacteria has advanced rapidly during the past decade (8, 21). The surface structures of the bacterial cell, the wall and the protoplasmic membrane, have been isolated and chemically characterized (8, 21). The internal anatomy of the bacterial cell has recently attracted much attention and the complexity of its structure has been emphasized by the discovery of membranous and vesicular organelles in a variety of bacteria (2, 3, 5-7, 9-11, 13-17, 22, 24, 25, 28). These cytoplasmic structures obviously correspond to the "peripheral bodies" first detected in the early thin sections of bacteria studied by Chapman and Hillier (1). These membranous organelles frequently show a continuity with the plasma membrane and have been called "mesosomes" by Fitz-James (2) to distinguish them from mitochondria of other cells (18). 1 Present address: Department of Microbiology, University of New South Wales, Kensington, N.S.W., Australia.
490
M . R . J . SALTON AND J. A. CHAPMAN
Particles c o r r e s p o n d i n g to the m e m b r a n o u s organelles o b s e r v e d in Mycobaeterium avium have been i s o l a t e d b y Y a m a g u c h i (28) b u t little c o u l d be c o n c l u d e d a b o u t the structure of these particles f r o m the thin sections. I n t r a c e l l u l a r particles of a b o u t 2500 A d i a m e t e r have been k n o w n to be present in Mierococcus lysodeitcticus for s o m e time. T h e y have been o b s e r v e d on lysis of cells with lysozyme (12), a n d on disi n t e g r a t i o n of cells for the i s o l a t i o n of walls (20). The susceptibility of M. lysodeikticus to lysis with lysozyme p r o v i d e s a gentle m e t h o d of cell disintegration, a n d thus offered attractive possibilities for the i s o l a t i o n a n d eventual b i o c h e m i c a l c h a r a c t e r i z a t i o n of the i n t r a c e l l u l a r organelles.
MATERIALS AND METHODS
Organism and growth conditions Micrococcus lysodeikticus (NCTC 2665) was grown on a medium consisting of 1% Bacto peptone, 0.1% Difco yeast extract, 1% NaC1, being modified from that described by Litwack and Pramer (14). One liter of the medium (pH 7.5) inoculated with 50 ml culture of the organism was incubated at 30°C in a rotating 5-I flask, and the cells were harvested by centrifugation after 16 hours' growth.
Preparation of membrane-mesosome fractions Isolation of the mesosome "particles" was attempted by the following procedures. (a) Conversion of intact ceils into protoplasts by incubation of thick suspensions of M. Iysodeikticus in 0.067 M phosphate buffer, p H 7.0, containing 1.5 M sucrose, with 50/zg crystalline egg-white lysozyme (Armour Laboratory)/ml at room temperature (c. 20°C). The cell wall of this organism is completely digested by lysozyme (20), such a property being an essential prerequisite for naked protoplast formation (26, 27). Protoplast formation, judged by complete osmotic fragility on dilution in distilled water, had occurred within 15 minutes under the above conditions of treatment with lysozyme. The membrane-mesosome fraction was isolated by centrifugation at 9000 x g for 20 minutes after lysing the protoplasts by diluting the suspensions with 2 volumes distilled water. Virtually all of the carotenoid pigment remained in the gel-like pellet obtained under these conditions. The deposit was suspended in 0.5 M sucrose-0.067 M p h o s p h a t e buffer and crystalline deoxyribonuclease (L. Light & Co.), added to give a final concentration of 20/~g/ml. After standing for 30 minutes at room temperature, the viscosity of the suspension had dropped considerably and the membrane-mesosome fraction was collected by centrifugation for 30 minutes at 36,000 x g, at 0 °. The deposits were washed twice with 0.5 M sucrose-phosphate buffer and the packed
FIGS. 1, 2 and 3. Thin sections of Micrococcus lysodeikticus cells showing the presence of intracellular membranous structures ("mesosomes" m). The organisms are surrounded by a plasma membrane (pro) and a thick outer cell wall (cw). The nuclear material (n), shown most clearly in Fig. 2, occurs in close proximity to the mesosomes. Stained with lanthanum nitrate. Figs. 1 and 2, x 105,000; Fig. 3, x 90,000.
492
M. R. J. SALTON AND J. A. CHAPMAN
pellet (36,000 ×g) was then ready for the preparative steps involved in thin sectioning for electron microscopy. (b) Lysates of M. lysodeikticus were prepared by direct lysis of the cells suspended in 0.5 M sucrose-0.067 M phosphate buffer, p H 7.0, with lysozyme as described for procedure (a). After incubation with deoxyribonuclease for 30 minutes at room temperature, the "particle" fraction was deposited and washed on the centrifuge as for method (a). (c) Membrane-mesosome fractions were similarly isolated from lysozyme lysates prepared in 0.067 M phosphate buffer, p H 7.0 without any sucrose present. The fractions deposited at 36,000 ×g were washed twice with 0.067 M phosphate buffer.
Fixatioa, embedding, and electron microscopy Deposits of the intact cells, lysed protoplasts and membrane-mesosome fractions were taken up in tryptone medium and fixed exactly as described by Kellenberger et al. (IlL All preparations were left in contact with the fixative (I ml) and 0.1 ml tryptone medium overnight at room temperature. Agar blocks of fixed materials were treated with either 0.5% uranyl acetate or with 1% lanthanum nitrate in buffer as described by Kellenberger et al. (11). The blocks were dehyrated in acetone following the procedure outlined by Kellenberger et al. (11) and were embedded in Araldite as described by Glauert and Glauert (4). Sections, about 500 • in thickness, were cut on a Huxley ultra-microtome and mounted on carbonfilmed grids. Specimens were examined in a Siemens Elmiskop I operating at 80 kV, using double condenser illumination and at an instrumental magnification of either × 20,000 or × 40,000. Micrographs were recorded on Ilford Special Lantern Plates (contrasty) with a n exposure time of 1 second or on Gevaert Diapositive Contrast Plates with an exposure time of 2 seconds. RESULTS A N D D I S C U S S I O N Thin sections of M. lysodeikticus cells shown in Figs. 1, 2 a n d 3 have revealed the presence of intracellular m e m b r a n o u s structures similar to those first seen so clearly in the c y t o p l a s m of Bacillus subtiIis sections p r e p a r e d b y R y t e r a n d K e l l e n b e r g e r (19) a n d in Streptomyces coelicolor studied by G l a u e r t a n d H o p w o o d (5), a n d subsequently in a variety of bacterial species (2, 3, 6, 7, 9, 10, 13, 17, 22, 24, 25, 28). F i t z - J a m e s (2) has p r o p o s e d the t e r m " m e s o s o m e " for these structures a n d we have a d o p t e d this n o m e n c l a t u r e until m o r e is k n o w n a b o u t the relationships of these organelles to m i t o c h o n d r i a . I n o u r sections of M. lysodeikticus we have observed o n l y one type of clearly defined m e s o s o m e organelle, the type m a d e up of a series of concentric m e m branes. M e s o s o m e s in the sections of Bacillus species by F i t z - J a m e s (2), Bacillus subtilis b y van I t e r s o n (25) a n d in Streptomyces coelicolor by G l a u e r t a n d H o p w o o d (5, 6) a p p e a r either as concentric m e m b r a n e s o r as c o m p l e x vesicular structures. FIGS. 4 and 5. The material obtained by fixation immediately after lysis of protoplasts of Micrococcus lysodeikticus. The most conspicuous structures present are the membranous structures (m) derived from the mesosomes and the plasma membranes. In addition fibrous material (f) and small spherical particles (sp) are found. Stained with uranyl acetate. × 90,000.
494
M. R. J. SALTON AND J. A. CHAPMAN
Whether these two types represent different stages in the development of the structure or whether the vesicular type arises from the folding up and compression of concentric membrane shells cannot be resolved at the moment. One other feature of the appearance of the thin sections of M. lysodeikticus shown in Figs. 1, 2 and 3 is the relatively thick wall possessed by the organism, at least under the growth conditions used. The average wall thickness for many Gram-positive bacteria is about 200 A (21) compared with 400-500 A for the cells shown in Figs. 1-3. The appearance of material obtained by fixation immediately after lysis of protoplasts of M. lysodeikticus is illustrated in Figs. 4 and 5. Membranous structures together with a background of electron-dense, particulate and fibrous material were found in this fraction. Some small spherical particles of unknown origin were also present in the lysed protoplast preparations. The typical appearance of the isolated "particulate" fraction from 34. lysodeikticus is shown in Figs. 6-9. The fractions isolated by all three methods (a, b, c) described above were indistinguishable from one another under electron microscopic observation. Thus what is normally regarded as a plasma (or protoplast) membrane fraction or a "large particle" fraction appears to be made up almost entirely of membrane structures of fairly uniform over-all thickness (75 A), there being anything from one to a maximum of 5 concentric shells. We therefore conclude that this fraction contains the unfolded membranes of the mesosome organelle as well as the plasma membrane, a conclusion compatible with the presence of continuous membranes appearing in thin section as circles of varying diameters. Yamaguchi (28) also isolated a "particulate fraction" from Mycobacterium aviurn composed largely of circular membrane structures, but the preparations did not show the system of concentric membrane shells described in this paper. Our results suggest that the mesosome is probably composed of 4 concentric spherical membranes in M. lysodeikticus and that in the intact cell the membranes are folded into a particle of dimensions smaller than that observed for the isolated structures. It appears likely that there is one mesosome organelle in each cell, judging from the results obtained with both intact cells and the isolated membrane-mesosome fractions. However, occasionally the large external membrane enclosed small membrane circles which were not concentrically arranged (see Fig. 7) and such an appearance could be due to mesosome duplication preceding the division of the plasma membrane or may be due simply to a breakdown of a single mesosome particle. FIG. 6. Typical appearance of the isolated particulate fraction from Micrococeus lysodeikticus, isolated by essentially the same procedures used for the isolation of plasma membranes. The fraction is made up almost entirely of membranous structures; in many cases these consist of a series of concentric shells. Diffuse membranes are due to oblique sectioning. Stained with uranyl acetate. x 55,000.
496
M. R. J. SALTONAND J. A. CHAPMAN
As shown in Figs. 8 and 9 each of the concentric shells has a typical double membrane appearance with two electron-dense layers bounding an electron-transparent layer, each layer being about 25 & in thickness. There is some evidence of particulate material attached to the outer surfaces of the electron-dense layers of this double membrane system. The double membrane frequently has a diffuse appearance, presumably due to oblique sectioning; the innermost shell in Fig. 9 shows this effect. Further investigations of the influence of environmental conditions during the isolation of the mesosome structures are clearly needed. The conspicuous unfolding or swelling of the structures during isolation has suggested that the effects of compounds found to be inhibitors of metabolic swelling of mitochondria may be useful in the isolation of these bacterial organelles and we are at present investigating this problem. The localization of enzymes of the electron-transport system in preparations variously described as "ghosts" (26), protoplast and plasma membranes (16, 23, 26), and particulate fractions (15, 16, 27) from Gram-positive bacteria capable of being transformed into bacterial protoplasts has been well established. Studies carried out in this laboratory in collaboration with Dr. J. Davies have also shown the presence of these enzyme systems in our m e m b r a n e - m e s o s o m e fractions. It is now almost certain that the activities described for the particle fraction isolated by Mathews and Sistrom (15) from Sarcina lutea reside in the m e m b r a n e - m e s o s o m e structures of the type we have isolated and structurally characterized. Finally, it should be emphasized that the co-separation of plasma membranes and mesosomes will inevitably complicate the interpretation of data on the localization of enzymes and the chemical composition of what has frequently been assumed to be a homogeneous preparation of protoplast membranes (8, 16, 27) unless of course both structures are of identical enzymic and chemical constitution. If, as Fitz-James (2) has suggested, all of the mesosomes originate as an invaginated growth of the plasma membrane, then there is a strong possibility that they would be composed of identical chemical and biochemical subunits. The authors would like to thank Mr. S. Grundy for assistance with the photography.
Fro. 7. Membranous structures in the particulate fraction showing a large external membrane enclosing several smaller non-concentric circles. The larger external membrane is probably the plasma membrane. Stained with lanthanum nitrate, x 90,000. FrGs. 8 and 9. Typical membranous structures at higher magnification, demonstrating the maximum number of concentric shells. The inner four double membranes are thought to arise from an unfolding of the mesosomes. Stained with uranyl acetate, x 145,000.
498
M. R. J. SALTON A N D J. A. CHAPMAN
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 1l. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
CHAPMAN,G. B. and HILLIER,J., d. Bacteriol. 66, 362 (1953). FITZ-JAMES, P. C., J. Biophys. Biochem. Cytol. 8, 507 (1960). GIESBRECHT,P., Zentr. Bakteriol. Parasitenk. Abt. I., Orig. 179, 538 (1960). GLAUERT,A. M. and GLAUERT, R. H., J. Biophys. Biochem. Cytol. 4, 191 (1958). GLAUERT, A. M. and HoPWOOD, D. A., J. Biophys. Biochem. Cytol. 6, 515 (1959). -ibM. 7, 479 (1960). GLAUERT,A. M., BRIEGER,E. M. and ALLEN, J. M., Exptl. Cell Research 22, 73 (1961). GUNSALUS,I. C. and STANIER,R. Y., The Bacteria, Vol. 1. Academic Press, New York, 1960. HAGEDORN,H., Deut. Botan. Ges. 73, 211 (1960). HICKMAN,D. D. and FRENKEL, A. W., J. Biophys. Biochem. Cytol. 6, 277 (1959). KELLENBERGER,E., RYTER, A. and S~CHAUD, J., J. Biophys. Biochem. Cytol. 4, 671 (1958). KERN, R. A., KINGKADE,M. J., KERN, S. F. and ]~EHRENS,O. K., J. Bacteriol. 61, 171 (1951). KOIKE, M. and TAKEYA, K., J. Biophys. Biochem. Cytol. 9, 597 (1961). LITWACK, G. and PRAMER, D., Proc. Soc. Exptl. Biol. Med. 91, 290 (1956). MATHEWS, M. M. and SISTROM,W. R., d. Bacteriol. 78, 778 (1959). MITCHELL,P., Ann. Rev. Microbiol. 13, 407 (1959). MOORE, R. T. and CHAPMAN, G. B., J. Bacteriol. 78, 878 (1959). ROBERTSON,J. D., Biochem. Soc. Symposia, Cambridge, Engl. 16, 3 (1959). RYTER, A. and KELLENBERGER,E., Z. Naturforsch. 13b, 597 (1958). SALTON, M. R. J., Nature 170, 746 (1952). -Microbial Cell Walls. John Wiley, New York, 1961. SHrNO~IARA,C., FUKUSm, K. and SuzuKI, J., J. Bacteriol. 74, 413 (1957). STORCK, R. and WACHSMAN,J. T., J. BacterioL 73, 784 (1957). TOKUYASO,K. and YAMADA, E., J. Biophys. Biochem. Cytol. 5, 123 (1959). VAN ITERSON,W., J. Biophys. Biochem. Cytol. 9, 183 (1961). WEIBULL, C., J. Bacteriol. 66, 696 (1953). -Ann. Rev. Microbiol. 12, 1 (1958). YAMAGUCHLJ., Science Repts Research Inst. T8hoku Univ. C9, 125 (1960).
489
Isolation of the Membrane-mesosome Structures from i~/icrococcus
lysodeikticus
M. R. J. SALTON1 and J. A. CHAPMAN
Department of Bacteriology and Rheumatism Research Centre, University of Manchester, England Received August 23, 1961 Micrococcus lysodeikticus cells possess a membranous organdie of about 2500 ~ diameter. This structure is similar to those found in other bacteria and the term "mesosome", which was proposed by Fitz-James to describe such organelles, has been adopted here. The mesosomes of Micrococcus lysodeiktieus have been isolated by differential centrifugation of lysed protoplasts and from lysates prepared by dissolution of the wall with lysozyme. The isolated mesosomes are composed of a series of concentric shells, each membrane being double and having an over-all thickness of about 75 ~. There are probably four concentric membranes forming the mesosome particle which on isolation is frequently surrounded by the plasma membrane forming a fifth shell. The maximum number of concentric membranes observed in the isolated membranemesosome fractious was 5. The present procedure does not permit a differential separation of plasma membrane from mesosome and it is concluded that previous protoplast membrane preparations of this organism contained the mesosome structures. With the isolation of the major structural components and organelles of the bacterial cell, our knowledge of the chemical and biochemical anatomy of bacteria has advanced rapidly during the past decade (8, 21). The surface structures of the bacterial cell, the wall and the protoplasmic membrane, have been isolated and chemically characterized (8, 21). The internal anatomy of the bacterial cell has recently attracted much attention and the complexity of its structure has been emphasized by the discovery of membranous and vesicular organelles in a variety of bacteria (2, 3, 5-7, 9-11, 13-17, 22, 24, 25, 28). These cytoplasmic structures obviously correspond to the "peripheral bodies" first detected in the early thin sections of bacteria studied by Chapman and Hillier (1). These membranous organelles frequently show a continuity with the plasma membrane and have been called "mesosomes" by Fitz-James (2) to distinguish them from mitochondria of other cells (18). 1 Present address: Department of Microbiology, University of New South Wales, Kensington, N.S.W., Australia.
490
M . R . J . SALTON AND J. A. CHAPMAN
Particles c o r r e s p o n d i n g to the m e m b r a n o u s organelles o b s e r v e d in Mycobaeterium avium have been i s o l a t e d b y Y a m a g u c h i (28) b u t little c o u l d be c o n c l u d e d a b o u t the structure of these particles f r o m the thin sections. I n t r a c e l l u l a r particles of a b o u t 2500 A d i a m e t e r have been k n o w n to be present in Mierococcus lysodeitcticus for s o m e time. T h e y have been o b s e r v e d on lysis of cells with lysozyme (12), a n d on disi n t e g r a t i o n of cells for the i s o l a t i o n of walls (20). The susceptibility of M. lysodeikticus to lysis with lysozyme p r o v i d e s a gentle m e t h o d of cell disintegration, a n d thus offered attractive possibilities for the i s o l a t i o n a n d eventual b i o c h e m i c a l c h a r a c t e r i z a t i o n of the i n t r a c e l l u l a r organelles.
MATERIALS AND METHODS
Organism and growth conditions Micrococcus lysodeikticus (NCTC 2665) was grown on a medium consisting of 1% Bacto peptone, 0.1% Difco yeast extract, 1% NaC1, being modified from that described by Litwack and Pramer (14). One liter of the medium (pH 7.5) inoculated with 50 ml culture of the organism was incubated at 30°C in a rotating 5-I flask, and the cells were harvested by centrifugation after 16 hours' growth.
Preparation of membrane-mesosome fractions Isolation of the mesosome "particles" was attempted by the following procedures. (a) Conversion of intact ceils into protoplasts by incubation of thick suspensions of M. Iysodeikticus in 0.067 M phosphate buffer, p H 7.0, containing 1.5 M sucrose, with 50/zg crystalline egg-white lysozyme (Armour Laboratory)/ml at room temperature (c. 20°C). The cell wall of this organism is completely digested by lysozyme (20), such a property being an essential prerequisite for naked protoplast formation (26, 27). Protoplast formation, judged by complete osmotic fragility on dilution in distilled water, had occurred within 15 minutes under the above conditions of treatment with lysozyme. The membrane-mesosome fraction was isolated by centrifugation at 9000 x g for 20 minutes after lysing the protoplasts by diluting the suspensions with 2 volumes distilled water. Virtually all of the carotenoid pigment remained in the gel-like pellet obtained under these conditions. The deposit was suspended in 0.5 M sucrose-0.067 M p h o s p h a t e buffer and crystalline deoxyribonuclease (L. Light & Co.), added to give a final concentration of 20/~g/ml. After standing for 30 minutes at room temperature, the viscosity of the suspension had dropped considerably and the membrane-mesosome fraction was collected by centrifugation for 30 minutes at 36,000 x g, at 0 °. The deposits were washed twice with 0.5 M sucrose-phosphate buffer and the packed
FIGS. 1, 2 and 3. Thin sections of Micrococcus lysodeikticus cells showing the presence of intracellular membranous structures ("mesosomes" m). The organisms are surrounded by a plasma membrane (pro) and a thick outer cell wall (cw). The nuclear material (n), shown most clearly in Fig. 2, occurs in close proximity to the mesosomes. Stained with lanthanum nitrate. Figs. 1 and 2, x 105,000; Fig. 3, x 90,000.
492
M. R. J. SALTON AND J. A. CHAPMAN
pellet (36,000 ×g) was then ready for the preparative steps involved in thin sectioning for electron microscopy. (b) Lysates of M. lysodeikticus were prepared by direct lysis of the cells suspended in 0.5 M sucrose-0.067 M phosphate buffer, p H 7.0, with lysozyme as described for procedure (a). After incubation with deoxyribonuclease for 30 minutes at room temperature, the "particle" fraction was deposited and washed on the centrifuge as for method (a). (c) Membrane-mesosome fractions were similarly isolated from lysozyme lysates prepared in 0.067 M phosphate buffer, p H 7.0 without any sucrose present. The fractions deposited at 36,000 ×g were washed twice with 0.067 M phosphate buffer.
Fixatioa, embedding, and electron microscopy Deposits of the intact cells, lysed protoplasts and membrane-mesosome fractions were taken up in tryptone medium and fixed exactly as described by Kellenberger et al. (IlL All preparations were left in contact with the fixative (I ml) and 0.1 ml tryptone medium overnight at room temperature. Agar blocks of fixed materials were treated with either 0.5% uranyl acetate or with 1% lanthanum nitrate in buffer as described by Kellenberger et al. (11). The blocks were dehyrated in acetone following the procedure outlined by Kellenberger et al. (11) and were embedded in Araldite as described by Glauert and Glauert (4). Sections, about 500 • in thickness, were cut on a Huxley ultra-microtome and mounted on carbonfilmed grids. Specimens were examined in a Siemens Elmiskop I operating at 80 kV, using double condenser illumination and at an instrumental magnification of either × 20,000 or × 40,000. Micrographs were recorded on Ilford Special Lantern Plates (contrasty) with a n exposure time of 1 second or on Gevaert Diapositive Contrast Plates with an exposure time of 2 seconds. RESULTS A N D D I S C U S S I O N Thin sections of M. lysodeikticus cells shown in Figs. 1, 2 a n d 3 have revealed the presence of intracellular m e m b r a n o u s structures similar to those first seen so clearly in the c y t o p l a s m of Bacillus subtiIis sections p r e p a r e d b y R y t e r a n d K e l l e n b e r g e r (19) a n d in Streptomyces coelicolor studied by G l a u e r t a n d H o p w o o d (5), a n d subsequently in a variety of bacterial species (2, 3, 6, 7, 9, 10, 13, 17, 22, 24, 25, 28). F i t z - J a m e s (2) has p r o p o s e d the t e r m " m e s o s o m e " for these structures a n d we have a d o p t e d this n o m e n c l a t u r e until m o r e is k n o w n a b o u t the relationships of these organelles to m i t o c h o n d r i a . I n o u r sections of M. lysodeikticus we have observed o n l y one type of clearly defined m e s o s o m e organelle, the type m a d e up of a series of concentric m e m branes. M e s o s o m e s in the sections of Bacillus species by F i t z - J a m e s (2), Bacillus subtilis b y van I t e r s o n (25) a n d in Streptomyces coelicolor by G l a u e r t a n d H o p w o o d (5, 6) a p p e a r either as concentric m e m b r a n e s o r as c o m p l e x vesicular structures. FIGS. 4 and 5. The material obtained by fixation immediately after lysis of protoplasts of Micrococcus lysodeikticus. The most conspicuous structures present are the membranous structures (m) derived from the mesosomes and the plasma membranes. In addition fibrous material (f) and small spherical particles (sp) are found. Stained with uranyl acetate. × 90,000.
494
M. R. J. SALTON AND J. A. CHAPMAN
Whether these two types represent different stages in the development of the structure or whether the vesicular type arises from the folding up and compression of concentric membrane shells cannot be resolved at the moment. One other feature of the appearance of the thin sections of M. lysodeikticus shown in Figs. 1, 2 and 3 is the relatively thick wall possessed by the organism, at least under the growth conditions used. The average wall thickness for many Gram-positive bacteria is about 200 A (21) compared with 400-500 A for the cells shown in Figs. 1-3. The appearance of material obtained by fixation immediately after lysis of protoplasts of M. lysodeikticus is illustrated in Figs. 4 and 5. Membranous structures together with a background of electron-dense, particulate and fibrous material were found in this fraction. Some small spherical particles of unknown origin were also present in the lysed protoplast preparations. The typical appearance of the isolated "particulate" fraction from 34. lysodeikticus is shown in Figs. 6-9. The fractions isolated by all three methods (a, b, c) described above were indistinguishable from one another under electron microscopic observation. Thus what is normally regarded as a plasma (or protoplast) membrane fraction or a "large particle" fraction appears to be made up almost entirely of membrane structures of fairly uniform over-all thickness (75 A), there being anything from one to a maximum of 5 concentric shells. We therefore conclude that this fraction contains the unfolded membranes of the mesosome organelle as well as the plasma membrane, a conclusion compatible with the presence of continuous membranes appearing in thin section as circles of varying diameters. Yamaguchi (28) also isolated a "particulate fraction" from Mycobacterium aviurn composed largely of circular membrane structures, but the preparations did not show the system of concentric membrane shells described in this paper. Our results suggest that the mesosome is probably composed of 4 concentric spherical membranes in M. lysodeikticus and that in the intact cell the membranes are folded into a particle of dimensions smaller than that observed for the isolated structures. It appears likely that there is one mesosome organelle in each cell, judging from the results obtained with both intact cells and the isolated membrane-mesosome fractions. However, occasionally the large external membrane enclosed small membrane circles which were not concentrically arranged (see Fig. 7) and such an appearance could be due to mesosome duplication preceding the division of the plasma membrane or may be due simply to a breakdown of a single mesosome particle. FIG. 6. Typical appearance of the isolated particulate fraction from Micrococeus lysodeikticus, isolated by essentially the same procedures used for the isolation of plasma membranes. The fraction is made up almost entirely of membranous structures; in many cases these consist of a series of concentric shells. Diffuse membranes are due to oblique sectioning. Stained with uranyl acetate. x 55,000.
496
M. R. J. SALTONAND J. A. CHAPMAN
As shown in Figs. 8 and 9 each of the concentric shells has a typical double membrane appearance with two electron-dense layers bounding an electron-transparent layer, each layer being about 25 & in thickness. There is some evidence of particulate material attached to the outer surfaces of the electron-dense layers of this double membrane system. The double membrane frequently has a diffuse appearance, presumably due to oblique sectioning; the innermost shell in Fig. 9 shows this effect. Further investigations of the influence of environmental conditions during the isolation of the mesosome structures are clearly needed. The conspicuous unfolding or swelling of the structures during isolation has suggested that the effects of compounds found to be inhibitors of metabolic swelling of mitochondria may be useful in the isolation of these bacterial organelles and we are at present investigating this problem. The localization of enzymes of the electron-transport system in preparations variously described as "ghosts" (26), protoplast and plasma membranes (16, 23, 26), and particulate fractions (15, 16, 27) from Gram-positive bacteria capable of being transformed into bacterial protoplasts has been well established. Studies carried out in this laboratory in collaboration with Dr. J. Davies have also shown the presence of these enzyme systems in our m e m b r a n e - m e s o s o m e fractions. It is now almost certain that the activities described for the particle fraction isolated by Mathews and Sistrom (15) from Sarcina lutea reside in the m e m b r a n e - m e s o s o m e structures of the type we have isolated and structurally characterized. Finally, it should be emphasized that the co-separation of plasma membranes and mesosomes will inevitably complicate the interpretation of data on the localization of enzymes and the chemical composition of what has frequently been assumed to be a homogeneous preparation of protoplast membranes (8, 16, 27) unless of course both structures are of identical enzymic and chemical constitution. If, as Fitz-James (2) has suggested, all of the mesosomes originate as an invaginated growth of the plasma membrane, then there is a strong possibility that they would be composed of identical chemical and biochemical subunits. The authors would like to thank Mr. S. Grundy for assistance with the photography.
Fro. 7. Membranous structures in the particulate fraction showing a large external membrane enclosing several smaller non-concentric circles. The larger external membrane is probably the plasma membrane. Stained with lanthanum nitrate, x 90,000. FrGs. 8 and 9. Typical membranous structures at higher magnification, demonstrating the maximum number of concentric shells. The inner four double membranes are thought to arise from an unfolding of the mesosomes. Stained with uranyl acetate, x 145,000.
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