Structural Features of Mesosomes

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STRUCTURAL FEATURES OF MESOSOMES

(CHONDRIOIDS) OF BACILLUS SUBTILIS

AFTER FREEZE-ETCHING

N. NANNINGA

From the I~aboratory of Electron Microscopy, the University of Amsterdam, Amsterdam, The Netherlands

ABSTRACT Frcezc-etched cells of Bacillus subtilis have been studied with thc clcctron microscope. The outer surface of the plasma mcmbranc, i.c. thc side facing thc cell wall, is covercd with numcrous granulcs and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to cnter the cell wall. The inner surface of the plasma membrane, i.e. thc side facing the cytoplasm, appears to be sparsely dotted with small particlcs measuring about 50 A. The envelope of mcsosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inncr surface appears smooth. Stalked particles, as described by othcr investigators after negative staining with phosphotungstic acid, wcrc not observed on any membranc surfacc in our material. Preparations wcrc also made of spccimcns prefixed in osmium tctroxide prior to frceze-ctching. Under these conditions the bacterial mcmbrancs appeared to bc surprisingly well preserved. In contrast to directly frozen, unfixcd cells, some osmium tetroxidc-fixed preparations showed a diffcrentiation in cytoplasm and nucleoplasm, which madc it possiblc to observe the close association of the mesosomc with the latter.

INTRODUCTION The freeze-etching technique represents an impor- freeze-etching technique is not yet so widely used tant method for the electron microscopic examina- as the method of thin-sectioning fixed and tion of the fine structure of organisms (18-20, 34). embedded specimens. Papers on the fine structure As outlined by Moor (20), the essential part of this of freeze-etched bacteria have included, among method is the quick freezing of the specimen in other subjects, the photosynthetic lamellae in order to protect it from being damaged by ice Rhodopseudomonas viridis (13), the cell envelope of crystals. Keeping the object in an environment FerrobaciUus ferrooxidans (21), the spore of Bacillus supplemented with 20% glycerol helps to prevent cereus (22), and the cytomembranes of Nitrosocystis growth of these crystals. Moor (20) was able to oceanus (23). No detailed studies had been show that yeast cells rapidly frozen under these published on bacterial mesosomes before the com- conditions continued to divide after thawing, and pletion of this article; since then, however, an thus he provided evidence that the fine structure article by Remsen has appeared (24). of the rapidly frozen cells is close to that of these In bacilli, complex membranous organelles cells in their natural state. described as chondrioids (27, 39) or mesosomes (9) Few studies of frcczc-ctched organisms or cell are commonly considered to arise as invaginations components have becn published so far because the of the plasma membrane, Such mesosomes are

251 dentical with the so-called peripheral bodies MgCI~, 0.25 M sucrose, and 20% glycerol. Freeze- described by Chapman and Hillier in 1953 (6) for etched preparations also were made of cells which Bacillus cereus. These bodies at that time revealed were grown without addition of glycerol, but which no visible fine detail, but they were considered to were fixed with osmium tetroxide according to the function in cell-wall synthesis. Membranous organ- method of Kellenberger et al. (16). elles have been observed in practically all bacteria Freeze-Etching studied so far (see reference 38). In Gram-positive bacilli these membranous structures have a special The procedure developed by Moor and Mfihle- thaler (19) was followed in all essentials. The etched organization, as described earlier for Bacillu~ sub- surface, at an object temperature of --100°C, was tilis (14, 15, 36, 37), B. megaterium (9, 12), B. cereus shadowed with platinum-carbon and reinforced with (9, 26), and B. medusa (9). In sections, "they can carbon. The cleaned replica was picked up on a appear as clusters of vesicles and tubules, as mem- Formvar-coated copper grid and, after being dried, branous whorls, or as a combination of these two was examined in the electron microscope. types of structure" (38). The functions of the organelle might be manifold. Its close association Fixation and Embedding with the cell wall suggests that the organelle has a Cells grown in heart-infusion broth were prefixed role in the synthesis of the wall (6, 12, 36, 37). in about 1% glutaraldehyde (31) in 0.067 ~ phos- Furthermore, evidence for the presence in the phate buffer pH 6.0, with 0.015 M MgC12 (PM- mesosome of reducing enzymes (17, 35, 39) and buffer) at about 4°C for 1 hr. After having been cytoehromes (8) has been presented. That the washed twice with PM-buffer, the ceils were fixed mesosome functions in the segregation of the divid- for 1 hr with 2% OsO4 in the same buffer plus try'p- ing nueleoid has been suggested (12) and con- tone : 9 volumes of fixative and 1 volume of tryptone (16). The next steps, viz. washing in acetate-Veronal sidered likely (28, 30). Sections of cells kept in a buffer, posttreatment with 0.5% uranyl acetate, de- hypertonic medium have provided evidence that hydration in acetone, and embedding in Vestopal W, the mesosomes are expelled from the cell interior were carried out according to the procedure of and accumulate as a collection of vesicles between Kellenberger et al. (16). the cell wall and the plasma membrane (10, 29). Thin sections were cut on an LKB ultratome ann The physiological conditions that determine the contrasted with lead citrate (25). In some sections development and the shape of mesosomes are not the OsO4 was removed by being bleached with yet fully understood. periodic acid (32). Afterwards, these sections were The author considered it of interest to supple- stained with lead citrate (25). ment the data derived from studies of fixed and Electron :~/licroscopy sectioned material and of negatively stained preparations (1,2) withobservationsmadeafterappliea- Electron micrographs were taken with either a tion of the freeze-etching technique. This paper Philips EM 200 or EM 300 electron microscope, describes mesosomes of cells from an ordinary operating at 80 kv, and with objective apertures of 40 and 30 /.~, respectively. medium supplemented with glycerol, of cells that, In contrast to the micrographs in other papers on after having been harvested, have been kept in a freeze-etching, the micrographs in the present paper glycerol-containing buffer made somewhat hyper- are printed in reverse, as is customary for shadowed tonic by means of sucrose, and of cells fixed in preparations. In all the electron micrographs pre- osmium tetroxide prior to freeze-etching. sented, the direction of shadowing is approximately from top to bottom (see arrow in lower right-hand MATERIALS AND METHODS corner of each micrograph). Cultivation for Freeze-Etching OBSERVATIONS B. subtilis, strain Marburg, was grown aerobically Initial difficulties were encountered in the interpre- in heart infusion broth (Difco Laboratories, Detroit, tation of the spatial arrangement of the structures Mich.) on a shaker at 35°C. For freeze-etching ex- periments ceils were grown for 4-5 hr in the above observed in the freeze-etched specimens. So that medium supplemented with 20% glycerol (13). After the perception of the three-dimensional structure the harvesting, the very thick suspension was either of the replica of the fractured bacteria could be used as such for the freeze-etching, or suspended in facilitated, the micrographs have been printed in 0.1 M phosphate buffer pH 6.0, containing 0.01 M reverse. Depending on the direction of shadowing

~52 TIlE JOURNAL OF CELL BIOLOGY • VOLUME 39, 1968 relative to the observer, structural elements certain granularity, probably in longitudinal arrays, appear either depressed or elevated. If the micro- and sometimes fibrils can be faintly (Figs. 1 and graph is then turned upside down, depressions be- 2) or more clearly (Fig. 3) recognized (arrows). come raised areas and vice versa. Consequently, For further explanation, see the Discussion. To the in the first place all the micrographs had to be so ar- left in Fig. 2 one sees a structure (M) that, by ranged that the shadows are approximately in the analogy with what is known from sectioned same direction. In the second place, the original material, can be identified as a mesosome (see direction of shadowing had to be known, In the below). In Fig. l, at the lower part of the preparations studied this direction could be deter- micrograph, the cross-fractured cell is bordered mined unambiguously by making use of knife by a concave, comparatively smooth surface scratches occasionally present in the fractured sparsely dotted with particles of the order of 50 A surface of the frozen specimen. These scratches are in diameter. This surface is thought to represent the always situated at a higher level than the parts inner face of the plasma membrane (ipm). Occa- from which the material has been removed by the sionally, fibrils can be seen to pass from the cyto- fracturing. When the micrographs are studied with plasm to this surface of the plasma membrane. the shadows pointed downwards, the original ele- Once one has become familiar with the sparsely vated and depressed parts are usually seen as such. dotted appearance of the inner surface of the However, after sufficiently accurate orientation of plasma membrane, it becomes possible to identify the micrographs has been made, the problem of see- this surface even in situations where the cell con- ing elevations and depressions reversed may occa- tent has been removed completely, as is the case at sionally reappear, owing to optical illusion. This ipm in Figs. 5, 6, 10, and 11. The outer surface of problem can then be overcome: if, for instance, a the plasma membrane (opm), i.e. the face that is structural element has its shadow at the lower side, turned toward the cell wall, is recognizable by its this element is protruding from its immediate convex appearance (Figs. 1, 2, 4, 6-9, 12, and 14); surroundings. it is dotted with far more particles and short Two observations indicate that we have suc- strands than the inner surface. The outer surface ceeded in obtaining fairly reliable preservation of (opm) can be seen to smoothly border the cross- the ultrastructure of our specimens. (a) The bacte- fractured cell wall (cw), and occasionally strands rial cells grown in the presence of glycerol and fro- from the outer surface of the plasma membrane zen at approximately -150°C resumed growth pass into the cell wall (arrows s in Figs. 1 and 14). when inoculated into fresh heart-infusion broth; The width of the strands and granules is approxi- this is in accordance with the results obtained by mately 50 A. The surface of the cell wall is rarely Moor (20) with frozen yeast cells. (b) No signs of revealed (see reference 5; see, however, Figs. 15 plasmolysis were seen in thin sections of bacteria and 16). It was practically always found to be cultivated in broth supplemented with glycerol; cross-fractured, and so far we have not been able this suggests that the interior of the cells had to distinguish various layers in the cell wall. reached equilibrium with the glycerol in their environment. The presence of glycerol, however, The Mesosome without Chemical Fixation does affect the growth rate of the cells. In the freeze-etched bacilli, as in sectioned With freeze-etching, structural details in bac- material, the mesosomes were found at the cell teria are in principle revealed in two ways, i.e., periphery, in contact with the septum separating by fracturing along existing faces (mostly mem- dividing cells, and also in the cell interior. Most of branes) or by cross-fracturing. However, both the mesosomes encountered were clusters of events can happen in one and the same cell (see for vesicles. The vesicles are not always round; many instance Fig. 1). Before the structural aspects of the of them appear bounded by sharply angled plane mesosome are discussed, some general features of surfaces, e.g., those in Figs. 2, 5, and 6. Fig. 2 freeze-etched B. subtilis will be described. shows a centrally located, complex meSosome (M) The cell content can be seen cross-fractured in in which a cluster of vesicles can be recognized Figs. 1-3. In contrast to the observations that can which appears to be connected to the plasma mem- be made in sections, no clear distinction is possible brane. The mesosome in Fig. 3 is similarly located here between cytoplasm and nucleoplasm. The and shows part of the mesosomal envelope. Meso- cell content (cc) appears to be characterized by a somes situated outside the plasma membrane (oM)

N. NANNINGA MesosomeStructure After Freeze-Etching 253 can be seen in Figs. 4, 6-8, 12, and 14 and in a (M) can be seen together with the inner surface of section in Fig. 13. At iM in Figs. 5 and 6 the vesicles the plasma membrane (ipm). It is not clear whether are inside the plasma membrane. In Fig. 6 at the vesicles are cross-fractured or the structures crw some vesicles can be seen in close contact with observed are imprints of vesicles in the inner sur- the cross-wall. face of the mesosomal envelope. There is a little Only rarely are vesicles seen as such, e.g., at iM knob in the center of each "vesicle cup." In Fig. and at crw in Fig. 6; in many cases one has the 11 the situation is almost identical with that in impression that the vesicle clusters are covered by Fig. 10, except that the mesosome appears to be a special envelope (see Figs. 4, 6-8, 12, and 14). composed mainly of tubules. In Fig. 12 such a In all figures the surface of this mesosomal enve- mesosome is seen at the outer surface of the plasma lope has a texture different from that of both the membrane, and it is covered by a smooth mesoso- inner and the outer surfaces of the plasma mem- mal envelope. The mesosomal tubules in Fig. 11 brane. In Figs. 4, 6-8, 12, and 14 the structure of are sharply bent, and it is noteworthy that heavy the outer surface of the plasma membrane can be ridges alternate with more delicate ones. compared with that of the supposedly outer surface Figs. 1-12 and 14 demonstrate cells preserved of the mesosomal envelope, whereas in the Figs. 6 in two different ways. The cells shown in Figs. I, and 9 so much of the mesosome seems to have been 2, 4--6, and 14 were freeze-etched directly after removed by the fracturing that the inner surface they had been collected from the glycerol-contain- of the mesosomal envelope (imm) might be ing broth, whereas those shown in Figs. 3 and 7-12, revealed. Particularly in Figs. 4, 6, and 7 the outer were harvested from the glycerol-containing me- surface of the mesosomal envelope appears to be dium and then were washed in 0.1 M phosphate studded with minute, blunt protrusions (p), which buffer pH 6.0 containing 0.01 M MgC12 and 0.25 are very different in size, distribution, and appear- M sucrose, besides glycerol. The morphologT and ance from the granules and short strands on both location of the mesosomes did not appear the outer and the inner surfaces of the plasma materially different in the two kinds of samples. membrane. The inner surface of the mesosomal envelope (irnm in Figs. 6 and 9) appears free of The Mesosome after Chemical Fixation protrusions; the outlines of the vesicles that have It was considered desirable to make a direct been removed fl'om it can still be recognized. comparison between freeze-etched cells and cells In Fig. 10, beautifully regular arrays of vesicles freeze-etched after chemical fixation. In addition,

FIGURE 1 Cross-fractured cell revealing the granular cell content (cc), the outer surface of the plasma membrane (opm), and the inner surface of the plasma membrane (ipm). Strands can be observed to pass from the cell content into the inner surface of the plasma membrane (arrows). At s, a strand can be observed to connect the outer surface of the plasma membrane with the cross-fractured cell wall (cw). Nuclcoplasm and cytoplasm can not be distinguished from each other (see Figs. 16-19). )< 78,000. FmURE ~ Cross-fractured cell showing cell content (cc), outer surface of the plasma membrane (opm), cross-fractured cell wall (cw), and a mesosome (M) composed of numerous vesicles. X 63,000. FmcnE 8 A mesosome (M) in a cross-fractured cell from a specimen that had been exposed to phosphate buffer containing 0.25 M sucrose (see Materials and Methods). The cell content appears to be rather fibrillar. Part of the mesosomal envelope is visible. Fibrils (arrows) can be seen in contact with the mesosome, co, cell content; cw, cell wall. >( 81,000. FIGURE 4 A mesosome (oM) located at the outer surface of the plasma membrane (opm). The mesosolne is partly covered by an envelope which has on its surface some blunt protrusions (p). cw, cell wall. >( 104,000. FIGURE 5 Extensive survey of the inner surface of the plasma membrane (ipm) on which the remains of a mesosome (iM) is still visible. X 104,000.

254 THE JOURNAL OF CELL BIOLOGY • VOLUME 39, 1968 N. NANNINGA Mesosome Structure After Freeze-Etching 255 Fig. 13 shows a section of a glutaraldehyde-fixed Figs. 15-19 illustrate the appearance of freeze- cell postfixed with osmium tetroxide. At the tip of etched B. subtilis from a sample prefixed according the cell in this section a cluster of vesicles can be to the method of Kellenberger et al. (16), but not seen in a situation which is almost identical with subjected to a posttreatment with uranyl acetate. that of a freeze-etched cell shown in Fig. 14. The In these specimens the nucleoplasm (N) is well two techniques (freeze-etching and sectioning) visible, although in a rather coarse texture (Figs. satisfactorily complement each other; for instance, 16-19). In these cells, which were not grown in the in Fig. 14 the plasma membrane and the mesosomal envelope are seen in extenso, whereas in Fig. presence of glycerol, the mesosomes were located 13 the plasma membrane is reduced to its sectioned in the same positions as in fixed and sectioned cells profile, and the mesosomal envelope cannot be (references 9, 12, 15, 37). Figs. 16, 18, and 19 show identified. In both figures the mesosome is situated the outer surface of the mesosomal envelope. The between the cell wall and the plasma membrane. structure of the surface of the outer envelope of In Fig. 14 it is seen in spatial relationship; in Fig. mesosomes of chemically prefixed cells differs 13 it is seen in only two dimensions. The periodic from that of either surface of the plasma mem- acid-bleached and lead citrate-poststained section brane in the same way that it differs from them in in Fig. 13 gives a very clear picture of the densely directly freeze-etched specimens (opm, Figs. 15, 16, stained ribosomes in their arrangement in the cyto- 18). In Fig. 16 the mesosome is connected at one plasm, and a few ribosomes also are seen in close end with the nucleoplasm, and at the other end association with the nucleoplasm. The densely stained ribosomes must correspond to the granular with the cross-wall (crw); in Figs. 18 and 19 the substructure in the freeze-etched cell content in mesosomes are in broad contact with the nucleo- Figs. 1 and 2. In Fig. 3, a cell exposed to phosphate plasm. A narrow extension of the mesosome in buffer with sucrose has cytoplasm of a somewhat Fig. 19 is in contact with the cell surface. In Figs. more fibrillar appearance. 15 and 16 the cell wall (cw) covering the plasma

FIGURE 6 Two cells separated by a cross wall (crw). In the left-hand cell the cell content has been removed to expose a cluster of mesosomal vesicles (iM) attached to the inner surface of the plasma membrane (ipm). The right-hand cell shows the outer surface of the plasma membrane (opm). Near the cross- wall (crw) a group of mesosomal vesicles is seen to be situated between the outer surface of the plasma membrane and the cross-fractured cell wall (cw). At oM some of the mesosomal vesicles have been broken away; their imprints thus are left on the inner surface of the mesosoliml envelope (imm). At p blunt protrusions are visible on the outer surface of the mesosomal envelope. )< 139,000.

256 '.['HE JOURNAL OF CELL BIOLOGY • VOLUME 39, 1968 FIGURE 7 Surface view of a mesosomal layer (oM) lying on the outer surface of the plasma membrane (opm). The mesosome seems to be covered by a membrane on which many blunt protrusions (p) can be seen. c~, cell wall. X 52,000.

FIGURE 8 Mesosome (oM) located at the outer surface of the plasma membrane (opm). Note regular array of vesicles. X 104,000. FmuuE 9 Similar situation as in Fig. 8, except that the mesosomal vesicles have been removed; this reveals the inner surface of the mesosomal envelope (imm). opm, outer surface of the plasma membrane; cw, cell wall. × 104,000.

Figs. 7-12 Cells from samples that had been held in 0.1 r~ phosphate buffer pH 6.0, containing 0.01 MgC]2, 0.25 M sucrose, and ~0% glycerol, after having been harvested from heart infusion broth supplied with 20% glycerol. membrane is preserved in extensive areas, a feature chemical prefixation with osmium tetroxide did which does not often occur in dirccfly frozen ceils, not materially affect the fine structure of either the or in chemically fixcd ones. plasma membrane or the mesosomal envelope in DISCUSSION freeze-etched bacilli. At the resolution obtained, In thc course of the prcsent study it was found that no fine structural differences could be distinguished

N. NANNINGA Mesosome Structure After Freeze-Etching 257 FIGURE 10 Part of a cell from which the cell content has been removed completely; this leaves behind the inner surface of the plasma membrane (i/rm) and traces of the mesosome (M). Whether the mesomal vesicles are cross-fractured or the vesicle cups are imprints in the inner surface of the mesosomal envelope is difficult to judge. Note the little knob in the centre of each vesicle cup. X 104,000.

FIGURE 11 Situation comparable to that seen in Fig. 10. The mesosome (M) is mainly composed of tubules. The tubules are bent in angles, and heavy ridges alternate with thinner ones. ipm, inner surface of the plasma membrane; cw, cross-fractured cell wall. X 104,000.

FIGURE l~ Mesosomal tubules (at oM) lying at the outer surface of the plasma membrane (opm). cc, cell content. X 104,000.

258 FIGURE 13 Glutaraldchyde- and osmium tctroxide-flxed cell. The sections were bleached with periodic acid (82) and afterwards were contrasted with lead citrate (25). Note the prominence of the ril)osomes, and their close proximity to the nucleoplasm (N). pm, plasma membrane; cy, cytoplasm~ cw, cell wall; M, mesosome (see Fig. 14). )< 130,000.

FmtmE 14 Freeze-etched cell in which the location of the mesosome (oM) is comparable with that in Fig. 13. opm, outer surface of the plasma membrane; s, strands passing from the plasma membrane into the cell wall (cw). X 130,000.

N. NANNINGA Mesosome Structure After Freeze-Etching 259 FIOURE 15-19 Cells fixed in osmium tetroxide (16) before freeze-etching. Under these conditions the nueleoplasm (N, Figs. 16-19) beeomes differentiated from the cytoplasm (cy) (See Figs. 1-3). The mesosomes (M) are in close contact with the nueleoplasm, and they extend from there to the eross-wall (erw, Fig. 16) or to the cell periphery (Figs. 18, arrow, and 19). The outer surface of the plasma membrane (opm, Figs. 15, 16, 18) is not appreciably altered by the osmium tetroxide fixation. Extensive areas of the eell wall (ew, Figs. 15, 16) are revealed in several places. X 75,000. between the plasma membranes or between the hand, although its external and internal surfaces mesosomal envelopes in freeze-etched specimens differ from one another, has a surface structure compared with prefixed, freeze-etched specimens. which is totally different from that of the plasma The outer surface of the plasma membrane has membrane. The mesosomal envelope therefore a rough texture consisting of numerous granules may not be considered to be a mere invagination and short strands, whereas its inner surface is of the plasma membrane; at best, it can be looked sparsely studded with irregularly shaped, small upon as a differentiation of the latter. If the enve- particles. The mesosomal envelope, on the other lope had been a simple invagination of the plasma

260 THE JOURNAL OF CF~LL BIOLOGY • VOLUME 39, 1968 membrane, the particles studding the surface of granules and granular strands, about 50 A in the plasma membrane would have been carried diameter, which are particularly prominent on the into the cell interior as well. Since this is not the outer surface of the cell membrane, is so far not case, it seems more reasonable to consider the meso- known. The only suggestion that can be made at somes to be a cell organelle distinct from but in present is that they might somehow correspond to continuity with the plasma membrane. This the so-called "bridges" (15) revealed in thin sec- distinction between mesosome and plasma mem- tions when the cell plasm retracts from the cell brane is not surprising in view of their different wall. The small particles observed on the inner chemical compositions, i.e. the content of reducing surface of the plasma membrane resemble those enzymes (17, 35, 39), the presence of cytochromes on the outer surface, but data regarding their (8), and the ratio of lipid to protein (11). significance are also lacking; perhaps they provide Birjuzova and Meissel (2) isolated mesosomal the points for the attachment of the ribosomal membranes from Micrococcus lysodeikticus and found strands to the cell membrane. that after negative staining with phosphotungstic Contrary to that seen in sections (Fig. 13), no acid these membranes appeared to be covered with clear distinction exists between the cytoplasm and numerous stalked particles ("mushroom-like sub- the nucleoplasm of directly frozen cells (cc in Figs. structures") which measured 70-80 A. Since these 1-3). In sectioned specimens, nucleic acid--con- authors found no particles on the plasma m em~ taining structures are revealed particularly by their brane, they considered the stalked particles to be the strong affinities to uranyl and lead compounds. In bacterial equivalents of the elementary particles of freeze-etched specimens, the situation is clearly the mitochondrion (7). In the present study on different: after quick freezing, both the cytoplasmic freeze-etched bacilli, stalked particles were not and the nucleoplasmic components remain in their observed on any of the mesosomal membranes, original dispersed state amidst various com- the mesosomal vesicles or the mesosomal envelope. pounds which hardly contribute to the electron Mushroom-like substructures of 70-80 A are in microscopic image seen in a chemically fixed fact even larger than the granules and the short section contrasted with heavy metals. In our expe- strands observed here on both surfaces of the plasma rience so far, the nucleoplasmic region was distin- membrane; this makes it very unlikely that they guishable from the cytoplasm only in some of the would have been overlooked had they been pres- specimens that were fixed with osmium tetroxide ent. Abram (1), working with phosphotungstic prior to freeze-etching (Figs. 16-19), probably acid-stained preparations of Bacillus stearothermoph- owing to a process of coagulation. ilus and other bacilli, noticed particles of about The locations of the mesosomes are the same in the same size ("structural units") on the cyto- the osmium tetroxide-fixed, freeze-etched cells plasmic face of the plasma membrane and on its and in sections (9, 12, 15, 37), i.e. one end of the infoldings. SjSstrand et al. (33), on the other hand, mesosome was often seen to be in close contact with arrived at the conclusion that, in negatively stained the nucleoplasm whereas the other end contacted membranes of mitochondria, the stalked particles either the cross-wall (Fig. 16) or the cell periphery arise as artifacts. (Figs. 18 and 19). In the glycerol-impregnated Earlier descriptions of the outer surface of the cells, both with and without 0.25 M sucrose, the plasma membrane of Ferrobacillus ferrooxidans (21) mesosomes were frequently observed in large areas and B. cereus (22), have indicated that in these two over the cell membrane where they protruded into bacteria the outer surface has a rough texture. the infrawall space. This result may be due to the However, the size of the particles on this surface in high osmolarity of the medium, but it needs further these organisms was reported to be about twice the investigation. Other investigators (10, ' 29) have size we have estimated for the particles in B. used more or less comparable media when hyper- subtilis. The inner as well as outer surfaces of the tonicity was desired. Their observations on sec- plasma membrane of Nitrosocystis oceanus (23), have tions differ from the present ones in that the meso- been observed, but no structural distinction has somal vesicles appear to be dispersed and not, or no been made between the two. In Escherichia coli longer, wrapped in a mesosomal membrane (10, (Unpublished observations.) the plasma mem- 29, 37). On the other hand, in many instances one brane appeared to be structurally identical with may infer, from published electron micrographs that described for B. subtilis. The significance of (Figs. 1 and 2 in reference 10, and Figs. 7 and 9

N. NANNINGA Mesosome Structure After Freeze-Etching 261 in reference 37) of. cells grown and fixed under firm almost entirely the most recent results of standard conditions, that mesosomal vesicles are Remsen (24). Interestingly, he observed the nu- also present in the infrawall space, although they clear material in freeze-etched old cells but not in are often not sufficiently conspicuous to have freeze-etched young ones, whereas the present attracted the attention of the various authors. work indicates that it was possible to visualize the The mesosomal envelope appears as a delicate nucleoplasm only in the young cells when these layer which (Fig. 13) is not easily observed in sec- cells were chemically fixed prior to freeze-etching. tions. The little knobs on its surface (Figs. 4, 6, 7) A study of the visibility of the nucleoplasm in form an interesting feature which needs further freeze-etched cells is in progress. It seems investigation. Whether the structures in Figs. l0 premature to suggest a model for the mesosome. and 11 are imprints of mesosomal vesicles or tu- Moreover, the location and morphology of the bules or structures split in halves as yet cannot be mesosomes may well depend on growth conditions decided with certainty. Up to the present time, that as yet have not been sufficiently analyzed. It cross-fractured cell membranes have failed to yield is of interest, however, to observe how well the clear indications of a layered substructure similar mesosomes in bacilli are preserved by Moor and to that which is often observed in electron micro- Mtihlethaler's technique (19) when applied to graphs of thin sections, and there have been no osmium tetroxide-fixed cells. Also, the plasma indications of a splitting of the membranes along membrane appeared little altered by the chemical an internal face (3, 4). The way in which the frac- fixation. turing occurs, either externally or internally, along Much remains to be learned about mesosomes, the membrane sheet probably depends on the however. Meanwhile the present study justifies the properties of the object (see reference 5). somewhat unexpected conclusion that the fine So far, no descriptions of mesosomes (or chon- structure of the mesosomal membranes differs drioids) in freeze-etched growing bacteria have from that of the plasma membrane. been published, but after this paper had been The author is indebted to Dr. Woutera van Iterson submitted for publication we learned about the for her valuable help in preparing the manuscript. very recent appearance of an article by Remsen The excellent technical assistance of Mrs. D. van (24) who previously had shown mesosomes in a Winkel-Konter, Mrs. J. Rapha~l-Snijer, and Mr. freeze-etched spore of Bacillus cereus (22). Our P. J. Barends is gratefully acknowledged. results insofar as they cover the same aspects, con- Receivedfor publication 22 April 1968.

REFERENCES

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N. NANNINGA Mesosome Structure After Freeze-Etching 263