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Electron microscopic cytochemical study of cell-wall polysaccharides in Bacillus subtilis and two strains of Bacillus megaterium
JOURNALOF ULTRASTRUCTURERESEARCH81, 66-77 (1982)
Electron Microscopic Cytochemical Study of Cell-Wall Polysaccharides in Bacillus subtilis and Two Strains of Bacillus megaterium CLAUDE FREHEL AND ANTOINETTE RYTER
Unit~ de Microscopie Electronique, D~partement de Biologie Moldculaire, Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris Cedex 15, France Received November 25, 1981, and in revised form June 1, 1982 Thin sections of Bacillus subtilis and B. megaterium cells were treated by two cytochemical techniques revealing polysaccharides: the phosphotungstic-acid (PTA) and the silver-proteinate (SP) stainings. These procedures were applied to exponentially growing cells as well as cells treated with chloramphenicol (CMP) or cells regrown after antibiotic elimination. It appeared that both stains mainly deposited on the cell wall, but did not give the same staining patterns. In vegetative and CMP-treated cells, PTA gave a homogeneous dark deposit, whereas with S P a deposit was only observed on the outer and inner wall faces. During cell-wall thinning, PTA-stained walls remained rather homogeneous, whereas the silver deposit spread throughout the cell-wall thickness. The meaning of these differences in relation to cell-wall polymer organization is discussed.
We have previously shown that in a D a p - L y s - strain of Bacillus megaterium a high-acid-precipitable peptidoglycan turnover occurs during growth. This release seems to be produced by lyric enzyme(s) excreted into the culture medium, and to correspond to the detachment of large pieces from the superficial wall layer (Frehel and Ryter, 1979; Frehel et al., 1980). One can wonder whether the filamentous layer surrounding the cell wall of this strain reflects the superficial enzymatic wall degradation or corresponds to a sort of capsule, the chemical composition of which would be different from the cell wall itself. To obtain more information on the cellwall constituents and their distribution in this envelope, we have used two cytochemical staining methods that reveal different polysaccharides: (1) the silver-proteinate (SP) method (Thiery, 1967) that was shown to reveal teichoic acid in bacteria (Rousseau and Hermier, 1975), and (2) the phosphotungstic-acid (PTA) method (Ramb o u r g , 1969) t h a t c o u l d r e a c t w i t h peptidoglycan (Rousseau and Hermier, 1975). This study was made with three strains
ofB. megaterium: strain MB, its D a p - L y s mutant with which the turnover study was made, and strain MA (Aubert et al., 1961). The first two strains are characterized by the fact that they never form long cellular chains contrary to other B. megaterium strains, which suggests a high lytic activity. The MA strain orB. megaterium forms long cellular chains. Moreover, strain 168 of B. subtilis was used for comparison because it is the most intensively studied bacillus species from the biocehemical and cytochemical point of view. These techniques were applied to growing cells and also to bacteria treated with chloramphenicol (CMP) and then transferred into fresh culture medium in which growth resumed. It is well known that protein synthesis inhibition induced by CMP leads to wall thickening in gram-positive bacteria (Shockman, 1965; Giesbrecht and Ruska, 1968; Frehel et al., 1971; Hughes et al., 1970). When the cells are resuspended in fresh medium devoid of antibiotic, the cell wall recovers its normal thickness during the first generation of growth. Electron microscope observations (Frehel et al., 1971) showed that the first step of wall thin66
0022-5320/82/100066-12502.00/0 Copyright O 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
STAINING OF CELL-WALL POLYSACCHARIDES
ning consists of the formation of many transverse cracks, suggesting the presence of a high lytic activity. The peptidoglycanturnover study made during this process indicated, however, that contrary to what was expected, wall thinning was not accompanied by an important release of wall material. Moreover, DAP incorporation that occurs during wall thinning and cell elongation remained lower than during normal growth (Frehel et al., 1980). These observations suggested that the bacterium uses the material in its thick wall for elongation, which implies a profound wall reorganization. The present cytochemical study confirmed this assumption, brought new data on the distribution of wall constituents, and showed that the filamentous outer layer of strain MB has the same composition as the wall itself and very probably reflects a high lytic degradation. MATERIALS AND METHODS
Bacterial Strains and Medium Experiments were performed with three different strains of B. megaterium: strain MA (Aubert et al., 1961), strain MB and a Dap-Lys- mutant strain isolated from strain MB in our laboratory (de Chastellier et al., 1975), and also with strain 168 of B. subtilis Marburg. Bacillus megaterium cells were grown in Salton synthetic medium (Aubert et al., 1961) supplemented with 0.2% glucose and 1% yeast extract; for the Dap-LysMB mutant strain mesodiaminopimelic acid and lysine (de Chastellier et al., 1975) were added. Cells were grown at 30°C under strong agitation; the doubting time was 35--40 rain. Bacillus subtilis 168 strain was grown in complex medium (Ryter, 1965) at 37°C under strong agitation; its doubling time was 30--40 rain.
Cell Treatments Plasmolysis. Exponentially growing cells were centrifuged and resuspended in phosphate buffer, pH 7, added to 0.5 M sucrose. They were incubated for 20 min at 30°C with gentle shaking; plasmolysis was monitored under the light microscope. Chlorarnphenicol treatment and resumption of growth. Exponentially growing ceils were treated for 2 hr with 20 to 40 tzg/ml of chloramphenicol (CMP). They were centrifuged and resuspended in four times the initial volume of fresh medium or partially spent medium (Frehel and Ryter, 1979). Regrowth was fol-
67
lowed by turbidimetry. Samples were withdrawn at the end of the CMP treatment, and at different times during growth resumption, and processed for electron microscopy.
Electron Microscopy Fixation and embedding. Samples were centrifuged and embedded in agar (Whitehouse et al., 1977) and fixed (l) overnight with 1% osmium tetroxide in Michaelis buffer (Ryter and KeUenberger, 1958) (Os fixation), (2) overnight with 1% osmium tetroxide in Michaelis buffer, and postfixed with uranyl acetate in the same buffer (Ryter and Kefienberger, 1958) (Os +Ua fixation), (3) overnight with 2.5% glutaraldehyde in Michaelis buffer, washed for 4 hr in the same buffer, and fixed overnight with osmium tetroxide as in 1 (GaOs fixation), (4) overnight with ghitaraldehyde, then osmium as in 3, and postfixed for 1 hr with uranyl acetate in Michaelis buffer (Ga--Os +Ua fixation). Fixation with glutaraldehyde alone was not performed because of the bad preservation of the cell ultrastructure. All samples were dehydrated with acetone and embedded in Epon. Sections cut with a diamond khife were contrasted with lead citrate or treated with polysaccharide stains. Staining of Polysaccharides Thin sections were gathered in plastic rings and floated on different reagents using one of the following techniques.
(i) Silver proteinate staining (SP) (Thiery, 1967). This technique is based on oxidation of 1-2a-glycol bonds into aldehydes by periodic acid (PA). Aldehydic groups then react with thiocarbohydrazide (TCH) to form hydrazones; the final treatment with silver proteinate (SP) results in a fine silver granulation on the 1-2a-glycol linkage. To eliminate nonspecific deposits because of Os fixation, the sections were deosmicated with 10 Vol hydrogen peroxide (HP) before periodate oxidation (Robertson et al., 1975). The standard silver staining procedure was as follows: (HP) 15 min; 1% periodic acid (PA) in distilled water 30 min; 0.2% thiocarbohydrazide (TCH) in 20% acetic acid 24 hr (B. subtilis), or 48 hr (B. megaterium MB), or 72 hr (B. megaterium MA); 1% silver proteinate (SP) in bidistilled water 30 rain (B. megaterium MB) or 35 min (B. subtilis; B. megaterium MA). After each incubation, sections were rinsed for 10 sec three times, and 10 rain three times with distilled water; moreover, after thiocarbohydrazide treatment they were rinsed with different concentrations of acetic acid, then with bidistilled water. The tests described below were done to check the specificity of the silver-staining p r o c e d u r e : (a) TCH+SP. Free aldehyde groups coming from ghataraldehyde or tissue-bound osmium present in the cells before PA oxidation could nonspecifically react with TCH and SP. Therefore, this control was done after a
68
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STAINING OF CELL-WALL POLYSACCHARIDES double fixation with glutaraldehyde and osmium tetroxide, and after a simple fixation with osmium tetroxide. (b) H P + T C H + S P . At this concentration, HP completely removes osmium from thin sections of fixed cells and does not oxidize glycols to aldehydes, ff nonspecific staining is due to osmium tetroxide bound to cell structures, this reaction will be negative; if it is due to free aldehydes present in the cell, staining will persist. (c) SP only. This reveals cell structures having a nonspecific affinity for silver. (d) PA+SP. This reaction checks for groups able to reduce SP after PA oxidation. These different controls were negative. (ii) Phosphotungstic acid staining at low p H as described by Rambourg (1971) and modified by Roland et al. (1972) (PTA). To date the staining mechanism is unknown, it was only proposed that it reveals hydroxyl groups. Thin sections were floated on 1% periodic acid for 15 to 30 min, rinsed with bidistilled water (10 sec three times and i0 rain three times), floated for 4 rain on phosphotungstic acid (PTA) in 10% chromic acid, and rinsed rapidly in bidistilled water. Controls were made by floating sections on PTA at pH 7. All sections were gathered on 400-mesh copper grids with or without a Formvar film. RESULTS
Because B. megaterium strain MB and its Dap-Lys- mutant presented the same morphological appearance and reacted identically to all staining procedures, the name MB refers to both strains in the following results.
Exponentially Growing Cells After the conventional lead-citrate staining, the cell wall of B. subtilis presented the trilamellar structure frequently described: two dense layers enclosing an electron translucent internal layer (Fig. 1). This appearance was found whatever the fixation procedure (Os, Os+Ua, Ga-Os+Ua, GaOs). The cell wall of B. megaterium MB showed a single and homogeneous stained
69
layer surrounded by a filamentous layer after Os-Ua or G a - O s + U a fixation (Fig. 2); if Ua postfixation was omitted, this outer filamentous layer was irregular and coarsely aggregated onto the cell wall (Fig. 3). The MA-strain cell wall also consisted of a homogeneously stained layer covered by dispersed filaments (Fig. 4), but no changes were found in the absence of Ua postfixation. (i) Silver proteinate staining (SP). To eliminate nonspecific staining due to osmium tetroxide bound to cell structure, s, sections were treated with HP prior to periodic acid oxidation. On thin sections stained according to this procedure, the silver deposit appeared mainly on the cell walls along their entire length including poles (Figs. 12-14). On the B. subtilis cell wall, the silver deposit formed a stratified structure as observed by Rousseau and Hermier (1975): a thin granulated layer and a thick outer granulated one enclosing a poorly stained central region (Figs. 5 and 12). A similar distribution was observed in the B. megaterium MB strain, the outer layer corresponding to the filamentous layer in 1Lhe Os+Ua" or G a - O s + U a fixed cells (Figs. 6 and 13) or to the coarse aggregated layer in the Os- or Ga-Os-fixed cells (Fig. 7). In strain MA the reaction was lighter, the outer filamentous layers showing dispersed silver deposit (Figs. 8 and 14). This outer granulated layer appeared after an incubation time of only 72 hr. During septation the silver granulation was found only along the inner wall face, the outer deposit appearing only at the onset of septum cleavage (Figs. 13 and 14). A light and diffuse granulation was observed inside some fully formed septa of B. subtilis (Fig. 12) (see arrowhead).
Fins. 1-4. Lead-citrate staining; Fig. 1, strain 168 of B. subtilis; Figs. 2 and 3, strain MB of B. megaterium, Os + Ua fixation (Fig. 2), Os fixation without Ua (Fig. 3); Fig. 4, strain MA ofB. megaterium. FIGs. 5-8. SP staining; Fig. 5, strain 168; Figs. 6 and 7, strain MB, Os + Ua fixation (Fig. 6), Os fixation without Ua (Fig. 7); Fig. 8, strain MA. FIGS. 9-11. Plasmolyzed cells stained with SP procedure. Fig. 9, strain 168; Fig. 10, strain MB; Fig. 11, strain MA. CM, cytoplasmic membrane.
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STAINING OF CELL-WALL POLYSACCHARIDES
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FIGS. 18-29. Thin sections of B. megaterium (strain MB) after a CMP treatment and during the first generation of growth resumption after antibiotic elimination; detail of the cell wall. Bar represents 0.15/xm. FIGS. 18, 20, 22, 24, 26, AND 28. PTA staining. FIGS. 19, 21, 23, 25, 27, AND 29. SP staining. FIGS. 18 AND 19. End of the CMP treatment.
STAINING OF CELL-WALL POLYSACCHARIDES
To see whether the inner silver granulated layer corresponded to the inner-cell-wall face or to the cytoplasmic membrane, silver staining was applied to plasmolyzed cells. In all strains, silver granulation deposited on the inner-cell-wall face (Figs. 9-11, see arrowheads) and not on the cytoplasmic membranes. (ii) Phosphotungstic acid + chromic acid staining (PTA). With this technique a dark deposit completely covered the cell wall including the poles and septa of the B. subtilis (Fig. 15) and the B. megaterium (Figs. 16 and 17) strains. The outer filamentous layer was also contrasted in strain MB; in strain MA the outer filaments were poorly contrasted with PTA, as compared to their staining with SP (Fig. 14). In the B. megaterium cells the staining became lighter during septum cleavage (Figs. 16 and 17, see arrowheads). No deposits were observed on the cytoplasmic membrane or mesosomes in. all strains. Chloramphenicol-Treated Cells and Growth Resumption
CMP treatment results in the thickening of the cell wall of B. subtilis and B. megaterium (Frehel et al., 1971, 1980). When cells are allowed to regrow in fresh medium devoid of antibiotic, transverse cracks caused by a lyric process progressively appear along the thick cell wall. This causes cell-wall thinning, with the recovery of its initial width by the end of the first generation (Frehel et al., 1971, 1980). As said under Materials and Methods, samples were fixed at the end of the CMP treatment and at different times during the first generation of regrowth. Thin sections were treated with both polysaccharide stains. (i) SP stain. After CMP treatment the cell
73
wall of B. subtilis and B. megaterium presented the stratified silver deposit observed in exponentially growing cells (Figs. 19, 31, and 35). The inner and outer layer, corresponding to the filamentous material in B. megaterium strain, appeared as thin as in exponentially growing cells, whereas the unstained central layer was much thicker. When growth resumed the thickness of the outer silver deposit increased in all strains by progressively spreading toward the inner layer. At the end of the first gteneration, only a thin central layer remained unstained (Figs. 21, 23, 33, and 37; Chart 1A). In the MA strain, the enlargement of the outer silver deposit was not so striking because of the low number of silver grains, but the granulation also appeared inside the central region (Fig. 37). These observations suggested that the spreading of silver granulation was related to the lyric activity responsible for the wall cracking and thinning. To verify this assumption CMP-treated bacteria (strain MB) were resuspended in partially spent medium in which the lytic activity enhances the enzymatic degradation of the cell wall (Frehel et al., 1980). In this case cell-wall thinning does not occur by transverse cracking, but by longitudinal cleavage leading to 1Lhe shedding of large scallops of cell wall (Frehel et al., 1980). In partially spent medium, silver grain spreading did not occur by 1the widening of the outer layer, but by the longitudinal extension of discrete granulated regions inside the unstained layer (Figs. 25 and 27, see arrowhead). This occurred during the first generation until the appearance of the cleavage zone between the old and the new cell wall (Fig. 29, Chart 1B). During this process, the old cell wall progressively became homogeneously stained. (ii) PTA strain. In all strains the CMP
FIGS. 20-23. Growth resumption in fresh medium; middle of the first generation (Figs. 20 and 21) and ,end of the first generation (Figs. 22 and 23). FIGS. 24-29. Growth resumption in partially spent medium; beginning of the first generation (Figs. 24 and 25), middle of the first generation (Figs. 26 and 27), and end of the first generation (Figs. 28 and 29).
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FIGS. 30-37. Strain 168 and strain M A treated with C M P and regrown after antibiotic elimination. Bar represents 0.1 /xm. FIGS. 30, 32, 34, AND 36. P T A staining. FIGS. 31, 33, 35, AND 37. SP staining. FIGS. 30 AND 31. B. subtil& treated with CMP. FIGS. 32 AND 33. E n d of the first generation of regrowth in B. subtilis.
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thick cell wall was strongly stained (Figs. 18, 30, and 34). During growth resumption in fresh medium, the cell wall remained entirely stained, but the stained material was progressively eliminated as the cell wall became thinner (Figs. 20, 22, 32, and 36). When strain MB was resuspended in partially spent medium, discrete longitudinal unstained regions appeared inside the thick cell wall as growth resumed (Fig. 24, see arrowhead); they spread longitudinally (Fig. 26) and at the end of the first generation the two cell wall layers separated from one another in these regions (Fig. 28). DISCUSSION
In this study PTA and SP procedures did not give the same staining patterns. In all strains PTA, which seems to react preferentially with peptidoglycan (Rouseau and Hermier, 1975; Frehel et al., 1982), gave a homogeneous staining of the cell wall as previously observed in B. subtilis (Rousseau and Hermier, 1975). This was observed in exponentially growing cells as well as after CMP treatment and wall thinning, and suggests a homogeneous distribution of peptidoglycan in the cell wall. Contrary to PTA, SP gave a tribanded structure in all strains with a much lighter staining in strain MA. In both B. megaterium strains the out-
FIGS. 34 AND 35. FIGS. 36 AND 37.
er layer covered by silver grains corresponded to the filamentous layer. As shown by plasmolysis experiments the thin inner layer was located on the inner face of the cell wall and not on the cytoplasmic membrane as opposed to what was found in Nocardia (Robertson et al., 1975) and Mycobacterium (Petitprez and Durieux, 1970) species. The location of the silver deposit did not depend upon the fixation procedure (Dubray, 1975); in strain MB, however, the outer filamentous layer was coarsely aggregated onto the wall when Ua postfixation was omitted. It was proposed that SP mainly reacts with teichoic acid in B. subtilis (Rousseau and Hermier, 1975; Frehel et al., 1982) and analogous polysaccharides in B. megaterium (Frehel et al., 1982). At first view the tribanded structure seemed to indicate that these nonpeptidoglycan polymers are mostly concentrated in the outer and inner cellwall layers as proposed by some workers (Nermut, 1967; Tsien et al., 1978). This assumption, however, is in disagreement with different data: (i) Doyle et al. (1975) have shown that Concanavalin-A receptor sites of teichoic acid increased during wall dlegradation by lysozyme, suggesting that this polymer was present throughout the wall. (ii) Mauck and Glaser (1972) reported that
Strain M A treated with CMP. E n d of the first generation of regrowth in strain MA.
76
F R E H E L AND RYTER
teichoic acid and peptidoglycan synthesis were coordinated; moreover, Archibald (1976) as well as Anderson et al. (1978) demonstrated that teichoic acid is first inserted as peptidoglycan in the inner cellwall layer and then migrates toward the outer cell surface. These data suggest that teichoic acid is present throughout the cell wall. (iii) At last, this cytochemical study showed that in CMP-treated cells the silver granulation occurred only on two thin inner and outer layers, although both teichoic acid and peptidoglycan continued to be synthesized (Hughes et al., 1970). It is thus unlikely that the absence of staining in the thick central zone corresponds to the absence of teichoic acid. In fact, our cytochemical study on cells regrown after a CMP treatment suggested that teichoic acid molecules (or analogous polysaccharides in B. m e g a t e r i u m ) were present throughout the cell wall; SP reacted, however, only with molecules accessible to the stain, that is to say, in regions where the lytic activity loosened their packing or modified their organization with peptidoglycan. Indeed, it was previously shown that cell-wall thinning was due to a lytic activity that produced transverse cracks or longitudinal cleavages of the wall (Frehel et al., 1980). Likewise, the silver granulation progressively spread into the wall as it cracked and thinned out or layered in the cleavage regions leading to the shedding of old wall. These observations therefore confirm the hypothesis of a reorganization of wall polymers during cell-wall thinning (Frehel et al., 1980). That SP reaction occurs in regions of high lytic activity is further suggested by the silver staining of the septum plane during septurn cleavage. It would also explain the stratified deposit observed in exponentially growing cells. At last, it is in good agreement with present knowledge on cell wall growth that shows that the inner layer corresponds to the insertion site of new wall material and the outer layer to a region of
active degradation leading to the release of old wall material (Pooley, 1976; Frehel and Ryter, 1979). SP reaction could have more easily occurred in these regions because the packing of wall components is different from that of the cell wall, as proposed by Millward and Reaveley (1974). This difference of packing is probably also responsible for the tribanded structure found in many gram-positive bacteria after Os and Ua fixation and lead-citrate staining. Whatever the cell wall constituent responsible for metal-salt binding (Matthews et al., 1979; Beveridge and Murray, 1980; Doyle et al., 1980), a looser packing probably promotes a better penetration and binding of metal inside the outer and inner cell-wall layers. This difference of packing could also explain the globular structures (Tsien et al., 1978) or the inner fracture plane (Sleytr et al. 1979) observed in freeze-fractured preparations of Streptococcus faecalis. This cytochemical study also contributes some data on the nature of the outer filamentous layer of the MA and MB strains. In strain MA this layer does not seem to contain peptidoglycan because it remained unstained with PTA. It therefore appears to be different from the wall and could correspond to a sort of capsule of which chemical analysis was made many years ago by Aubert and Millet (1950). In contrast, the filamentous layer of strain MB contained both peptidoglycan and SP-reacting nonpeptidoglycan polymer, which means that its composition would be identical to that of the remainder of the wall. It is thus tempting to think that its filamentous structure reflects the lytic activity that leads to the release of acid precipitable wall material. As previously shown (Frehel and Ryter, 1979), this material collected after precipitation was stained with both PTA and silver proteinate. In view of the present study two main conclusions can be drawn. First, teichoic acid or other nonpeptidoglycan-polysaccharides and peptidoglycan seem not to be
STAINING OF CELL-WALL POLYSACCHARIDES
separated in distinct layers. Second, the fuzzy outer filamentous layer appearing on the cell wall is probably produced by a lytic process in strains MB whereas it is probably a capsule in strain MA because of its distinct chemical nature. The authors thank R. Daty for her excellent technical assistance. This work was supported by grants from the Centre National de la Recherche Scientifique (Laboratoire Associ6 269 and ATP A1-5020), the Fondation pour la Recherche M6dicale, and from INSERM (contract 811 023).
REFERENCES ANDERSON, A. J., GREEN, R. S., STURMAN, A. J., AND ARCHIBALD, A. R. (1978) J. Bacteriol. 136, 886-899. ARCHIBALD, A. (1976) J. Bacteriol. 127, 956-960. AUBERT, J.-P., AND MILLET, J. (1950) Ann. Inst. Pasteur (Paris) 79, 468469. AUBERT, J.-P., MILLET, J., PINEAU, E., AND MILHAUD, G. (1961) Biochem. Biophys. Acta 51, 529537. BEVERIDGE, T. J., AND MURRAY, R. G. E. (1980) d. Bacteriol. 141, 876-887. DE CHASTELLIER, C., HELLIO, R., AND RYTER, A. (1975) J. Bacteriol. 123, 1184-1186. DOYLE, R. J., MACDANIEL, M. L., HELMAN, J. R., AND STREIPS, U. N. (1975) J. Bacteriol. 122, 152158. DOYLE, R. J., MATTHEWS, M. H., AND STREIPS, U. N. (1980) J. Bacteriol. 143, 471-480. DUBRAY, G. (1975) J. Microsc. (Paris) 24, 59-74. FREHEL, C., BEAUFILS, A.-M., AND RYTER, A. (1971) Ann. Inst. Pasteur (Paris) 121, 139-148.
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FREHEL, C., AND RYTER, A. (1979) J. Bacteriol. 137, 947-955. FREHEL, C., ROBBE, P., TINELLI, R., AND RYTER, A. (1982) J. Ultrastruct. Res., in press. FREHEL, C., DE CHASTELLIER, C., AND RYTER, A. (1980) Canad. J. Microbiol. 26, 308-317. GIESBRECHT, R., AND RUSKA, n . (1968) Klin. Wochenschr. 46, 575-582. HUGHES, R. C., TANNER, R. J., AND STOKES, E. (19'70) Biochem. J. 120, 159-170. MAUCK, J., AND GLASER, L. (1972) J. Biol. Chem. 247, 1180-1187. MILLWARD, G. R., AND REAVELEY, O A. (1974) J. Ultrastruct. Res. 46, 309-326. NERMUT, M. V. (1967) J. Gen. Microbiol. 49, 503512. PETITPREZ, A., AND DURIEUX, J. C. (1970) J. Microsc. (Paris) 9, 263-272. POOLEY, n . H. (1976)J. Bacteriol. 125, 1139-1147. RAMBOURG, A. (1969) J. Microsc. (Paris) 8, 325-342. RAMBOURG, A. (1971) Int. Rev. Cytol. 31, 57-114. ROBERTSON, J. G., LITTLETON, P., WILLIAMSON, K. I., AND BATT, R. D. (1975) J. Ultrastruct. Res. 52, 321-325. ROLAND, J. C., LEMBI, C. A., AND MORRE, D. J. (1972) Stain Technol. 47, 195-200. ROUSSEAU, M., AND HERMIER, J. (1975) J. Microsc. Biol. Cell. 23, 237-238. RYTER, A. (1965) Ann. Inst. Pasteur (Paris) 108, 4060. RYTER, A., AND KELLENBERGER, E. (1958) Z. Naturforsch. 13, 597-605. SHOCKMAN, G. D. (1965) Bacteriol. Rev. 29, 345-358. SLEYTR, U. B., SHOCKMAN, G. D., AND HIGGINS, M. L. (1979) J. Bacteriol. 139, 299-301. TmERY, J. B. (1967) J. Microsc. (Paris) 6, 987-1018. TSIEN, H. C., SCHOCKMAN, G. D., AND HIGGINS, M. L. (1978) J. Bacteriol. 133, 372-386. WHITEHOUSE, R. L. S., BENICHOU, J.-C., AND RYTER, A. (1977) Biol. Cell. 30, 155-158.
Electron Microscopic Cytochemical Study of Cell-Wall Polysaccharides in Bacillus subtilis and Two Strains of Bacillus megaterium CLAUDE FREHEL AND ANTOINETTE RYTER
Unit~ de Microscopie Electronique, D~partement de Biologie Moldculaire, Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris Cedex 15, France Received November 25, 1981, and in revised form June 1, 1982 Thin sections of Bacillus subtilis and B. megaterium cells were treated by two cytochemical techniques revealing polysaccharides: the phosphotungstic-acid (PTA) and the silver-proteinate (SP) stainings. These procedures were applied to exponentially growing cells as well as cells treated with chloramphenicol (CMP) or cells regrown after antibiotic elimination. It appeared that both stains mainly deposited on the cell wall, but did not give the same staining patterns. In vegetative and CMP-treated cells, PTA gave a homogeneous dark deposit, whereas with S P a deposit was only observed on the outer and inner wall faces. During cell-wall thinning, PTA-stained walls remained rather homogeneous, whereas the silver deposit spread throughout the cell-wall thickness. The meaning of these differences in relation to cell-wall polymer organization is discussed.
We have previously shown that in a D a p - L y s - strain of Bacillus megaterium a high-acid-precipitable peptidoglycan turnover occurs during growth. This release seems to be produced by lyric enzyme(s) excreted into the culture medium, and to correspond to the detachment of large pieces from the superficial wall layer (Frehel and Ryter, 1979; Frehel et al., 1980). One can wonder whether the filamentous layer surrounding the cell wall of this strain reflects the superficial enzymatic wall degradation or corresponds to a sort of capsule, the chemical composition of which would be different from the cell wall itself. To obtain more information on the cellwall constituents and their distribution in this envelope, we have used two cytochemical staining methods that reveal different polysaccharides: (1) the silver-proteinate (SP) method (Thiery, 1967) that was shown to reveal teichoic acid in bacteria (Rousseau and Hermier, 1975), and (2) the phosphotungstic-acid (PTA) method (Ramb o u r g , 1969) t h a t c o u l d r e a c t w i t h peptidoglycan (Rousseau and Hermier, 1975). This study was made with three strains
ofB. megaterium: strain MB, its D a p - L y s mutant with which the turnover study was made, and strain MA (Aubert et al., 1961). The first two strains are characterized by the fact that they never form long cellular chains contrary to other B. megaterium strains, which suggests a high lytic activity. The MA strain orB. megaterium forms long cellular chains. Moreover, strain 168 of B. subtilis was used for comparison because it is the most intensively studied bacillus species from the biocehemical and cytochemical point of view. These techniques were applied to growing cells and also to bacteria treated with chloramphenicol (CMP) and then transferred into fresh culture medium in which growth resumed. It is well known that protein synthesis inhibition induced by CMP leads to wall thickening in gram-positive bacteria (Shockman, 1965; Giesbrecht and Ruska, 1968; Frehel et al., 1971; Hughes et al., 1970). When the cells are resuspended in fresh medium devoid of antibiotic, the cell wall recovers its normal thickness during the first generation of growth. Electron microscope observations (Frehel et al., 1971) showed that the first step of wall thin66
0022-5320/82/100066-12502.00/0 Copyright O 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
STAINING OF CELL-WALL POLYSACCHARIDES
ning consists of the formation of many transverse cracks, suggesting the presence of a high lytic activity. The peptidoglycanturnover study made during this process indicated, however, that contrary to what was expected, wall thinning was not accompanied by an important release of wall material. Moreover, DAP incorporation that occurs during wall thinning and cell elongation remained lower than during normal growth (Frehel et al., 1980). These observations suggested that the bacterium uses the material in its thick wall for elongation, which implies a profound wall reorganization. The present cytochemical study confirmed this assumption, brought new data on the distribution of wall constituents, and showed that the filamentous outer layer of strain MB has the same composition as the wall itself and very probably reflects a high lytic degradation. MATERIALS AND METHODS
Bacterial Strains and Medium Experiments were performed with three different strains of B. megaterium: strain MA (Aubert et al., 1961), strain MB and a Dap-Lys- mutant strain isolated from strain MB in our laboratory (de Chastellier et al., 1975), and also with strain 168 of B. subtilis Marburg. Bacillus megaterium cells were grown in Salton synthetic medium (Aubert et al., 1961) supplemented with 0.2% glucose and 1% yeast extract; for the Dap-LysMB mutant strain mesodiaminopimelic acid and lysine (de Chastellier et al., 1975) were added. Cells were grown at 30°C under strong agitation; the doubting time was 35--40 rain. Bacillus subtilis 168 strain was grown in complex medium (Ryter, 1965) at 37°C under strong agitation; its doubling time was 30--40 rain.
Cell Treatments Plasmolysis. Exponentially growing cells were centrifuged and resuspended in phosphate buffer, pH 7, added to 0.5 M sucrose. They were incubated for 20 min at 30°C with gentle shaking; plasmolysis was monitored under the light microscope. Chlorarnphenicol treatment and resumption of growth. Exponentially growing ceils were treated for 2 hr with 20 to 40 tzg/ml of chloramphenicol (CMP). They were centrifuged and resuspended in four times the initial volume of fresh medium or partially spent medium (Frehel and Ryter, 1979). Regrowth was fol-
67
lowed by turbidimetry. Samples were withdrawn at the end of the CMP treatment, and at different times during growth resumption, and processed for electron microscopy.
Electron Microscopy Fixation and embedding. Samples were centrifuged and embedded in agar (Whitehouse et al., 1977) and fixed (l) overnight with 1% osmium tetroxide in Michaelis buffer (Ryter and KeUenberger, 1958) (Os fixation), (2) overnight with 1% osmium tetroxide in Michaelis buffer, and postfixed with uranyl acetate in the same buffer (Ryter and Kefienberger, 1958) (Os +Ua fixation), (3) overnight with 2.5% glutaraldehyde in Michaelis buffer, washed for 4 hr in the same buffer, and fixed overnight with osmium tetroxide as in 1 (GaOs fixation), (4) overnight with ghitaraldehyde, then osmium as in 3, and postfixed for 1 hr with uranyl acetate in Michaelis buffer (Ga--Os +Ua fixation). Fixation with glutaraldehyde alone was not performed because of the bad preservation of the cell ultrastructure. All samples were dehydrated with acetone and embedded in Epon. Sections cut with a diamond khife were contrasted with lead citrate or treated with polysaccharide stains. Staining of Polysaccharides Thin sections were gathered in plastic rings and floated on different reagents using one of the following techniques.
(i) Silver proteinate staining (SP) (Thiery, 1967). This technique is based on oxidation of 1-2a-glycol bonds into aldehydes by periodic acid (PA). Aldehydic groups then react with thiocarbohydrazide (TCH) to form hydrazones; the final treatment with silver proteinate (SP) results in a fine silver granulation on the 1-2a-glycol linkage. To eliminate nonspecific deposits because of Os fixation, the sections were deosmicated with 10 Vol hydrogen peroxide (HP) before periodate oxidation (Robertson et al., 1975). The standard silver staining procedure was as follows: (HP) 15 min; 1% periodic acid (PA) in distilled water 30 min; 0.2% thiocarbohydrazide (TCH) in 20% acetic acid 24 hr (B. subtilis), or 48 hr (B. megaterium MB), or 72 hr (B. megaterium MA); 1% silver proteinate (SP) in bidistilled water 30 rain (B. megaterium MB) or 35 min (B. subtilis; B. megaterium MA). After each incubation, sections were rinsed for 10 sec three times, and 10 rain three times with distilled water; moreover, after thiocarbohydrazide treatment they were rinsed with different concentrations of acetic acid, then with bidistilled water. The tests described below were done to check the specificity of the silver-staining p r o c e d u r e : (a) TCH+SP. Free aldehyde groups coming from ghataraldehyde or tissue-bound osmium present in the cells before PA oxidation could nonspecifically react with TCH and SP. Therefore, this control was done after a
68
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STAINING OF CELL-WALL POLYSACCHARIDES double fixation with glutaraldehyde and osmium tetroxide, and after a simple fixation with osmium tetroxide. (b) H P + T C H + S P . At this concentration, HP completely removes osmium from thin sections of fixed cells and does not oxidize glycols to aldehydes, ff nonspecific staining is due to osmium tetroxide bound to cell structures, this reaction will be negative; if it is due to free aldehydes present in the cell, staining will persist. (c) SP only. This reveals cell structures having a nonspecific affinity for silver. (d) PA+SP. This reaction checks for groups able to reduce SP after PA oxidation. These different controls were negative. (ii) Phosphotungstic acid staining at low p H as described by Rambourg (1971) and modified by Roland et al. (1972) (PTA). To date the staining mechanism is unknown, it was only proposed that it reveals hydroxyl groups. Thin sections were floated on 1% periodic acid for 15 to 30 min, rinsed with bidistilled water (10 sec three times and i0 rain three times), floated for 4 rain on phosphotungstic acid (PTA) in 10% chromic acid, and rinsed rapidly in bidistilled water. Controls were made by floating sections on PTA at pH 7. All sections were gathered on 400-mesh copper grids with or without a Formvar film. RESULTS
Because B. megaterium strain MB and its Dap-Lys- mutant presented the same morphological appearance and reacted identically to all staining procedures, the name MB refers to both strains in the following results.
Exponentially Growing Cells After the conventional lead-citrate staining, the cell wall of B. subtilis presented the trilamellar structure frequently described: two dense layers enclosing an electron translucent internal layer (Fig. 1). This appearance was found whatever the fixation procedure (Os, Os+Ua, Ga-Os+Ua, GaOs). The cell wall of B. megaterium MB showed a single and homogeneous stained
69
layer surrounded by a filamentous layer after Os-Ua or G a - O s + U a fixation (Fig. 2); if Ua postfixation was omitted, this outer filamentous layer was irregular and coarsely aggregated onto the cell wall (Fig. 3). The MA-strain cell wall also consisted of a homogeneously stained layer covered by dispersed filaments (Fig. 4), but no changes were found in the absence of Ua postfixation. (i) Silver proteinate staining (SP). To eliminate nonspecific staining due to osmium tetroxide bound to cell structure, s, sections were treated with HP prior to periodic acid oxidation. On thin sections stained according to this procedure, the silver deposit appeared mainly on the cell walls along their entire length including poles (Figs. 12-14). On the B. subtilis cell wall, the silver deposit formed a stratified structure as observed by Rousseau and Hermier (1975): a thin granulated layer and a thick outer granulated one enclosing a poorly stained central region (Figs. 5 and 12). A similar distribution was observed in the B. megaterium MB strain, the outer layer corresponding to the filamentous layer in 1Lhe Os+Ua" or G a - O s + U a fixed cells (Figs. 6 and 13) or to the coarse aggregated layer in the Os- or Ga-Os-fixed cells (Fig. 7). In strain MA the reaction was lighter, the outer filamentous layers showing dispersed silver deposit (Figs. 8 and 14). This outer granulated layer appeared after an incubation time of only 72 hr. During septation the silver granulation was found only along the inner wall face, the outer deposit appearing only at the onset of septum cleavage (Figs. 13 and 14). A light and diffuse granulation was observed inside some fully formed septa of B. subtilis (Fig. 12) (see arrowhead).
Fins. 1-4. Lead-citrate staining; Fig. 1, strain 168 of B. subtilis; Figs. 2 and 3, strain MB of B. megaterium, Os + Ua fixation (Fig. 2), Os fixation without Ua (Fig. 3); Fig. 4, strain MA ofB. megaterium. FIGs. 5-8. SP staining; Fig. 5, strain 168; Figs. 6 and 7, strain MB, Os + Ua fixation (Fig. 6), Os fixation without Ua (Fig. 7); Fig. 8, strain MA. FIGS. 9-11. Plasmolyzed cells stained with SP procedure. Fig. 9, strain 168; Fig. 10, strain MB; Fig. 11, strain MA. CM, cytoplasmic membrane.
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STAINING OF CELL-WALL POLYSACCHARIDES
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STAINING OF CELL-WALL POLYSACCHARIDES
To see whether the inner silver granulated layer corresponded to the inner-cell-wall face or to the cytoplasmic membrane, silver staining was applied to plasmolyzed cells. In all strains, silver granulation deposited on the inner-cell-wall face (Figs. 9-11, see arrowheads) and not on the cytoplasmic membranes. (ii) Phosphotungstic acid + chromic acid staining (PTA). With this technique a dark deposit completely covered the cell wall including the poles and septa of the B. subtilis (Fig. 15) and the B. megaterium (Figs. 16 and 17) strains. The outer filamentous layer was also contrasted in strain MB; in strain MA the outer filaments were poorly contrasted with PTA, as compared to their staining with SP (Fig. 14). In the B. megaterium cells the staining became lighter during septum cleavage (Figs. 16 and 17, see arrowheads). No deposits were observed on the cytoplasmic membrane or mesosomes in. all strains. Chloramphenicol-Treated Cells and Growth Resumption
CMP treatment results in the thickening of the cell wall of B. subtilis and B. megaterium (Frehel et al., 1971, 1980). When cells are allowed to regrow in fresh medium devoid of antibiotic, transverse cracks caused by a lyric process progressively appear along the thick cell wall. This causes cell-wall thinning, with the recovery of its initial width by the end of the first generation (Frehel et al., 1971, 1980). As said under Materials and Methods, samples were fixed at the end of the CMP treatment and at different times during the first generation of regrowth. Thin sections were treated with both polysaccharide stains. (i) SP stain. After CMP treatment the cell
73
wall of B. subtilis and B. megaterium presented the stratified silver deposit observed in exponentially growing cells (Figs. 19, 31, and 35). The inner and outer layer, corresponding to the filamentous material in B. megaterium strain, appeared as thin as in exponentially growing cells, whereas the unstained central layer was much thicker. When growth resumed the thickness of the outer silver deposit increased in all strains by progressively spreading toward the inner layer. At the end of the first gteneration, only a thin central layer remained unstained (Figs. 21, 23, 33, and 37; Chart 1A). In the MA strain, the enlargement of the outer silver deposit was not so striking because of the low number of silver grains, but the granulation also appeared inside the central region (Fig. 37). These observations suggested that the spreading of silver granulation was related to the lyric activity responsible for the wall cracking and thinning. To verify this assumption CMP-treated bacteria (strain MB) were resuspended in partially spent medium in which the lytic activity enhances the enzymatic degradation of the cell wall (Frehel et al., 1980). In this case cell-wall thinning does not occur by transverse cracking, but by longitudinal cleavage leading to 1Lhe shedding of large scallops of cell wall (Frehel et al., 1980). In partially spent medium, silver grain spreading did not occur by 1the widening of the outer layer, but by the longitudinal extension of discrete granulated regions inside the unstained layer (Figs. 25 and 27, see arrowhead). This occurred during the first generation until the appearance of the cleavage zone between the old and the new cell wall (Fig. 29, Chart 1B). During this process, the old cell wall progressively became homogeneously stained. (ii) PTA strain. In all strains the CMP
FIGS. 20-23. Growth resumption in fresh medium; middle of the first generation (Figs. 20 and 21) and ,end of the first generation (Figs. 22 and 23). FIGS. 24-29. Growth resumption in partially spent medium; beginning of the first generation (Figs. 24 and 25), middle of the first generation (Figs. 26 and 27), and end of the first generation (Figs. 28 and 29).
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75
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thick cell wall was strongly stained (Figs. 18, 30, and 34). During growth resumption in fresh medium, the cell wall remained entirely stained, but the stained material was progressively eliminated as the cell wall became thinner (Figs. 20, 22, 32, and 36). When strain MB was resuspended in partially spent medium, discrete longitudinal unstained regions appeared inside the thick cell wall as growth resumed (Fig. 24, see arrowhead); they spread longitudinally (Fig. 26) and at the end of the first generation the two cell wall layers separated from one another in these regions (Fig. 28). DISCUSSION
In this study PTA and SP procedures did not give the same staining patterns. In all strains PTA, which seems to react preferentially with peptidoglycan (Rouseau and Hermier, 1975; Frehel et al., 1982), gave a homogeneous staining of the cell wall as previously observed in B. subtilis (Rousseau and Hermier, 1975). This was observed in exponentially growing cells as well as after CMP treatment and wall thinning, and suggests a homogeneous distribution of peptidoglycan in the cell wall. Contrary to PTA, SP gave a tribanded structure in all strains with a much lighter staining in strain MA. In both B. megaterium strains the out-
FIGS. 34 AND 35. FIGS. 36 AND 37.
er layer covered by silver grains corresponded to the filamentous layer. As shown by plasmolysis experiments the thin inner layer was located on the inner face of the cell wall and not on the cytoplasmic membrane as opposed to what was found in Nocardia (Robertson et al., 1975) and Mycobacterium (Petitprez and Durieux, 1970) species. The location of the silver deposit did not depend upon the fixation procedure (Dubray, 1975); in strain MB, however, the outer filamentous layer was coarsely aggregated onto the wall when Ua postfixation was omitted. It was proposed that SP mainly reacts with teichoic acid in B. subtilis (Rousseau and Hermier, 1975; Frehel et al., 1982) and analogous polysaccharides in B. megaterium (Frehel et al., 1982). At first view the tribanded structure seemed to indicate that these nonpeptidoglycan polymers are mostly concentrated in the outer and inner cellwall layers as proposed by some workers (Nermut, 1967; Tsien et al., 1978). This assumption, however, is in disagreement with different data: (i) Doyle et al. (1975) have shown that Concanavalin-A receptor sites of teichoic acid increased during wall dlegradation by lysozyme, suggesting that this polymer was present throughout the wall. (ii) Mauck and Glaser (1972) reported that
Strain M A treated with CMP. E n d of the first generation of regrowth in strain MA.
76
F R E H E L AND RYTER
teichoic acid and peptidoglycan synthesis were coordinated; moreover, Archibald (1976) as well as Anderson et al. (1978) demonstrated that teichoic acid is first inserted as peptidoglycan in the inner cellwall layer and then migrates toward the outer cell surface. These data suggest that teichoic acid is present throughout the cell wall. (iii) At last, this cytochemical study showed that in CMP-treated cells the silver granulation occurred only on two thin inner and outer layers, although both teichoic acid and peptidoglycan continued to be synthesized (Hughes et al., 1970). It is thus unlikely that the absence of staining in the thick central zone corresponds to the absence of teichoic acid. In fact, our cytochemical study on cells regrown after a CMP treatment suggested that teichoic acid molecules (or analogous polysaccharides in B. m e g a t e r i u m ) were present throughout the cell wall; SP reacted, however, only with molecules accessible to the stain, that is to say, in regions where the lytic activity loosened their packing or modified their organization with peptidoglycan. Indeed, it was previously shown that cell-wall thinning was due to a lytic activity that produced transverse cracks or longitudinal cleavages of the wall (Frehel et al., 1980). Likewise, the silver granulation progressively spread into the wall as it cracked and thinned out or layered in the cleavage regions leading to the shedding of old wall. These observations therefore confirm the hypothesis of a reorganization of wall polymers during cell-wall thinning (Frehel et al., 1980). That SP reaction occurs in regions of high lytic activity is further suggested by the silver staining of the septum plane during septurn cleavage. It would also explain the stratified deposit observed in exponentially growing cells. At last, it is in good agreement with present knowledge on cell wall growth that shows that the inner layer corresponds to the insertion site of new wall material and the outer layer to a region of
active degradation leading to the release of old wall material (Pooley, 1976; Frehel and Ryter, 1979). SP reaction could have more easily occurred in these regions because the packing of wall components is different from that of the cell wall, as proposed by Millward and Reaveley (1974). This difference of packing is probably also responsible for the tribanded structure found in many gram-positive bacteria after Os and Ua fixation and lead-citrate staining. Whatever the cell wall constituent responsible for metal-salt binding (Matthews et al., 1979; Beveridge and Murray, 1980; Doyle et al., 1980), a looser packing probably promotes a better penetration and binding of metal inside the outer and inner cell-wall layers. This difference of packing could also explain the globular structures (Tsien et al., 1978) or the inner fracture plane (Sleytr et al. 1979) observed in freeze-fractured preparations of Streptococcus faecalis. This cytochemical study also contributes some data on the nature of the outer filamentous layer of the MA and MB strains. In strain MA this layer does not seem to contain peptidoglycan because it remained unstained with PTA. It therefore appears to be different from the wall and could correspond to a sort of capsule of which chemical analysis was made many years ago by Aubert and Millet (1950). In contrast, the filamentous layer of strain MB contained both peptidoglycan and SP-reacting nonpeptidoglycan polymer, which means that its composition would be identical to that of the remainder of the wall. It is thus tempting to think that its filamentous structure reflects the lytic activity that leads to the release of acid precipitable wall material. As previously shown (Frehel and Ryter, 1979), this material collected after precipitation was stained with both PTA and silver proteinate. In view of the present study two main conclusions can be drawn. First, teichoic acid or other nonpeptidoglycan-polysaccharides and peptidoglycan seem not to be
STAINING OF CELL-WALL POLYSACCHARIDES
separated in distinct layers. Second, the fuzzy outer filamentous layer appearing on the cell wall is probably produced by a lytic process in strains MB whereas it is probably a capsule in strain MA because of its distinct chemical nature. The authors thank R. Daty for her excellent technical assistance. This work was supported by grants from the Centre National de la Recherche Scientifique (Laboratoire Associ6 269 and ATP A1-5020), the Fondation pour la Recherche M6dicale, and from INSERM (contract 811 023).
REFERENCES ANDERSON, A. J., GREEN, R. S., STURMAN, A. J., AND ARCHIBALD, A. R. (1978) J. Bacteriol. 136, 886-899. ARCHIBALD, A. (1976) J. Bacteriol. 127, 956-960. AUBERT, J.-P., AND MILLET, J. (1950) Ann. Inst. Pasteur (Paris) 79, 468469. AUBERT, J.-P., MILLET, J., PINEAU, E., AND MILHAUD, G. (1961) Biochem. Biophys. Acta 51, 529537. BEVERIDGE, T. J., AND MURRAY, R. G. E. (1980) d. Bacteriol. 141, 876-887. DE CHASTELLIER, C., HELLIO, R., AND RYTER, A. (1975) J. Bacteriol. 123, 1184-1186. DOYLE, R. J., MACDANIEL, M. L., HELMAN, J. R., AND STREIPS, U. N. (1975) J. Bacteriol. 122, 152158. DOYLE, R. J., MATTHEWS, M. H., AND STREIPS, U. N. (1980) J. Bacteriol. 143, 471-480. DUBRAY, G. (1975) J. Microsc. (Paris) 24, 59-74. FREHEL, C., BEAUFILS, A.-M., AND RYTER, A. (1971) Ann. Inst. Pasteur (Paris) 121, 139-148.
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FREHEL, C., AND RYTER, A. (1979) J. Bacteriol. 137, 947-955. FREHEL, C., ROBBE, P., TINELLI, R., AND RYTER, A. (1982) J. Ultrastruct. Res., in press. FREHEL, C., DE CHASTELLIER, C., AND RYTER, A. (1980) Canad. J. Microbiol. 26, 308-317. GIESBRECHT, R., AND RUSKA, n . (1968) Klin. Wochenschr. 46, 575-582. HUGHES, R. C., TANNER, R. J., AND STOKES, E. (19'70) Biochem. J. 120, 159-170. MAUCK, J., AND GLASER, L. (1972) J. Biol. Chem. 247, 1180-1187. MILLWARD, G. R., AND REAVELEY, O A. (1974) J. Ultrastruct. Res. 46, 309-326. NERMUT, M. V. (1967) J. Gen. Microbiol. 49, 503512. PETITPREZ, A., AND DURIEUX, J. C. (1970) J. Microsc. (Paris) 9, 263-272. POOLEY, n . H. (1976)J. Bacteriol. 125, 1139-1147. RAMBOURG, A. (1969) J. Microsc. (Paris) 8, 325-342. RAMBOURG, A. (1971) Int. Rev. Cytol. 31, 57-114. ROBERTSON, J. G., LITTLETON, P., WILLIAMSON, K. I., AND BATT, R. D. (1975) J. Ultrastruct. Res. 52, 321-325. ROLAND, J. C., LEMBI, C. A., AND MORRE, D. J. (1972) Stain Technol. 47, 195-200. ROUSSEAU, M., AND HERMIER, J. (1975) J. Microsc. Biol. Cell. 23, 237-238. RYTER, A. (1965) Ann. Inst. Pasteur (Paris) 108, 4060. RYTER, A., AND KELLENBERGER, E. (1958) Z. Naturforsch. 13, 597-605. SHOCKMAN, G. D. (1965) Bacteriol. Rev. 29, 345-358. SLEYTR, U. B., SHOCKMAN, G. D., AND HIGGINS, M. L. (1979) J. Bacteriol. 139, 299-301. TmERY, J. B. (1967) J. Microsc. (Paris) 6, 987-1018. TSIEN, H. C., SCHOCKMAN, G. D., AND HIGGINS, M. L. (1978) J. Bacteriol. 133, 372-386. WHITEHOUSE, R. L. S., BENICHOU, J.-C., AND RYTER, A. (1977) Biol. Cell. 30, 155-158.