Vol. 139, No. 1
JOURNAL OF BACTERIOLOGY, July 1979, p. 299-301 0021-9193/79/07-0299/03$02.00/0
NOTES Demonstration of an Internal Fracture Plane in Cell Walls of Streptococcus faecalis and Streptococcus mutans UWE B. SLEYTR,' GERALD D. SHOCKMAN,2 AND MICHAEL L. HIGGINS2*
Institute of Food Technology, Agriculture University, Vienna, Austria,' and Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140'
Received for publication 11 April 1979
The proposed lower internal density of the gram-positive wall was confirmed by observing an internal fracture plane in the walls of Streptococcus faecalis and Streptococcus mutans. However, the granular surfaces produced by this cleavage appeared to be more of a reflection of distortion during preparation than of subunit construction.
Studies of freeze-fractures of isolated cell fractions of Streptococcus faecalis have indicated that the cross-fractured wall was made up of two rows of irregularly arranged granules separated by a central zone of somewhat lower density (5). The conclusions of this earlier study agreed with those of others (1, 2) in suggesting that the cell wall polymers found in gram-positive organisms are concentrated along the outer and inner faces of the cell wall. Here we confirm and extend this view by showing for the first time that a central internal fracture plane can be seen in wall preparations of S. faecalis which contain no cryoprotectant. In addition, we also have shown that these fracture planes can be demonstrated in another gram-positive organism, Streptococcus mutans. Mid-exponential-phase cultures of S. faecalis ATCC 9790 and S. mutans strain Ingbritt, grown in chemically defined media, were chilled and broken with a Braun MSK cell homogenizer as described previously (5). Wall fractions were partially purified by differential centrifugation, washed four times with 4°C water, frozen in liquid Freon 22, and freeze-fractured with a Balzers 360M freeze microtome. The specimen temperature at the time of fracture was -103°C, and then fractured samples were etched for 30 s before being replicated. Platinum-carbon was evaporated with electron guns. The carbon backing layer was deposited by thermal evaporation. The outer convex and inner concave wall surfaces of both S. faecalis and S. mutans were revealed by etching fractured wall preparations. These exposed surfaces showed smooth, apparently structureless wall faces (Fig. 1 and 2). Since both preparations were made by suspending
walls of either organism in double-distilled water, it would seem that these smooth surfaces are not the result of the superimposition of an interfering eutectic layer. Both wall fractions showed internal fracture planes. In contrast to the smooth texture of the outer and inner surfaces of the walls, in preparations from both organisms the internal concave and convex fracture faces adjacent to the fracture plane were quite rough and granular (Fig. 1 and 2). Although frequently seen in cell wall preparations of both organisms, internal fracture faces were somewhat more common in the isolated walls of S. faecalis. These internal fracture planes were not observed in our hands when glycerol was added to walls before freezing. It may be that in the absence of glycerol, ice crystal growth during freezing within interior regions of the cell wall renders the structure susceptible to internal fracturing. Also, it is conceivable that noncovalent bonds established between glycerol molecules and wall polymers could reduce the probability of internal fractures by providing internal stabilization. The granules seen on the internal fracture faces of both wall preparations seemed to have the same random distribution and heterogeneity of size as reported for similar structures seen before in cross-fractures of walls of S. faecalis (5). However, the granules observed on the scalloped ridges of cross-fractured walls (see the multiple black arrowheads in Fig. 2a) indicate that considerable structural alterations occur during the freeze-cleaving, or subsequent preparation steps, or both. That these granules frequently extend some distance above the ridge suggests that substantial deformation had taken
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place during the fracture process. It is conceiv- longer tenable to consider cell walls of gramable that this might result from the energy that positive bacteria as constructed of homogenemust be dissipated during the fracturing process. ously packed macromolecules. In this regard, a large number of different types of polymers have been shown to undergo plastic This work was supported by Public Health Service Redeformation during the fracture process (3). Fur- search grants DE 03487 from the National Institute of Dental thermore, the deformed molecules may have Research and AI 05044 and AI 10971 from the National of Allergy and Infectious Diseases and a grant from significantly changed by heating during evapo- Institute the National Science Foundation (PCM76-11021 A01). During ration of the replica, leading to the final granular the period of this investigation, U. B. Sleytr was a recipient of image seen on these fracture faces. Such defor- a Senior Foreign Dental Scientist Fellowship from the Amermations to other biological polymeric structures ican Association for Dental Research. have been reported (3, 4). Thus, it would seem that the appearance (or LITERATURE CITED increase in the prominence) of these granules 1. Garland, J. M., A. R. Archibald, and J. Baddiley. results from the freeze-fracture process and sug1975. An electron microscopic study of the location of teichoic acid and its contribution to staining reactions gests that the granules should not be taken as in walls of Streptococcus faecalis 8191. J. Gen. Microan indication of the presence of a subunit strucbiol. 89:73-86. ture, at least at this time. Nonetheless, the ob- 2. Millward, G. R., and D. A. Reaveley. 1974. Electron servation of granules in higher numbers on microscopic observations on the cell walls of some Gram-positive bacteria. J. Ultrastruct. Res. 46:309-326. either side of a central fracture plane is consist1977. Plastic deforent with the idea that wall polymers are concen- 3. Sleytr, U. B., and A. W. Robards. review. J. Microsc. mation during freeze-cleaving: a trated in an apparently heterogeneous manner (Oxford) 110:1-25. along the outer and inner planes of the walls. 4. Sleytr, U. B., and A. W. Robards. 1977. Freeze-fracturing: a review of methods and results. J. Microsc. (OxThe observed unequal distribution of wall comford) 111:77-100. ponents could be a reflection of hydrated wall H. C., G. D. Shockman, and M. L. Higgins. structure or have resulted from events occurring 5. Tsien, 1978. Structural arrangement of polymers within the during either wall isolation or any of the steps of wall of Streptococcus faecalis. J. Bacteriol. 133:372386. the freeze-fracture process. In any case, it is no
FIG. 1. S. faecium ATCC 9790 cell wall fragments showing both the inner (rs) and outer (a3) surfaces and the concave (F) and convex (f) fracture faces. I, Ice. The arrows in the upper right-hand corner of this and the following micrographs indicate the direction of Pt-C-shadowing. Bar in all micrographs represents 100 nm. FIG. 2. S. mutans strain IB cell wall fragments showing the fine granular structure of the inner (rS) surface (a) and the outer (ak) surface (b). Fig. 2b shows a portion of a wall fragment, which, at the time of freezing, was folded in a concave manner toward what had been the principal axis of the cell. A coarse globular structure can be seen on both the concave (P in a) and convex (P in b) fracture faces. Note that the granules at the ridge (R), indicated by arrows, cast a shadow onto the etched surface (TS) in (a), indicating that they extend to a higher level than the etched surface.
JOURNAL OF BACTERIOLOGY, July 1979, p. 299-301 0021-9193/79/07-0299/03$02.00/0
NOTES Demonstration of an Internal Fracture Plane in Cell Walls of Streptococcus faecalis and Streptococcus mutans UWE B. SLEYTR,' GERALD D. SHOCKMAN,2 AND MICHAEL L. HIGGINS2*
Institute of Food Technology, Agriculture University, Vienna, Austria,' and Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140'
Received for publication 11 April 1979
The proposed lower internal density of the gram-positive wall was confirmed by observing an internal fracture plane in the walls of Streptococcus faecalis and Streptococcus mutans. However, the granular surfaces produced by this cleavage appeared to be more of a reflection of distortion during preparation than of subunit construction.
Studies of freeze-fractures of isolated cell fractions of Streptococcus faecalis have indicated that the cross-fractured wall was made up of two rows of irregularly arranged granules separated by a central zone of somewhat lower density (5). The conclusions of this earlier study agreed with those of others (1, 2) in suggesting that the cell wall polymers found in gram-positive organisms are concentrated along the outer and inner faces of the cell wall. Here we confirm and extend this view by showing for the first time that a central internal fracture plane can be seen in wall preparations of S. faecalis which contain no cryoprotectant. In addition, we also have shown that these fracture planes can be demonstrated in another gram-positive organism, Streptococcus mutans. Mid-exponential-phase cultures of S. faecalis ATCC 9790 and S. mutans strain Ingbritt, grown in chemically defined media, were chilled and broken with a Braun MSK cell homogenizer as described previously (5). Wall fractions were partially purified by differential centrifugation, washed four times with 4°C water, frozen in liquid Freon 22, and freeze-fractured with a Balzers 360M freeze microtome. The specimen temperature at the time of fracture was -103°C, and then fractured samples were etched for 30 s before being replicated. Platinum-carbon was evaporated with electron guns. The carbon backing layer was deposited by thermal evaporation. The outer convex and inner concave wall surfaces of both S. faecalis and S. mutans were revealed by etching fractured wall preparations. These exposed surfaces showed smooth, apparently structureless wall faces (Fig. 1 and 2). Since both preparations were made by suspending
walls of either organism in double-distilled water, it would seem that these smooth surfaces are not the result of the superimposition of an interfering eutectic layer. Both wall fractions showed internal fracture planes. In contrast to the smooth texture of the outer and inner surfaces of the walls, in preparations from both organisms the internal concave and convex fracture faces adjacent to the fracture plane were quite rough and granular (Fig. 1 and 2). Although frequently seen in cell wall preparations of both organisms, internal fracture faces were somewhat more common in the isolated walls of S. faecalis. These internal fracture planes were not observed in our hands when glycerol was added to walls before freezing. It may be that in the absence of glycerol, ice crystal growth during freezing within interior regions of the cell wall renders the structure susceptible to internal fracturing. Also, it is conceivable that noncovalent bonds established between glycerol molecules and wall polymers could reduce the probability of internal fractures by providing internal stabilization. The granules seen on the internal fracture faces of both wall preparations seemed to have the same random distribution and heterogeneity of size as reported for similar structures seen before in cross-fractures of walls of S. faecalis (5). However, the granules observed on the scalloped ridges of cross-fractured walls (see the multiple black arrowheads in Fig. 2a) indicate that considerable structural alterations occur during the freeze-cleaving, or subsequent preparation steps, or both. That these granules frequently extend some distance above the ridge suggests that substantial deformation had taken
299
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VOL. 139, 1979
NOTES
301
place during the fracture process. It is conceiv- longer tenable to consider cell walls of gramable that this might result from the energy that positive bacteria as constructed of homogenemust be dissipated during the fracturing process. ously packed macromolecules. In this regard, a large number of different types of polymers have been shown to undergo plastic This work was supported by Public Health Service Redeformation during the fracture process (3). Fur- search grants DE 03487 from the National Institute of Dental thermore, the deformed molecules may have Research and AI 05044 and AI 10971 from the National of Allergy and Infectious Diseases and a grant from significantly changed by heating during evapo- Institute the National Science Foundation (PCM76-11021 A01). During ration of the replica, leading to the final granular the period of this investigation, U. B. Sleytr was a recipient of image seen on these fracture faces. Such defor- a Senior Foreign Dental Scientist Fellowship from the Amermations to other biological polymeric structures ican Association for Dental Research. have been reported (3, 4). Thus, it would seem that the appearance (or LITERATURE CITED increase in the prominence) of these granules 1. Garland, J. M., A. R. Archibald, and J. Baddiley. results from the freeze-fracture process and sug1975. An electron microscopic study of the location of teichoic acid and its contribution to staining reactions gests that the granules should not be taken as in walls of Streptococcus faecalis 8191. J. Gen. Microan indication of the presence of a subunit strucbiol. 89:73-86. ture, at least at this time. Nonetheless, the ob- 2. Millward, G. R., and D. A. Reaveley. 1974. Electron servation of granules in higher numbers on microscopic observations on the cell walls of some Gram-positive bacteria. J. Ultrastruct. Res. 46:309-326. either side of a central fracture plane is consist1977. Plastic deforent with the idea that wall polymers are concen- 3. Sleytr, U. B., and A. W. Robards. review. J. Microsc. mation during freeze-cleaving: a trated in an apparently heterogeneous manner (Oxford) 110:1-25. along the outer and inner planes of the walls. 4. Sleytr, U. B., and A. W. Robards. 1977. Freeze-fracturing: a review of methods and results. J. Microsc. (OxThe observed unequal distribution of wall comford) 111:77-100. ponents could be a reflection of hydrated wall H. C., G. D. Shockman, and M. L. Higgins. structure or have resulted from events occurring 5. Tsien, 1978. Structural arrangement of polymers within the during either wall isolation or any of the steps of wall of Streptococcus faecalis. J. Bacteriol. 133:372386. the freeze-fracture process. In any case, it is no
FIG. 1. S. faecium ATCC 9790 cell wall fragments showing both the inner (rs) and outer (a3) surfaces and the concave (F) and convex (f) fracture faces. I, Ice. The arrows in the upper right-hand corner of this and the following micrographs indicate the direction of Pt-C-shadowing. Bar in all micrographs represents 100 nm. FIG. 2. S. mutans strain IB cell wall fragments showing the fine granular structure of the inner (rS) surface (a) and the outer (ak) surface (b). Fig. 2b shows a portion of a wall fragment, which, at the time of freezing, was folded in a concave manner toward what had been the principal axis of the cell. A coarse globular structure can be seen on both the concave (P in a) and convex (P in b) fracture faces. Note that the granules at the ridge (R), indicated by arrows, cast a shadow onto the etched surface (TS) in (a), indicating that they extend to a higher level than the etched surface.
