Archives of
Microbiology
Arch. Microbiol. 107, 313- 320 (1976)
9 by Springer-Verlag 1976
The Fine Structure of Micrococcus radiophilus and Micrococcus radioproteolyticus U. B. S L E Y T R 1, M. T. S I L V A 2, M. K O C U R 3, and N. F. L E W I S 4 1Strangeways Research Laboratory, Cambridge CBI 4RN, England* ZCentro de Microscopia Electr6nica and Centro de Estudos de Bioquimica do I.A.C. Universidade do Porto, Porto, Portugal 3Czechoslovak Collection of Microorganisms, University of J. E. Purkyn~, tL Obrfincfl mini 10, Brno, (~SSR** 4 Biochemistry and Food Technology Division, Phabha Atomic Research Centre, Trombay, Bombay 400 085, India
Abstract. The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freeze-etching techniques and the two species were f o u n d to be similar in the fine structure. The only significant difference was in the appearance o f the surfaces o f the cell walls in freeze-etched preparations. Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different f r o m that o f other micrococci and Gram-positive cocci, it is recomm e n d e d that they be reclassified into a new genus.
Key words: Micrococcus radiophilus - Micrococcus radioproteolyticus - Bacterial cell walls - Fine structure -
Electron m i c r o s c o p y - T a x o n o m y .
Three species o f radiation-resistant micrococci, M.
radiodurans ( D u g g a n et al., 1959), M. radiophilus (Lewis, 1973), and M. radioproteolyticus ( K o b a t a k e et al., 1973) have been described. These species are very similar in their pigments and m o s t biochemical characteristics ( K o b a t a k e et al., 1973). Electron microscopic studies o f M. radiodurans have shown that the fine structure o f the cell wall is different f r o m that o f other Gram-positive cocci (Thornley etal., 1965; W o r k and Griffiths, 1968; Sleytr et al., 1973). To date studies have n o t been m a d e o f the ultrastructure o f the celt walls o f two other radio-resistant micrococci, and consequently the p u r p o s e o f the present w o r k was to examine the ultrastructure o f M. radiophilus and M. radioproteolyticus and c o m p a r e these observations with those already m a d e on M. radiodurans. * Permanent address: Department of Biochemical Technology, University of Agriculture, Peter-Jordan-Str. 82, A-I190 Vienna,
Austria. ** Address for offprint requests.
MATERIAL AND METHODS Organisms and Growth Conditions. The strains ltsed were Micrococcus radiophitus CCM 2564 (RBD), isolated by one of us (N. L.) and Micrococcus radioproteotyticus CCM 2703, which was supplied by M. Kobatake, National Institm for Hygiene, Tokyo, Japan.
The strains were grown in Difco Brain Heart trrfiision Broth (BH1) at 30~ with shaking or o~ BHI agar at 30~ for 16 h. Cells were harvested in the logarithmic or stationary please of growth. Electron Microscopy. For the uttrathin sectioning, the bacteria were fixed either (1) by tile procedure of Ryter and Kellenberger (R-K)
(1958) without prefixation (Silva, ~971) with or without subsequent postfixation with uranyl acetate, or (2) in 2.5 % glutaraldehyde (TAAB, London) in 0.I M cacocFylatebuffer, pH 7.0, for I h at room temperature, followed by the procedure of Ryter and Kellenberger (1958) without intermediate washing. The fixed samples were processed for electron micr~ascopyas described by Silva and Kocur (1972). The freeze-etching of intact cells of both strains was carried out in an EPA 100 (Leybold-Heraeus, Cologne, West Germany) freezeetching apparatus, bythe method of Sleytr and Umrath (1974), but complementary replicas were not examined. Platinum carbon replicas were eteaned with 30% chromic acid (CrO3) and washed in distilled water. Cleaned replicas were picked up on Formvarcoated copper grids. Thin sections and replicas were examined in a Philips 301 electron microscope operating at 80 kV with a 50 lain objective aperture.
RESULTS AND DISCUSSION
Electron Microscopy of Thin Sections Cells o f both Micrococcus radiophilus and M. radioproteolyticus are approximately spherical and 1 . 0 1.6 ~tm in diameter. They occur in pairs, tetrads, and irregular clumps (Figs. 1 c and 7). The cell wall profile o f both organisms is irreguIar in outline (Figs. 1 c and 7). In most sections o f intact or slightly autolyzed cells, three different cell wall layers can usually be distinguished (Figs. 1 a and 7). The innermost layer adjacent to the plasma m e m b r a n e is a dense layer (d), which corresponds to the rigid, peptidoglycan-containing
314
Arch. Microbiol., Vol. 107 (1976) Fig. 1a - c Thin sections of Micrococcus radiophilus. (a) The envelope of a partially lysed cell. • 84000. (b) An oblique section through the cell wall near a septation site showing a striated pattern. R-K fixation without uranyl post fixation, x 68 000. (c) Two cells in the process of division with growing septa (s). The innermost dense layer is the only one of the three visible layers of the cell wall to be involved in septum formation. The intermediate layer and the outer membrane surround groups of cells. The cytoplasmic membrane is only visible in a few areas. • 50000
Symbols used in Figures 1-13: cp cytoplasm; w cell wall; pm cytoplasmic membrane (p~n concave view, p~n convex view); om outer membrane; i intermediate layer; d dense layer; l storage material; n nucleus; pp polyphosphate granule; s septum; f fimbriae; fi fibrils; b blebs. The arrow in the lower right-hand corners of micrographs of freeze-etch replicas indicates the direction of shadowing
layer o f the cell wall. It has been observed in m a n y Gram-negative bacteria as well as in M . radiodurans (Thornley et al., 1965; W o r k and Griffiths, 1968). The next layer, the intermediate layer (i), is not always clearly visible. This layer is trypsin-sensitive in M. radiodurans and shows a pattern o f c o m p a r t m e n t s in negatively stained preparations. The outermost layer, the outer m e m b r a n e (ore), appears as a unit m e m brane and has an irregular wavy profile, Thus the cell envelope is similar to that o f a Gram-negative bacterium although the dense layer (d) is thicker than in m o s t Gram-negative bacteria. In b o t h organisms, the innermost layer is the only one o f the three visible
layers o f the cell wall to be involved in septum form a t i o n (Figs. 1 c and 7). The intermediate layer and the outer m e m b r a n e s u r r o u n d groups o f cells as observed in thin sections o f M . radiodurans (Thornley et al., 1965). The cytoplasmic m e m b r a n e is rarely observed in intact cells after either fixation procedure. This seems to be due to p o o r preservation rather than to a masking effect o f the dense cytoplasm as suggested by T h o r n l e y et al. (1965). The m e m b r a n e is m o r e clearly seen in partially lysed cells (Fig. 1 a); when visible, it has the usual appearance as an unit m e m brane. The cytoplasm is unusually dense and contains a b u n d a n t light areas (1; Figs. 1 c and 7). These
U. B. Sleytr et a l . Structure of M. radiophilus and M. radioproteolyticus
315
Figs. 2 - 6 Electron micrographs of replicas of frozenetched cells of M. radiophilus Fig. 2 Dividing cells. The fracture plane has revealed concave (p~n) and convex (p~n) views of the cytoplasmic membrane. Both septa (s) o f the tetrade can be seen. x 55000 Fig. 3 A dividing cell. Fracture has taken place within the plasma membrane, revealing the convex of the plasma membrane face (p~n) adjacent to the cytoplasm. The etched outer surface of the cell wail (w) can also be seen. In a cross fracture of the cell wall, two layers can be distinguished (arrows). x 30000 Fig. 4 Cross fractured cell with polyphosphate (pp) granules which reveal a delicate granular structure, x 50000
areas give a strong positive reaction with Thi6ry's procedure (Thi6ry, 1967) indicating that they contain polysaccharide (unpublished observation). These areas are much more clearly visible in bacteria fixed initially in OsO4 than in glutaraldehyde. Polyphosphate granules were also observed (Fig.7); they sometimes appeared as empty areas. The nuclei have the usual structure found in prokaryotic cells, consisting of arrays of fine filaments (Figs. 1 c and 7).
Electron Microscopy of Freeze-Etched Cells Examination of freeze-etched preparations of M. radiophilus and M. radioproteolytieus confirms that the cells occur predominately in tetrads or pairs (Figs. 2, 3, and 8). After low-angle shadowing the surface of the cell wall of M. radiophilus shows a fine granular structure with randomly distributed thin strands of timbriae-like material (Fig. 5). The cell wall surface of M. radioproteolyticus (Figs. 8 and 9) reveals a charac-
316
Arch. Microbiol., Vot. 107 (1976) Fig. 5 Part of a frozen-etched cell shadowed at a low angle. The structure of the surface of the cell wall (w) and fimbriae (D are visible Compare with the cell wall in Figure 6 which was shadowed at a larger angle and in which no details of the cell wall surface are visible. x 110000
Fig. 6 A freeze-etched preparation. A slightly oblique fracture shows the existence of three layers in the cell envelope (arrows), the numerous fibrils (fi) at the cross fractured cell wall arise by plastic deformation of wall components, x 110000
Fig. 7 Thin section of dividing cells of M. radioproteolyticus. Note the presence of polyphosphate (pp) and storage material (l). R-K fixation with uranyl post-fixation. • 50000
U. B. Sleytr et al. : Structure of M. radiophilusand M. radioproteolyticus
317
Figs. 8 - 12 Electron micrographs of frozen-etched cells of M. radioproteolyticus in different stages of division Fig. 8 A cell in an early stage of division. The surface of the cell wall (w) revealed by etching can be seen. The characteristic "rod-like" surface structures are randomly distributed, x 80000 Fig. 9 A later stage in cell division. The area of constriction is free of rod-like structures but blebs (b) are visible-at the septation site. The cell wall shows three layers (arrows). in a region where it is cross fractured, x 60000 Fig. I 0 A cross fracture through the region of septation of two cells. Except for a small anular region (a) the cells have completely separated, x 60000
teristic structure c o m p o s e d o f an a r r a y o f curved rodlike elevations. The average diameter o f the " r o d s " is 30 n m and they are up to 900 n m long. Similar structures have only been observed previously in M. cryophilus (Sleytr and K o c u r , 1971) and the " r o d s " in this organism were less p r o n o u n c e d and considerably shorter. The similarity between the m o r p h o l o g y and dimensions o f the rods f o u n d in M. radioproteolyticus and the rod-like elevations on the inner surface o f the cell wall adjacent t o the cytoplasm in n u m e r o u s filam e n t o u s and non-filamentous fungi ( M o o r and Mtihlethaler, 1963; R e m s e n et al., 1967; Sleytr et al., 1969) is striking.
The layer with a hexagonal pattern observed by Thornley et al. (1965) and W o r k and Griffiths (1968) in negatively-stained preparations and by Sleytr et al. (1973) in replicas o f freeze-etched specimens on the outer surface o f the cell wall o f M. radiodurans is not visible in cell walls o f M. radiophilus and M. radio-
proteolyticus. At high magnification, cross-fractured cell walls appear triple layered, similar to M. radiodurans (Sleytr et al., 1973). These layers are most distinct in sligthly oblique fractures (Figs. 6 and 9). However, in fractures almost perpendicular to the surface, only two layers o f the cell wall can be distinguished (Figs. 3,11, and 12).
318
Arch. Microbiol., Vol. 107 (1976) Figs. 11 and 12 The final stage of cell division. The annular region separates leading to the formation of fibrils Oct) which still join the two daughter cells. In a cross fracture of the cell wall, two layers can be distinguished (arrows). The blebs (b) at the septation site are still present, x 60000 Fig. 13 The pole of a newly separated cell. Blebs (b) are still visible but no rodlike structures have yet been formed. x 80000
No fracture planes are observed within the layers of the cell envelope external to the cytoplasmic membrane. Frequently, fibrils are seen, extending from the broken edges of the cell wail and lying over the fracture plane of.the cytoplasmic membrane (Figs. 5, 6, and 9). These have been interpreted as artefacts produced by plastic deformation of wall components (Sleytr, 1975). The cytoplasmic membrane fractures internally into two parts (Figs. 2 and 12). Both fracture faces show the typical irregularly-arranged membrane particles, the convex face (p~m) containing more particles than the concave face (p~m). The cytoplasmic membranes
of M. radiophilus and M. radioproteolyticus are thus similar in appearance to the cytoplasmic membranes of the other Gram-positive and Gram-negative bacteria examined by freeze-etching (Remsen and Watson, 1972). Rod-like membrane invaginations similar to those seen in Micrococcus denitrificans, M. halodenitrifieans (Sleytr and Kocur, 1973) and M. diversus (Kocur and Sleytr, 1974) are not observed. Cross-fractured cells o f M: radiophilus and M. M. radioproteolyticus reveal the presence of polyphosphate granules which have a delicate granular structure (Fig.4). Similar dense bodies have also been
U. B. Sleytr et al. : Structure of M. radiophilus and M. radioproteolyticus
observed in the cytoplasm of M. radiodurans (Sleytr et al., 1973) and other bacteria. Thornley et al. (1965) reported that these polymetaphosphate granules are frequently not preserved during sectioning and appear as empty areas. They suggest that these dense bodies probably correspond to the refractile granules observed in the phase-contrast microscope. Whereas cells of M. radiophilus do not show any changes in cell wall surface structure during septation, freeze etched preparations of M. radioproteolyticus revealed a significant alteration of the distribution of the "rod-like" structures. The changes observed during the process of cell growth and septum formation in M. radioproteolyticus are illustrated in Figs. 8 - 13. In non-dividing cells and in cells at early stages of septation, the "rods" appear randomly distributed on the cell surface (Fig. 8). With progressing septation and constriction of the cells the apparently newly-formed cell surfaces appear to be relatively free of rods and show only 12 nm particles in a random distribution (Figs.9,11, and 12). As in other dividing organisms, a surplus of material is shed in the form of blebs (b) at the septum (Sleytr and Thornley, 1973; Burdett and Murray, 1974). In the final stage of septation the cells are completely separated except for a very small annular area (Fig. 10). When the cells finally separated they are still joined by fine fibrils (Figs. ll and 12). Figure 13 is interpreted as end-on view of a newly formed cell pole. These areas are free of rod-like structures but exhibit frequently a few blebs. In contrast to M. radioproteolyticus, no extrusion of blebs of cell wall material is observed in freezeetched preparations of M. radiophilus and M. radiodurans (Sleytr et al., 1973). Thus this study of thin-sectioned and freezeetched cells of M. radiophilus and M. radioproteolyticus by electron microscopy has shown that their cell walls have a similar structure to M. radiodurans (Thornley et al., 1965; Work and Griffiths, 1968 ; Sleytr et al., 1973), except for the outermost surface layers. In addition, chemical analyses on the cell walls have shown that all these three species possess a peptidoglycan of the L-Orn-Gly2 type (Ghuysen, 1968; Schleifer and Kandler, 1972; K. H. Schleifer, pers. comm.). They differ in cell wall ultrastructure and resistance to gamma radiation so distinctly from the species of the genus Micrococcus that it is recommended that they be placed in a new separate genus. However, their relationships should be confirmed by DNA hybridization studies.
Acknowledgements. We wish to thank Dr. A. M. Glauert for helpful discussions during preparation of the manuscript and Misses
319 E. Pohoralek, M. Irene Barros, Paula Macedo, and Mr. R. Parker, for their technical assistance. This investigation was supported by the "Fonds zur F6rderung tier wissenschaftlichen Forschung Osterreichs'" (Proj. Nr. 2402) and by grant PFR/1 from the I.A.C., Lisbon, Portugal.
REFERENCES Burdett, 1. D. J., Murray, R. G. E. : Electron microscope study of septum formation in Escherichia colt strain B and B/r during synchronous growth. J. Bact. 119, 1039 1056 (1974) Duggan, D. E., Anderson, A. W., Elliker, P. R., Cain, R. F. : Ultraviolet exposure studies on a gamma radiation resistant Micrococcus isolated from food. Food Res. 24, 376-382 (1959) Ghuysen, J. M. : Use of bacteriologic enzymes in determination of wall structure and their role in cell metabolism. Bact. Rev. 32, 4 2 5 - 4 6 4 (1968) Kobatake, M., Tanabe, S., Hasegawa, S.: Nouveau Micrococcus radiordsistant fi pigment rouge, isol6 de f6ces de Lama glama, at son utilisation comme indicateur microbiologique de la radiost~rilisation. C. R. Soc. Biol. (Paris) 167, 1506-1510 (1973) Kocur, M., Sleytr, U. B.: Structure of Mierococcus diversus after freeze-etching. Microbios 10, 7 1 - 73 (1974) Lewis, N. F. : Radio-resistant Micrococcus radiophilus sp. nov. isolated from irradiated Bombay duck (Harpodon neherus). Curt. Sci. 42, 504 (1973) Moor, H., Mtihlethaler, K.: Fine structure in frozen-etched yeast cells. J. Cell Biol. 17, 609-628 (1963) Remsen, C. C., Hess, W. M., Sassen, M. M. A. : Fine structure of germinating Penieillium mega,v~orum conidia. Protoplasma (Wien) 64, 439-451 (1967) Remsen, C. C., Watson, S. W.: Freeze-etching of bacteria. Int. Rev. Cytol. 33, 253-296 (,1972) Ryter, A., Kellenberger, E.: Etude au microscope electronique de plasmas contenant de I'acide d6soxyribonucl6ique. I. Les nucl6oides des bactdries en croissance active. Z. Naturforsch. 13b, 5 9 7 - 605 (1958) Schleifer, K. H., Kandler, O.: Peptidoglycan types of baclcrial cell walls and their taxonomic implication. Bact. Rev. 36, 407 - 477 (1972) Silva, M. T.: Changes induced in the ultrastructure of the cyloplasmic and intracytoplasmic membranes of several Grampositive bacteria by variations in OsO4 fixation. J. Microscopy 93, 2 2 7 - 2 3 2 (1971) Silva, M. T., Kocur, M. : The fine structure of Mierocoecus cvaneus. Arch. Mikrobiol. 86, 2 1 1 - 2 2 0 (1972) Sleytr, U. B. : Plastic deformation during freeze-cleaving. Proc. Roy. Microscop. Soc. 10, 103 (1975) Sleytr, U. B., Adam, H., Klaushofer, H.: Die Feinstruktur dcr Konidien yon Aspergillus niger, V. Tiegh., dargestellt mit Hilfe der Gefrierfitztechnik. Mikroskopie 25, 320 331 (1969) Sleytr, U. B., Kocur, M. : Structure of Mierococcus eryophilus after freeze-etching. Arch. Mikrobiol. 78, 3 5 3 - 359 (I 971 ) Sleytr, U. B., Kocur, M.: Structure of Microeoecus denitr(ficans and M. halodenitr(/~cans revealed by freeze-etching. J. appt. Bact, 36, 1 9 - 2 2 (1973) Sleytr, U. B., Kocur, M., Glauert, A. M., Thornley, M. J. : A study by freeze-etching of the fine structure of Microcoeeus radiodurans. Arch. Mikrobiol. 94, 7 7 - 8 7 (1973) Sleytr, U. B., Thornley, M. J.: Freeze-etching of the cell envelope of an Acinetobacter species which carries a regular array of surface subunits. J. Bact. il6, 1383-1397 (1973)
320 Sleytr, U. B., Umrath, W. : A simple fracturing device i'or obtaining complementary replicas of freeze-fractured and freeze-etched suspensions and tissue fragments. J. Microscopy 101, 177-186 (1974) Thi6ry, J. P. : Mise en 6vidence des polysaccharides sur couples fine en microscopie 61ectronique. J. Microscopie 6, 987-1018 (1967)
Arch. Microbiol., Vol. 107 (1976) Thornley, M. J., Horne, R. W., Glauert, A. M. : The fine structure of Micrococcus radiodurans. Arch. Mikrobiol. 51, 267-287 (1965) Work, E., Griffiths, H. : Morphology and chemistry of cell walls of Micrococcus radiodurans. J. Bact. 95, 6 4 1 - 657 (1968)
Received September 19, 1975
Microbiology
Arch. Microbiol. 107, 313- 320 (1976)
9 by Springer-Verlag 1976
The Fine Structure of Micrococcus radiophilus and Micrococcus radioproteolyticus U. B. S L E Y T R 1, M. T. S I L V A 2, M. K O C U R 3, and N. F. L E W I S 4 1Strangeways Research Laboratory, Cambridge CBI 4RN, England* ZCentro de Microscopia Electr6nica and Centro de Estudos de Bioquimica do I.A.C. Universidade do Porto, Porto, Portugal 3Czechoslovak Collection of Microorganisms, University of J. E. Purkyn~, tL Obrfincfl mini 10, Brno, (~SSR** 4 Biochemistry and Food Technology Division, Phabha Atomic Research Centre, Trombay, Bombay 400 085, India
Abstract. The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freeze-etching techniques and the two species were f o u n d to be similar in the fine structure. The only significant difference was in the appearance o f the surfaces o f the cell walls in freeze-etched preparations. Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different f r o m that o f other micrococci and Gram-positive cocci, it is recomm e n d e d that they be reclassified into a new genus.
Key words: Micrococcus radiophilus - Micrococcus radioproteolyticus - Bacterial cell walls - Fine structure -
Electron m i c r o s c o p y - T a x o n o m y .
Three species o f radiation-resistant micrococci, M.
radiodurans ( D u g g a n et al., 1959), M. radiophilus (Lewis, 1973), and M. radioproteolyticus ( K o b a t a k e et al., 1973) have been described. These species are very similar in their pigments and m o s t biochemical characteristics ( K o b a t a k e et al., 1973). Electron microscopic studies o f M. radiodurans have shown that the fine structure o f the cell wall is different f r o m that o f other Gram-positive cocci (Thornley etal., 1965; W o r k and Griffiths, 1968; Sleytr et al., 1973). To date studies have n o t been m a d e o f the ultrastructure o f the celt walls o f two other radio-resistant micrococci, and consequently the p u r p o s e o f the present w o r k was to examine the ultrastructure o f M. radiophilus and M. radioproteolyticus and c o m p a r e these observations with those already m a d e on M. radiodurans. * Permanent address: Department of Biochemical Technology, University of Agriculture, Peter-Jordan-Str. 82, A-I190 Vienna,
Austria. ** Address for offprint requests.
MATERIAL AND METHODS Organisms and Growth Conditions. The strains ltsed were Micrococcus radiophitus CCM 2564 (RBD), isolated by one of us (N. L.) and Micrococcus radioproteotyticus CCM 2703, which was supplied by M. Kobatake, National Institm for Hygiene, Tokyo, Japan.
The strains were grown in Difco Brain Heart trrfiision Broth (BH1) at 30~ with shaking or o~ BHI agar at 30~ for 16 h. Cells were harvested in the logarithmic or stationary please of growth. Electron Microscopy. For the uttrathin sectioning, the bacteria were fixed either (1) by tile procedure of Ryter and Kellenberger (R-K)
(1958) without prefixation (Silva, ~971) with or without subsequent postfixation with uranyl acetate, or (2) in 2.5 % glutaraldehyde (TAAB, London) in 0.I M cacocFylatebuffer, pH 7.0, for I h at room temperature, followed by the procedure of Ryter and Kellenberger (1958) without intermediate washing. The fixed samples were processed for electron micr~ascopyas described by Silva and Kocur (1972). The freeze-etching of intact cells of both strains was carried out in an EPA 100 (Leybold-Heraeus, Cologne, West Germany) freezeetching apparatus, bythe method of Sleytr and Umrath (1974), but complementary replicas were not examined. Platinum carbon replicas were eteaned with 30% chromic acid (CrO3) and washed in distilled water. Cleaned replicas were picked up on Formvarcoated copper grids. Thin sections and replicas were examined in a Philips 301 electron microscope operating at 80 kV with a 50 lain objective aperture.
RESULTS AND DISCUSSION
Electron Microscopy of Thin Sections Cells o f both Micrococcus radiophilus and M. radioproteolyticus are approximately spherical and 1 . 0 1.6 ~tm in diameter. They occur in pairs, tetrads, and irregular clumps (Figs. 1 c and 7). The cell wall profile o f both organisms is irreguIar in outline (Figs. 1 c and 7). In most sections o f intact or slightly autolyzed cells, three different cell wall layers can usually be distinguished (Figs. 1 a and 7). The innermost layer adjacent to the plasma m e m b r a n e is a dense layer (d), which corresponds to the rigid, peptidoglycan-containing
314
Arch. Microbiol., Vol. 107 (1976) Fig. 1a - c Thin sections of Micrococcus radiophilus. (a) The envelope of a partially lysed cell. • 84000. (b) An oblique section through the cell wall near a septation site showing a striated pattern. R-K fixation without uranyl post fixation, x 68 000. (c) Two cells in the process of division with growing septa (s). The innermost dense layer is the only one of the three visible layers of the cell wall to be involved in septum formation. The intermediate layer and the outer membrane surround groups of cells. The cytoplasmic membrane is only visible in a few areas. • 50000
Symbols used in Figures 1-13: cp cytoplasm; w cell wall; pm cytoplasmic membrane (p~n concave view, p~n convex view); om outer membrane; i intermediate layer; d dense layer; l storage material; n nucleus; pp polyphosphate granule; s septum; f fimbriae; fi fibrils; b blebs. The arrow in the lower right-hand corners of micrographs of freeze-etch replicas indicates the direction of shadowing
layer o f the cell wall. It has been observed in m a n y Gram-negative bacteria as well as in M . radiodurans (Thornley et al., 1965; W o r k and Griffiths, 1968). The next layer, the intermediate layer (i), is not always clearly visible. This layer is trypsin-sensitive in M. radiodurans and shows a pattern o f c o m p a r t m e n t s in negatively stained preparations. The outermost layer, the outer m e m b r a n e (ore), appears as a unit m e m brane and has an irregular wavy profile, Thus the cell envelope is similar to that o f a Gram-negative bacterium although the dense layer (d) is thicker than in m o s t Gram-negative bacteria. In b o t h organisms, the innermost layer is the only one o f the three visible
layers o f the cell wall to be involved in septum form a t i o n (Figs. 1 c and 7). The intermediate layer and the outer m e m b r a n e s u r r o u n d groups o f cells as observed in thin sections o f M . radiodurans (Thornley et al., 1965). The cytoplasmic m e m b r a n e is rarely observed in intact cells after either fixation procedure. This seems to be due to p o o r preservation rather than to a masking effect o f the dense cytoplasm as suggested by T h o r n l e y et al. (1965). The m e m b r a n e is m o r e clearly seen in partially lysed cells (Fig. 1 a); when visible, it has the usual appearance as an unit m e m brane. The cytoplasm is unusually dense and contains a b u n d a n t light areas (1; Figs. 1 c and 7). These
U. B. Sleytr et a l . Structure of M. radiophilus and M. radioproteolyticus
315
Figs. 2 - 6 Electron micrographs of replicas of frozenetched cells of M. radiophilus Fig. 2 Dividing cells. The fracture plane has revealed concave (p~n) and convex (p~n) views of the cytoplasmic membrane. Both septa (s) o f the tetrade can be seen. x 55000 Fig. 3 A dividing cell. Fracture has taken place within the plasma membrane, revealing the convex of the plasma membrane face (p~n) adjacent to the cytoplasm. The etched outer surface of the cell wail (w) can also be seen. In a cross fracture of the cell wall, two layers can be distinguished (arrows). x 30000 Fig. 4 Cross fractured cell with polyphosphate (pp) granules which reveal a delicate granular structure, x 50000
areas give a strong positive reaction with Thi6ry's procedure (Thi6ry, 1967) indicating that they contain polysaccharide (unpublished observation). These areas are much more clearly visible in bacteria fixed initially in OsO4 than in glutaraldehyde. Polyphosphate granules were also observed (Fig.7); they sometimes appeared as empty areas. The nuclei have the usual structure found in prokaryotic cells, consisting of arrays of fine filaments (Figs. 1 c and 7).
Electron Microscopy of Freeze-Etched Cells Examination of freeze-etched preparations of M. radiophilus and M. radioproteolytieus confirms that the cells occur predominately in tetrads or pairs (Figs. 2, 3, and 8). After low-angle shadowing the surface of the cell wall of M. radiophilus shows a fine granular structure with randomly distributed thin strands of timbriae-like material (Fig. 5). The cell wall surface of M. radioproteolyticus (Figs. 8 and 9) reveals a charac-
316
Arch. Microbiol., Vot. 107 (1976) Fig. 5 Part of a frozen-etched cell shadowed at a low angle. The structure of the surface of the cell wall (w) and fimbriae (D are visible Compare with the cell wall in Figure 6 which was shadowed at a larger angle and in which no details of the cell wall surface are visible. x 110000
Fig. 6 A freeze-etched preparation. A slightly oblique fracture shows the existence of three layers in the cell envelope (arrows), the numerous fibrils (fi) at the cross fractured cell wall arise by plastic deformation of wall components, x 110000
Fig. 7 Thin section of dividing cells of M. radioproteolyticus. Note the presence of polyphosphate (pp) and storage material (l). R-K fixation with uranyl post-fixation. • 50000
U. B. Sleytr et al. : Structure of M. radiophilusand M. radioproteolyticus
317
Figs. 8 - 12 Electron micrographs of frozen-etched cells of M. radioproteolyticus in different stages of division Fig. 8 A cell in an early stage of division. The surface of the cell wall (w) revealed by etching can be seen. The characteristic "rod-like" surface structures are randomly distributed, x 80000 Fig. 9 A later stage in cell division. The area of constriction is free of rod-like structures but blebs (b) are visible-at the septation site. The cell wall shows three layers (arrows). in a region where it is cross fractured, x 60000 Fig. I 0 A cross fracture through the region of septation of two cells. Except for a small anular region (a) the cells have completely separated, x 60000
teristic structure c o m p o s e d o f an a r r a y o f curved rodlike elevations. The average diameter o f the " r o d s " is 30 n m and they are up to 900 n m long. Similar structures have only been observed previously in M. cryophilus (Sleytr and K o c u r , 1971) and the " r o d s " in this organism were less p r o n o u n c e d and considerably shorter. The similarity between the m o r p h o l o g y and dimensions o f the rods f o u n d in M. radioproteolyticus and the rod-like elevations on the inner surface o f the cell wall adjacent t o the cytoplasm in n u m e r o u s filam e n t o u s and non-filamentous fungi ( M o o r and Mtihlethaler, 1963; R e m s e n et al., 1967; Sleytr et al., 1969) is striking.
The layer with a hexagonal pattern observed by Thornley et al. (1965) and W o r k and Griffiths (1968) in negatively-stained preparations and by Sleytr et al. (1973) in replicas o f freeze-etched specimens on the outer surface o f the cell wall o f M. radiodurans is not visible in cell walls o f M. radiophilus and M. radio-
proteolyticus. At high magnification, cross-fractured cell walls appear triple layered, similar to M. radiodurans (Sleytr et al., 1973). These layers are most distinct in sligthly oblique fractures (Figs. 6 and 9). However, in fractures almost perpendicular to the surface, only two layers o f the cell wall can be distinguished (Figs. 3,11, and 12).
318
Arch. Microbiol., Vol. 107 (1976) Figs. 11 and 12 The final stage of cell division. The annular region separates leading to the formation of fibrils Oct) which still join the two daughter cells. In a cross fracture of the cell wall, two layers can be distinguished (arrows). The blebs (b) at the septation site are still present, x 60000 Fig. 13 The pole of a newly separated cell. Blebs (b) are still visible but no rodlike structures have yet been formed. x 80000
No fracture planes are observed within the layers of the cell envelope external to the cytoplasmic membrane. Frequently, fibrils are seen, extending from the broken edges of the cell wail and lying over the fracture plane of.the cytoplasmic membrane (Figs. 5, 6, and 9). These have been interpreted as artefacts produced by plastic deformation of wall components (Sleytr, 1975). The cytoplasmic membrane fractures internally into two parts (Figs. 2 and 12). Both fracture faces show the typical irregularly-arranged membrane particles, the convex face (p~m) containing more particles than the concave face (p~m). The cytoplasmic membranes
of M. radiophilus and M. radioproteolyticus are thus similar in appearance to the cytoplasmic membranes of the other Gram-positive and Gram-negative bacteria examined by freeze-etching (Remsen and Watson, 1972). Rod-like membrane invaginations similar to those seen in Micrococcus denitrificans, M. halodenitrifieans (Sleytr and Kocur, 1973) and M. diversus (Kocur and Sleytr, 1974) are not observed. Cross-fractured cells o f M: radiophilus and M. M. radioproteolyticus reveal the presence of polyphosphate granules which have a delicate granular structure (Fig.4). Similar dense bodies have also been
U. B. Sleytr et al. : Structure of M. radiophilus and M. radioproteolyticus
observed in the cytoplasm of M. radiodurans (Sleytr et al., 1973) and other bacteria. Thornley et al. (1965) reported that these polymetaphosphate granules are frequently not preserved during sectioning and appear as empty areas. They suggest that these dense bodies probably correspond to the refractile granules observed in the phase-contrast microscope. Whereas cells of M. radiophilus do not show any changes in cell wall surface structure during septation, freeze etched preparations of M. radioproteolyticus revealed a significant alteration of the distribution of the "rod-like" structures. The changes observed during the process of cell growth and septum formation in M. radioproteolyticus are illustrated in Figs. 8 - 13. In non-dividing cells and in cells at early stages of septation, the "rods" appear randomly distributed on the cell surface (Fig. 8). With progressing septation and constriction of the cells the apparently newly-formed cell surfaces appear to be relatively free of rods and show only 12 nm particles in a random distribution (Figs.9,11, and 12). As in other dividing organisms, a surplus of material is shed in the form of blebs (b) at the septum (Sleytr and Thornley, 1973; Burdett and Murray, 1974). In the final stage of septation the cells are completely separated except for a very small annular area (Fig. 10). When the cells finally separated they are still joined by fine fibrils (Figs. ll and 12). Figure 13 is interpreted as end-on view of a newly formed cell pole. These areas are free of rod-like structures but exhibit frequently a few blebs. In contrast to M. radioproteolyticus, no extrusion of blebs of cell wall material is observed in freezeetched preparations of M. radiophilus and M. radiodurans (Sleytr et al., 1973). Thus this study of thin-sectioned and freezeetched cells of M. radiophilus and M. radioproteolyticus by electron microscopy has shown that their cell walls have a similar structure to M. radiodurans (Thornley et al., 1965; Work and Griffiths, 1968 ; Sleytr et al., 1973), except for the outermost surface layers. In addition, chemical analyses on the cell walls have shown that all these three species possess a peptidoglycan of the L-Orn-Gly2 type (Ghuysen, 1968; Schleifer and Kandler, 1972; K. H. Schleifer, pers. comm.). They differ in cell wall ultrastructure and resistance to gamma radiation so distinctly from the species of the genus Micrococcus that it is recommended that they be placed in a new separate genus. However, their relationships should be confirmed by DNA hybridization studies.
Acknowledgements. We wish to thank Dr. A. M. Glauert for helpful discussions during preparation of the manuscript and Misses
319 E. Pohoralek, M. Irene Barros, Paula Macedo, and Mr. R. Parker, for their technical assistance. This investigation was supported by the "Fonds zur F6rderung tier wissenschaftlichen Forschung Osterreichs'" (Proj. Nr. 2402) and by grant PFR/1 from the I.A.C., Lisbon, Portugal.
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320 Sleytr, U. B., Umrath, W. : A simple fracturing device i'or obtaining complementary replicas of freeze-fractured and freeze-etched suspensions and tissue fragments. J. Microscopy 101, 177-186 (1974) Thi6ry, J. P. : Mise en 6vidence des polysaccharides sur couples fine en microscopie 61ectronique. J. Microscopie 6, 987-1018 (1967)
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Received September 19, 1975
