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Ultrastructure of Mycoplasma gallisepticum isolate 1056
© 1970 by Academic Press, Inc.
318
J. ULTRASTRUCTURE RESEARCH 33, 3 1 8 - 3 3 1 (1970)
Ultrastructure
of
MycoplasmagallisepticumI s o l o t e
1056 ~
T. C. ALLEN, J. O. STEVENS, E. R. FLORANCE, AND R. O. HAMPTON
Department of Botany and Plant Pathology, Department of Veterinary Medicine, Oregon State University, and Crops Research Division, Agriculture Research Service, United States Department of Agriculture, Corvallis, Oregon 97331 Received March 6, 1970 Cells of Mycoplasma gallisepticum isolate 1056, were bound by unit membranes, were elongated, pear-shaped, or spherical, and contained 14-15 nm ribosomes, cylindrical polysomes, fibrillar networks, highly structured blebs, and infrableb regions. Small independent cells similar in size to inclusions within typical cells of M. gallisepticum isolate 1056 were observed. Both the independent cells and inclusions may represent elementary bodies. Distinctive connections between adjacent cells of isolate 1056 are described, as well as huge cells 3-4 times as large as the average-sized cells. The u l t r a s t r u c t u r e of Mycoplasma gallisepticum consists of a unit m e m b r a n e , n u c l e a r region with D N A fibrils, 14 n m r i b o s o m e s , helical polysomes, highly s t r u c t u r e d blebs, a n d i n f r a b l e b regions (1, 5, 8). O n l y one u l t r a s t r u c t u r a l difference was f o u n d b e t w e e n M. gallisepticum strains A5969 a n d $6, the d e v e l o p m e n t of f i l a m e n t o u s structures by strain $6 (1). T h e present study was u n d e r t a k e n to d e t e r m i n e the u l t r a s t r u c t u r e of M. gall# septicum isolate 1056. MATERIALS AND METHODS Dr. J. Kilian, Department of Veterinary Medicine, Oregon State University recovered a mycoplasma from a chicken trachea in 1957. This isolate, designated 1056, has been serially passed over 500 times in Yoder's T H Y broth medium; Difco tryptose phosphate broth, 15 % horse serum, and 5 % yeast autolyzate p H 7.8-8.0 (H. W. Yoder, personal communication). It has been plaque purified at least once a year and eight successive colony inhibition tests have shown that this mycoplasma is Mycoplasma gallisepticum. M. gall# septicum isolate 1056 is a stable pathogen with virulence to avian species equal to, or exceeding that of M. gallisepticum strains A5969 and S6. F o r our studies, cultures of isolate 1056 in Yoder's T H Y broth medium were incubated at 37°C. After 2 days' incubation, mycoplasma cells were pelleted from broth culture in a Servall RC2-B centrifuge at 0°C at 20 000 g for 20 minutes. The supernatant was replaced z Technical Paper No. 2868, Oregon Agricultural Experiment Station, Corvallis, Oregon 97331.
MYCOPLASMA GALLISEPTICUM ULTRASTRUCTURE
319
with 2 % glutaraldehyde in 0.125 M phosphate buffer, pH 7.0. After 90 minutes in glutaraldehyde, the pellet was washed overnight in 0.125 M phosphate buffer, pH 7.0. The buffer then was removed and the pellet was covered with 1-2 % osmium tetroxide in 0.125 M phosphate buffer for 3.5 hours. Thereafter, the pellet was washed in three 15-minute changes in 50 % acetone, soaked overnight in 70 % acetone saturated with uranyl acetate and further dehydrated in 100 % acetone (7). Pellets were embedded in an Araldite 6005-Epon 812 mixture and sections were cut with a diamond knife on a MT-2 Servall Porter-Blum ultramicrotome. Sections were collected on Formvar-coated 200-mesh copper grids and stained 20 minutes in lead citrate, pH 12.0 (9). Mycoplasma cells also were pelleted as described earlier and fixed for 3 minutes in 4 % glutaraldehyde in 0.125 M phosphate buffer, pH 7.0, or in 1% sodium cacodylate. Fixation prevented the formation of protruding filaments observed in our initial unfixed preparations. Fixed specimens to be stained were treated with phosphotungstic acid, pH 3.5 or 4.0. Fixed specimens to be metal shadowed were washed twice in distilled water after fixation, placed upon a Formvar-coated grid and shadowed with a platinum-palladium (80:20) alloy. All preparations were examined with a Philips EM-300 electron microscope operating at 60 kV. Photographic prints were made with a Durst S-45EM enlarger with a mercury lamp point light source. OBSERVATIONS
Suspensions from pellets Individual cells of M. gallisepticum isolate 1056 were variable in shape, but were primarily pear-shaped. This shape was consistent in suspended, shadowed (Fig. 1) and stained (Fig. 2) preparations and in thin sections (Figs. 3-6). Cell roundness was indicated by the length and shape of shadow (Fig. 1). The cell shown is 564 n m in length, 290 n m in m a x i m u m diameter, 97 n m in m i n i m u m diameter, and has a 129 n m bleb. Both shadowed and P T A stained preparations contained aggregates of cells. Cells within the aggregates were close to each other but did not appear to be connected (Fig. 2). These cells are 658-1036 n m in length, 294-476 n m in m a x i m u m diameter, and have 98-140 n m blebs. Several cells appear to contain inclusions 140-224 n m in size. Also present are small cells 56-224 n m in diameter.
Pellet sections Sections of cells in pellets of isolate 1056 appeared r o u n d to pear-shaped (Fig. 3). They were b o u n d by a unit m e m b r a n e that when sectioned tangentially, appeared as a diffuse b r o a d gray line. The pear-shaped cell section shown in Fig. 3 is longisectioned t h r o u g h a bleb. This bleb is 139 n m in diameter and is adjacent to a granular infrableb region. A fibrillar nuclear region occurs in the center of the cell and is bordered by polysomes. Individual ribosomes are 14 n m in diameter. This cell is 403 × 863 n m in size. Some sectioned cells had unit-membrane-bound inclusions. The central cell in Fig.
320
ALLEN ET AL.
Fla. 1. Glutaraldehyde fixed pear-shaped cell of Mycoplasma gallisepticum isolate 1056 metal shadowed with a platinum: palladium alloy. Note the length of shadow. × 62 100.
4 has a 41 × 220 n m inclusion a n d two blebs. W h a t appear to be four small cells; 116, 116, 116, a n d 180 × 215 n m in size; occur a r o u n d this central cell. I n four cases pairs of cells appeared to be c o n n e c t e d t h r o u g h their blebs. Two of these connections are illustrated (Figs. 5 a n d 6). The j o i n e d cells i n Fig. 5 are similar i n size, 578 × 866 n m a n d 658 × 912 n m , a n d b o t h c o n t a i n e d polysomes a n d u n i t m e m b r a n e - b o u n d inclusions. N o inclusions are present in Fig. 6, a n d the cells are dissimilar in size. Cells of M. gallisepticum isolate 1056 were examined in serial sections of pellets. F o r identification, cells are labeled 1-9 o n the p h o t o m i c r o g r a p h s (Fig. 7 A - C ) . Cell 1 FIG. 2. Glutaraldehyde-fixed cells stained with phosphotungstic acid, pH 4.0. Although aggregated, no connections are evident between cells. Inclusions (IN) are possessed by at least four cells. Also present are small cells (SC). x 50 840. FIa. 3. Longitudinal section of a cell following centrifugation; embedded in plastic. Shown is the unit membrane (UM), bleb (BL), infrableb region (IR), fibrils thought to be DNA (DF), and polysomes (PS). x 151 140. FIG. 4. Section through a cell with two blebs (BL) and through four small cells (SC). An inclusion (IN) with a distinct unit membrane (UM) is within the larger cell. × 134 970.
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FIG. 5. Section showing two cells, equal-sized, connected at their blebs, inclusions (IN) and polysomes (PS) occur within the cells. × 45 100. FIa. 6. Section showing two unequal-sized ceils apparently connected through their blebs. No inclusions are present, x 66 060.
has a unit membrane-bound
i n c l u s i o n t h a t is c l o s e s t t o m e d i a n c r o s s s e c t i o n i n t h e
m i d d l e s e c t i o n ( F i g . 7 B). I n t h i s s e c t i o n , t h e i n c l u s i o n h a s a d i s t i n c t m e m b r a n e
and
is s l i g h t l y l a r g e r (185 n m ) t h a n i n t h e f i r s t s e c t i o n (150 × 178 n m , Fig. 7 A ) , o r i n t h e t h i r d s e c t i o n (127 x 162 n m , Fig. 7 C ) . G r a n u l a r
material and a small dense struc-
t u r e is p r e s e n t w i t h i n t h e i n c l u s i o n . Cells 2 a n d 3 h a d b e e n s e c t i o n e d t h r o u g h t w o
FIG. 7. Serial sections of centrifuged cells. Cells are labeled 1-9 for identification. × 62170. (A) Cell 1 contains an indistinct inclusion (IN) and cell 2 has an indistinct bleb (BL). Longitudinal and transverse section of polysomes occur in most cells while cell 7 contains distinct cross sections of polysomes (PSX). (B) Cell 1 has a cross section of an inclusion (IN) that contains granular material and a dense whorl. Two blebs (BL), one protruding, occur in cells 2 and 3. One polysome (PS) 25 ribosomes long, extends into the center of cell 6. Cell 7 again has polysome cross sections (PSX). Cell 8 appears as a gray amorphous mass. Cell 9 is a small cell that occurs only in this section. ((2) The inclusion (IN) in cell 1 and the bleb (BL) in cell 2 are again indistinct. Cells 2 and 5 are connected. A polysome (PS) is within connection. Polysome cross sections (PSX) are still evident in cell 7. Note the presence of 4-5 ribosomes in each cross section. Cell 8 now shows contents similar to other cells.
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sides and the middle of the bleb. Median longitudinal sections of both blebs occur in the second section (Fig. 7B). Although the internal structure of the blebs in both cells is distinct, the bleb of cell 2 protrudes much more than what is assumed to be a rudimentary bleb in cell 3. Cell 4 contains distinct D N A strands. Cell 2 is joined to cell 5 (Fig. 7C). A polysome occurs within the cytoplasmic connection. Polysomes are present in most of the cells. One polysome in cell 6 extends into the center of the cell (Fig. 7B). This polysome is 25 ribosomes in length. Cross sections of polysomes appear as rings of four to five ribosomes, as seen most clearly in cell 7. The rings are in identical positions in all three sections of cell 7 and are therefore assumed to represent successive cross sections. Therefore, the central polysome in cell 6 contains approximately 100 ribosomes. Cell 8 is sectioned at its surface and appears as a gray amorphous mass (Fig. 7B). Internal structure is distinguishable in the next section (Fig. 7 C). At least one 64 nm small cell, cell 9, occurs in Fig. 7 B. An occasional huge cell was seen in sections of pellets (Fig. 8). This cell, 1 283 × 2 369 n m in size, is considerably larger than the average-sized adjacent cells. It contains more than 100 polysomes in longitudinal transverse, and cross section. At least three of the polysomes are 30 ribosomes in length.
DISCUSSION
M. gallisepticum isolate 1056 cells appeared to be elongated, pear-shaped, and spherical as has been reported for M. gallisepticum strain A5969 (1, 5) and strain $6 (1). Spherical cells were considered to be an artifact (5, 6) or to be a specific growth stage (1). Based on observation of serial sections, we favor the latter idea and point out that a cross section of an elongated or a pear-shaped cell also would appear spherical. Filaments protruded from unfixed cells of isolate 1056 and from fixed cells of M. gallisepticum strain $6 (1). No filaments were associated with cells of isolate 1056 in our fixed preparations, although unfixed preparations of 1056 and other strains of M. gallisepticum can contain cells with filaments (2, 5). Observations by Reuss (10) clearly demonstrated that the filaments in his unfixed preparations of mycoplasmas were artifacts caused by distortion during the drying process on the grid. Small cells were observed following all methods of preparation of isolate 1056. Bernstein-Ziv (1) considered similar cells to be buds or daugher cells following divi-
FIG. 8. Section of a huge cell in a pellet of isolate 1056. This cell is at least 3-4 times as large as the adjacent cells. Over 100 polysomes occur within the huge cell. Several polysomes (PS) are 30 ribosomes long. × 71 640.
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sion of normal-sized cells. Maniloff and Morowitz (6) felt that they were detached blebs that represented aberrant nonviable bodies unrelated to the normal cellular life cycle of strain A5969. Blebs of 1056, 98-140 nm in diameter, are within the size range of the small cells, 56-215 nm. However, the size of unit membrane-bound inclusions in 1056 cells, 41-244 nm, is almost identical to the size of the small cells. To our knowledge, the presence of inclusions within M. gallisepticurn cells has not been reported. Bernstein-Ziv (1) did observe "bubbles" appearing on the top of several negatively stained unfixed cells of strain $6, but thought these probably were stain artifacts. Similar configurations were seen in negatively stained cells as well as sectioned cells of isolate 1056. They would therefore appear not to be stain artifacts, although they could be fixation artifacts, vacuoles, and/or elementary bodies. Domermuth et al. (3) described 120-190 nm elementary bodies for two strains of M. gallisepticum, but found no vacuoles, and others reported that their strains of M. gallisepticurn contained no elementary bodies (1, 6). Polysomes occurred within cells of isolate 1056. Apparently, they appear in M. gallisepticum cells only in the logarithmic growth phase when cell activity is high (1, 4). Polysomes were present in all shapes of cells of isolate 1056. Maniloff and Morowitz (6) observed them only in spherical cells of strain A5969. Most of the polysomes in strain A5969 were ten ribosomes long (4), and the largest number of ribosomes found in a row was 30 (1). Polysomes within a huge cell of isolate 1056 also extended 30 ribosomes in length, but the longest polysome observed in average-sized cells of 1056 was 25 ribosomes long. In cross section, polysomes of isolate 1056 contained four or five ribosomes as originally described for M. gallisepticurn strain A 5969 (4). This ribosome arrangement indicates that the polysomes are helical, as first suggested by Maniloff et al. (4). Maniloff et al. (4) also were the first to describe the ultrastructure of blebs of M. gallisepticum. Blebs of isolate 1056 are identical to this description. The role of the bleb in cell division was not examined by us, although connections between blebs of two cells were noted. Another type of intercellular connection that resembled conjugation was shown. Neither of these types of intercellular connections have been reported for M. gallisepticum. The occurrence of several huge cells in isolate 1056 cultures is unique since they have not been reported in the literature on M. gallisepticurn. These huge cells had the ultrastructural features of M. gaIlisepticum but were 3-4 times as large as th e averagesized cells. The ultrastructure studies of isolate 1056 help to substantiate its identity as M. gallisepticurn. To determine whether it is a distinctly different strain of M. gallisepticurn will require additional research. Therefore, at present, we prefer to call it an isolate instead of a strain.
MYCOPLASMA
GALLISEPTICUM
ULTRASTRUCTURE
331
This work was supported in part by National Science Foundation Grant BG-5677, Public Health Service Institutional Grant awarded to Oregon State University, and Grant-in-aid from USDA, ARS, ADPD, agreement no. 12-14-100-10, 349 (45).
REFERENCES 1. BERNSTEIN-ZIv,R., Can. J. MicrobioL 15, 1125 (1969). 2. CHU, H. P. and HORNE, R. W., Ann. N . Y . Acad. Sei. 143, 190 (1967). 3. DOMERMUTH, C. H., NIELSON, M. H., FREUNDT, E. A. and BmcH-ANDZRSON, A., J. Bacteriol. 88, 727 (1964). 4. MANILOFF,J., MOROWITZ,H. J. and BARRNETT,R. J., J. Cell Biol. 25, 139 (1965). 5. - Y. Baeteriol. 90, 193 (1965). 6. MANILOFF,J. and MOROWITZ,H. J., Ann. N . Y . Acad. Sei. 143, 59 (1967). 7. MILNE, R. G., Virology 28, 79 (1966). 8. MOROWITZ,H. J. and MANILOrF, J., J. BacterioL 91, 1638 (1966). 9. REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). 10. REUSS, K., J. Bacteriol. 93, 490 (1967).
2 2 - 7 0 1 8 3 1 J . Ultrastructure Research
318
J. ULTRASTRUCTURE RESEARCH 33, 3 1 8 - 3 3 1 (1970)
Ultrastructure
of
MycoplasmagallisepticumI s o l o t e
1056 ~
T. C. ALLEN, J. O. STEVENS, E. R. FLORANCE, AND R. O. HAMPTON
Department of Botany and Plant Pathology, Department of Veterinary Medicine, Oregon State University, and Crops Research Division, Agriculture Research Service, United States Department of Agriculture, Corvallis, Oregon 97331 Received March 6, 1970 Cells of Mycoplasma gallisepticum isolate 1056, were bound by unit membranes, were elongated, pear-shaped, or spherical, and contained 14-15 nm ribosomes, cylindrical polysomes, fibrillar networks, highly structured blebs, and infrableb regions. Small independent cells similar in size to inclusions within typical cells of M. gallisepticum isolate 1056 were observed. Both the independent cells and inclusions may represent elementary bodies. Distinctive connections between adjacent cells of isolate 1056 are described, as well as huge cells 3-4 times as large as the average-sized cells. The u l t r a s t r u c t u r e of Mycoplasma gallisepticum consists of a unit m e m b r a n e , n u c l e a r region with D N A fibrils, 14 n m r i b o s o m e s , helical polysomes, highly s t r u c t u r e d blebs, a n d i n f r a b l e b regions (1, 5, 8). O n l y one u l t r a s t r u c t u r a l difference was f o u n d b e t w e e n M. gallisepticum strains A5969 a n d $6, the d e v e l o p m e n t of f i l a m e n t o u s structures by strain $6 (1). T h e present study was u n d e r t a k e n to d e t e r m i n e the u l t r a s t r u c t u r e of M. gall# septicum isolate 1056. MATERIALS AND METHODS Dr. J. Kilian, Department of Veterinary Medicine, Oregon State University recovered a mycoplasma from a chicken trachea in 1957. This isolate, designated 1056, has been serially passed over 500 times in Yoder's T H Y broth medium; Difco tryptose phosphate broth, 15 % horse serum, and 5 % yeast autolyzate p H 7.8-8.0 (H. W. Yoder, personal communication). It has been plaque purified at least once a year and eight successive colony inhibition tests have shown that this mycoplasma is Mycoplasma gallisepticum. M. gall# septicum isolate 1056 is a stable pathogen with virulence to avian species equal to, or exceeding that of M. gallisepticum strains A5969 and S6. F o r our studies, cultures of isolate 1056 in Yoder's T H Y broth medium were incubated at 37°C. After 2 days' incubation, mycoplasma cells were pelleted from broth culture in a Servall RC2-B centrifuge at 0°C at 20 000 g for 20 minutes. The supernatant was replaced z Technical Paper No. 2868, Oregon Agricultural Experiment Station, Corvallis, Oregon 97331.
MYCOPLASMA GALLISEPTICUM ULTRASTRUCTURE
319
with 2 % glutaraldehyde in 0.125 M phosphate buffer, pH 7.0. After 90 minutes in glutaraldehyde, the pellet was washed overnight in 0.125 M phosphate buffer, pH 7.0. The buffer then was removed and the pellet was covered with 1-2 % osmium tetroxide in 0.125 M phosphate buffer for 3.5 hours. Thereafter, the pellet was washed in three 15-minute changes in 50 % acetone, soaked overnight in 70 % acetone saturated with uranyl acetate and further dehydrated in 100 % acetone (7). Pellets were embedded in an Araldite 6005-Epon 812 mixture and sections were cut with a diamond knife on a MT-2 Servall Porter-Blum ultramicrotome. Sections were collected on Formvar-coated 200-mesh copper grids and stained 20 minutes in lead citrate, pH 12.0 (9). Mycoplasma cells also were pelleted as described earlier and fixed for 3 minutes in 4 % glutaraldehyde in 0.125 M phosphate buffer, pH 7.0, or in 1% sodium cacodylate. Fixation prevented the formation of protruding filaments observed in our initial unfixed preparations. Fixed specimens to be stained were treated with phosphotungstic acid, pH 3.5 or 4.0. Fixed specimens to be metal shadowed were washed twice in distilled water after fixation, placed upon a Formvar-coated grid and shadowed with a platinum-palladium (80:20) alloy. All preparations were examined with a Philips EM-300 electron microscope operating at 60 kV. Photographic prints were made with a Durst S-45EM enlarger with a mercury lamp point light source. OBSERVATIONS
Suspensions from pellets Individual cells of M. gallisepticum isolate 1056 were variable in shape, but were primarily pear-shaped. This shape was consistent in suspended, shadowed (Fig. 1) and stained (Fig. 2) preparations and in thin sections (Figs. 3-6). Cell roundness was indicated by the length and shape of shadow (Fig. 1). The cell shown is 564 n m in length, 290 n m in m a x i m u m diameter, 97 n m in m i n i m u m diameter, and has a 129 n m bleb. Both shadowed and P T A stained preparations contained aggregates of cells. Cells within the aggregates were close to each other but did not appear to be connected (Fig. 2). These cells are 658-1036 n m in length, 294-476 n m in m a x i m u m diameter, and have 98-140 n m blebs. Several cells appear to contain inclusions 140-224 n m in size. Also present are small cells 56-224 n m in diameter.
Pellet sections Sections of cells in pellets of isolate 1056 appeared r o u n d to pear-shaped (Fig. 3). They were b o u n d by a unit m e m b r a n e that when sectioned tangentially, appeared as a diffuse b r o a d gray line. The pear-shaped cell section shown in Fig. 3 is longisectioned t h r o u g h a bleb. This bleb is 139 n m in diameter and is adjacent to a granular infrableb region. A fibrillar nuclear region occurs in the center of the cell and is bordered by polysomes. Individual ribosomes are 14 n m in diameter. This cell is 403 × 863 n m in size. Some sectioned cells had unit-membrane-bound inclusions. The central cell in Fig.
320
ALLEN ET AL.
Fla. 1. Glutaraldehyde fixed pear-shaped cell of Mycoplasma gallisepticum isolate 1056 metal shadowed with a platinum: palladium alloy. Note the length of shadow. × 62 100.
4 has a 41 × 220 n m inclusion a n d two blebs. W h a t appear to be four small cells; 116, 116, 116, a n d 180 × 215 n m in size; occur a r o u n d this central cell. I n four cases pairs of cells appeared to be c o n n e c t e d t h r o u g h their blebs. Two of these connections are illustrated (Figs. 5 a n d 6). The j o i n e d cells i n Fig. 5 are similar i n size, 578 × 866 n m a n d 658 × 912 n m , a n d b o t h c o n t a i n e d polysomes a n d u n i t m e m b r a n e - b o u n d inclusions. N o inclusions are present in Fig. 6, a n d the cells are dissimilar in size. Cells of M. gallisepticum isolate 1056 were examined in serial sections of pellets. F o r identification, cells are labeled 1-9 o n the p h o t o m i c r o g r a p h s (Fig. 7 A - C ) . Cell 1 FIG. 2. Glutaraldehyde-fixed cells stained with phosphotungstic acid, pH 4.0. Although aggregated, no connections are evident between cells. Inclusions (IN) are possessed by at least four cells. Also present are small cells (SC). x 50 840. FIa. 3. Longitudinal section of a cell following centrifugation; embedded in plastic. Shown is the unit membrane (UM), bleb (BL), infrableb region (IR), fibrils thought to be DNA (DF), and polysomes (PS). x 151 140. FIG. 4. Section through a cell with two blebs (BL) and through four small cells (SC). An inclusion (IN) with a distinct unit membrane (UM) is within the larger cell. × 134 970.
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FIG. 5. Section showing two cells, equal-sized, connected at their blebs, inclusions (IN) and polysomes (PS) occur within the cells. × 45 100. FIa. 6. Section showing two unequal-sized ceils apparently connected through their blebs. No inclusions are present, x 66 060.
has a unit membrane-bound
i n c l u s i o n t h a t is c l o s e s t t o m e d i a n c r o s s s e c t i o n i n t h e
m i d d l e s e c t i o n ( F i g . 7 B). I n t h i s s e c t i o n , t h e i n c l u s i o n h a s a d i s t i n c t m e m b r a n e
and
is s l i g h t l y l a r g e r (185 n m ) t h a n i n t h e f i r s t s e c t i o n (150 × 178 n m , Fig. 7 A ) , o r i n t h e t h i r d s e c t i o n (127 x 162 n m , Fig. 7 C ) . G r a n u l a r
material and a small dense struc-
t u r e is p r e s e n t w i t h i n t h e i n c l u s i o n . Cells 2 a n d 3 h a d b e e n s e c t i o n e d t h r o u g h t w o
FIG. 7. Serial sections of centrifuged cells. Cells are labeled 1-9 for identification. × 62170. (A) Cell 1 contains an indistinct inclusion (IN) and cell 2 has an indistinct bleb (BL). Longitudinal and transverse section of polysomes occur in most cells while cell 7 contains distinct cross sections of polysomes (PSX). (B) Cell 1 has a cross section of an inclusion (IN) that contains granular material and a dense whorl. Two blebs (BL), one protruding, occur in cells 2 and 3. One polysome (PS) 25 ribosomes long, extends into the center of cell 6. Cell 7 again has polysome cross sections (PSX). Cell 8 appears as a gray amorphous mass. Cell 9 is a small cell that occurs only in this section. ((2) The inclusion (IN) in cell 1 and the bleb (BL) in cell 2 are again indistinct. Cells 2 and 5 are connected. A polysome (PS) is within connection. Polysome cross sections (PSX) are still evident in cell 7. Note the presence of 4-5 ribosomes in each cross section. Cell 8 now shows contents similar to other cells.
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sides and the middle of the bleb. Median longitudinal sections of both blebs occur in the second section (Fig. 7B). Although the internal structure of the blebs in both cells is distinct, the bleb of cell 2 protrudes much more than what is assumed to be a rudimentary bleb in cell 3. Cell 4 contains distinct D N A strands. Cell 2 is joined to cell 5 (Fig. 7C). A polysome occurs within the cytoplasmic connection. Polysomes are present in most of the cells. One polysome in cell 6 extends into the center of the cell (Fig. 7B). This polysome is 25 ribosomes in length. Cross sections of polysomes appear as rings of four to five ribosomes, as seen most clearly in cell 7. The rings are in identical positions in all three sections of cell 7 and are therefore assumed to represent successive cross sections. Therefore, the central polysome in cell 6 contains approximately 100 ribosomes. Cell 8 is sectioned at its surface and appears as a gray amorphous mass (Fig. 7B). Internal structure is distinguishable in the next section (Fig. 7 C). At least one 64 nm small cell, cell 9, occurs in Fig. 7 B. An occasional huge cell was seen in sections of pellets (Fig. 8). This cell, 1 283 × 2 369 n m in size, is considerably larger than the average-sized adjacent cells. It contains more than 100 polysomes in longitudinal transverse, and cross section. At least three of the polysomes are 30 ribosomes in length.
DISCUSSION
M. gallisepticum isolate 1056 cells appeared to be elongated, pear-shaped, and spherical as has been reported for M. gallisepticum strain A5969 (1, 5) and strain $6 (1). Spherical cells were considered to be an artifact (5, 6) or to be a specific growth stage (1). Based on observation of serial sections, we favor the latter idea and point out that a cross section of an elongated or a pear-shaped cell also would appear spherical. Filaments protruded from unfixed cells of isolate 1056 and from fixed cells of M. gallisepticum strain $6 (1). No filaments were associated with cells of isolate 1056 in our fixed preparations, although unfixed preparations of 1056 and other strains of M. gallisepticum can contain cells with filaments (2, 5). Observations by Reuss (10) clearly demonstrated that the filaments in his unfixed preparations of mycoplasmas were artifacts caused by distortion during the drying process on the grid. Small cells were observed following all methods of preparation of isolate 1056. Bernstein-Ziv (1) considered similar cells to be buds or daugher cells following divi-
FIG. 8. Section of a huge cell in a pellet of isolate 1056. This cell is at least 3-4 times as large as the adjacent cells. Over 100 polysomes occur within the huge cell. Several polysomes (PS) are 30 ribosomes long. × 71 640.
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sion of normal-sized cells. Maniloff and Morowitz (6) felt that they were detached blebs that represented aberrant nonviable bodies unrelated to the normal cellular life cycle of strain A5969. Blebs of 1056, 98-140 nm in diameter, are within the size range of the small cells, 56-215 nm. However, the size of unit membrane-bound inclusions in 1056 cells, 41-244 nm, is almost identical to the size of the small cells. To our knowledge, the presence of inclusions within M. gallisepticurn cells has not been reported. Bernstein-Ziv (1) did observe "bubbles" appearing on the top of several negatively stained unfixed cells of strain $6, but thought these probably were stain artifacts. Similar configurations were seen in negatively stained cells as well as sectioned cells of isolate 1056. They would therefore appear not to be stain artifacts, although they could be fixation artifacts, vacuoles, and/or elementary bodies. Domermuth et al. (3) described 120-190 nm elementary bodies for two strains of M. gallisepticum, but found no vacuoles, and others reported that their strains of M. gallisepticurn contained no elementary bodies (1, 6). Polysomes occurred within cells of isolate 1056. Apparently, they appear in M. gallisepticum cells only in the logarithmic growth phase when cell activity is high (1, 4). Polysomes were present in all shapes of cells of isolate 1056. Maniloff and Morowitz (6) observed them only in spherical cells of strain A5969. Most of the polysomes in strain A5969 were ten ribosomes long (4), and the largest number of ribosomes found in a row was 30 (1). Polysomes within a huge cell of isolate 1056 also extended 30 ribosomes in length, but the longest polysome observed in average-sized cells of 1056 was 25 ribosomes long. In cross section, polysomes of isolate 1056 contained four or five ribosomes as originally described for M. gallisepticurn strain A 5969 (4). This ribosome arrangement indicates that the polysomes are helical, as first suggested by Maniloff et al. (4). Maniloff et al. (4) also were the first to describe the ultrastructure of blebs of M. gallisepticum. Blebs of isolate 1056 are identical to this description. The role of the bleb in cell division was not examined by us, although connections between blebs of two cells were noted. Another type of intercellular connection that resembled conjugation was shown. Neither of these types of intercellular connections have been reported for M. gallisepticum. The occurrence of several huge cells in isolate 1056 cultures is unique since they have not been reported in the literature on M. gallisepticurn. These huge cells had the ultrastructural features of M. gaIlisepticum but were 3-4 times as large as th e averagesized cells. The ultrastructure studies of isolate 1056 help to substantiate its identity as M. gallisepticurn. To determine whether it is a distinctly different strain of M. gallisepticurn will require additional research. Therefore, at present, we prefer to call it an isolate instead of a strain.
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This work was supported in part by National Science Foundation Grant BG-5677, Public Health Service Institutional Grant awarded to Oregon State University, and Grant-in-aid from USDA, ARS, ADPD, agreement no. 12-14-100-10, 349 (45).
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