Vol. 15, No. 4 Printed in U.S.A.
JOURNAL OF VIROLOGY, Apr. 1975, p. 894-897 Copyright 0 1975 American Society for Microbiology
Bacteriophage-Associated Spherical Bodies in Bacteroides fragilis RICHARD P. SILVER,* DAVID G. CHASE, FRANCIS P. TALLY, AND SHERWOOD L. GORBACH Infectious Disease Section and Cell Biology Laboratory, Veterans Administration Hospital, Sepulveda, California 91343,* and Department of Medicine, University of California School of Medicine, Los Angeles, California 90024
Received for publication 8 January 1975
Unique spherical bodies with multilayered walls were observed by electron microscopy in cells of a single strain of Bacteroides fragilis subsp. fragilis. Phage-like particles were present in the same cells, both free in the cytoplasm and within the spheres. The proportion of cells containing the phage-associated spherical structures ranged from less than 0.01% to about 7% depending on the culture conditions. Phage particles of morphological type B and spherical bodies were also found free in the medium surrounding the cells. Spherical bodies with discontinuities in their walls, through which phage-like particles sometimes appeared to be escaping, were also found both intra- and extracellularly. The biological significance of these distinctive spherical structures is a matter of conjecture.
Bacteroides fragilis is an anaerobic nonspore-forming gram-negative bacillus. It is a prominent constituent of the human colonic flora, outnumbering coliforms at least 1000 to 1 (3). There is an increasing awareness of the importance of Bacteroides fragilis in infectious processes, and it is now recognized as a significant human pathogen (4). However, relative to the common intestinal aerobes very little is known about the basic biology of this important microorganism. In the course of studies on the genetics, molecular biology, and ultrastructure of certain colonic anaerobes, we observed a unique spherical body associated with phagelike particles in a single strain of Bacteroides fragilis. The present communication provides a morphological description of this structure.
postfixed, dehydrated, and embedded by methods previously described (2). For negative staining, drops of diluted cell suspensions were placed on Teflon blocks. Grids were floated in sequence on drops of cell suspension, fixative (phosphate-buffered 3% glutaraldehyde), 1% ammonium acetate, and 2% uranyl acetate for 60 s each.
MATERIALS AND METHODS Bacteroides fragilis subsp. fragilis (American Type Culture Collection no. 23745) was used in this study. Broth media was prereduced brain heart infusion broth supplemented with yeast extract (0.5%), hemin (0.0005%) and menadione (0.00001%) (Scott Laboratories). Tubes were inoculated under a stream of N2 gas and incubated at 37 C. Agar plates (brain heart infusion agar with 5% sheep blood) were incubated at 37 C in the Gas-Pak anaerobic system (Baltimore
Biological Laboratories). For thin sectioning, cells in broth cultures were pelleted at 8000 x g for 10 min and flooded with fixative (phosphate-buffered 3% glutaraldehyde) or were collected with a loop from the surface of agar plates and transferred in clumps directly to fixative. Cells were fixed for 1 to 2 h. These preparations were 894
RESULTS
The phage-associated spherical bodies (Fig. 1A) were observed in thin sections of Bacteroides fragilis subsp. fragilis (ATCC 23745), both in our laboratory stock strain as well as in a lyophilized culture of the same strain from the American Type Culture Collection. We failed to detect the spherical structure in seven other strains of B. fragilis subsp. fragilis. As many as 20 spherical bodies were counted in a longitudinal thin section of a single intact cell. They were also found in cell ghosts (Fig. IB) or free in the medium (Fig. 1C to E), but were most frequent in intact cells. Serial sectioning verified that the structures under consideration were indeed spherical. The spheres were 300 nm in diameter and comprised an outer multilayered wall 60 nm thick and a central space with heterogenous contents. The contents included objects which corresponded in size, shape, and density to the heads of phage-like particles present in the cell cytoplasm (Fig. 2A) and in the medium (Fig. 2B). These particles were always present in intact spheres and were of two types according to electron density. Spheres contained either pale
.
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c FIG. 1. (A) Intact cell containing phage-like particles and spherical bodies with multilayered walls. Some of the particles are free, whereas others are sequestered within the spherical structures. One spherical body contains dense phage-like particles; the other three contain pale particles. x100,000. (B) Cell ghost containing spherical bodies and phage-like particles. A tail is seen on the particle at the far left. x52,000. (C) Spherical body free in the medium. x86,000. (D) Free spherical body with a discontinuity in its wall. x86,000. (E) An open spherical body closely apposed to the cell wall of a bacterium. Within the sphere an empty phage capsid is attached to the bacterium by a tail. A second attached tail can be seen. x86,000. 895
896
SILVER ET AL.
J. VIROL.
W..
u
4. ..
*A
.4--
.14 V ,.
.:..A
i.
*B.w~~~-S+.Sx >s ;.-
.
~; ~~~~~C
Li)
FIG. 2. (A) Intact bacterial cell containing spherical structures and large numbers of phage-like particles. x43,000. (B) Phage particle attached by a tail to a bacterial cell. x86,000. (C) Open spherical body within a bacterial cell. x52,000. (D) Negatively stained free phage particle. x 150,000.
or dense particles exclusively, although both pale and dense containing spheres were commonly found in the same cell (Fig. IA). A maximum of' 17 alternating dark and light layers could be resolved in the wall of the spherical body (Fig. IC). Allowing for minor variations caused by differences in focus and plane of sectioning, it appeared that the number of layers and the thickness and relative density of each layer was the same in all spheres. A single significant exception to this was a variation in the density of the innermost layer, which was usually very dense in spheres containing the dense phage-like particles and less dense in those containing pale phage-like particles (Fig. IA). Occasional cells contained incomplete spheres from which the phage-like particles appeared to be escaping (Fig. 2C). Incomplete spheres were also present in the medium; some were empty fragments of' various sizes, whereas others still contained phage-like particles (Fig. ID and IE). That these particles were related to phage was particularly evident in Fig. IE, which shows an incomplete sphere partially enclosing an empty-headed phage attached to the surface of' a bacterium. The morphology of the phage-like particles
(Fig. IE, 2B, and 2D) placed them in Bradley's group B (1). They had long, noncontractile tails, (185 nm long by 15 nm wide) and polyhedral, probably octahedral, heads (50 to 60 nm in diameter). Both empty and f'ull heads were f'ound in the medium, and this leads us to assume that the intracellular pale and dense particles represent capsids devoid of', or full of', nucleic acid, respectively. The spherical bodies were f'irst observed at very low frequencies (usually less than 0.01'.%) in cells harvested from broth cultures 6 h after dilution with fresh medium. The f'requency increased dramatically to approximately 7%' in cells grown for 60 h on the surface of' blood agar
plates.
DISCUSSION We have described an unusual biological system in which a complex spherical structure is formed in conjunction with the spontaneous expression of' what is presumably temperate phage. To our knowledge. the only other comparable system is the production of' refractile bodies in kappa, the bacterial endosymbiont of' killer strains of Paramecium aurelia (6). Like the spherical structures described here, ref'ractile bodies are associated with phage-like parti-
VOL. 15, 1975
PHAGE-ASSOCIATED BODIES IN B. FRAGILIS
cles and vary in frequency with culture conditions (6). Both structures are also lamellar. However, refractile bodies are hollow cylinders formed from a single coiled ribbon of homogeneous composition (5), whereas spheres comprise multiple layers of differing thickness and density, suggesting a more complex composition. The biological significance of phageassociated spheres in a single strain of Bacteroides fragilis subsp. fragilis is still obscure. None of our observations indicate how or when, relative to the assembly of phage particles, the spheres are formed. We do not know if the genetic information for the production of spheres is carried by the phage, by the bacterium, or both. Three possible functions come to mind, none of which can be definitely excluded. The presence of phage-like particles within the spheres raises the possibility that they are involved in phage morphogenesis. On the other hand, Fig. IE and our observations on negatively stained material indicate that extracellular spheres often occur in close apposition to the cell wall of intact bacteria. This suggests that spheres might play some role in phage dissemination, perhaps by retarding inactivation of phage in a hostile extracellular environment. For either of these functions the spheres would probably be products of the phage genome. Another possibility is that the spheres are produced by the bacterial cells as a defense
897
mechanism to retard the spread of phage. However, it is difficult to imagine the bacteria evolving an elaborate and clearly imperfect defense mechanism against an agent to which it is already immune because of lysogeny. Employing standard methods of phage induction such as mitomycin C and UV light, we failed to enhance the proportion of cells expressing spherical bodies and associated phage. Attempts to find strains of Bacteroides fragilis sensitive to the phage have also been unsuccessful. ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of Suni Kloss. This work was supported by Institutional Research Funds, Veterans Administration Hospital, Sepulveda, Calif. LITERATURE CITED 1. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 2. Chase, D. G., and L. Pik6. 1973. Expression of A- and C-type particles in early mouse embryos. J. Natl. Cancer Inst. 51:1971-1975. 3. Gorbach, S. L. 1971. Intestinal microflora. Gastroenterology 60:1110-1129. 4. Gorbach, S. L., and J. G. Bartlett. 1974. Anaerobic infections. N. Engl. J. Med. 290:1177-1184, 1237-1245, 1289-1294. 5. Preer, J. R., Jr., and L. B. Preer. 1967. Virus-like bodies in killer paramecia. Proc. Natl. Acad. Sci. U.S.A. 58:1774-1781. 6. Preer, J. R., Jr., L. B. Preer, and A. Jurand. 1974. Kappa and other endosymbionts in iParamecium aurelia.. Bacteriol. Rev. 38:113-163.
JOURNAL OF VIROLOGY, Apr. 1975, p. 894-897 Copyright 0 1975 American Society for Microbiology
Bacteriophage-Associated Spherical Bodies in Bacteroides fragilis RICHARD P. SILVER,* DAVID G. CHASE, FRANCIS P. TALLY, AND SHERWOOD L. GORBACH Infectious Disease Section and Cell Biology Laboratory, Veterans Administration Hospital, Sepulveda, California 91343,* and Department of Medicine, University of California School of Medicine, Los Angeles, California 90024
Received for publication 8 January 1975
Unique spherical bodies with multilayered walls were observed by electron microscopy in cells of a single strain of Bacteroides fragilis subsp. fragilis. Phage-like particles were present in the same cells, both free in the cytoplasm and within the spheres. The proportion of cells containing the phage-associated spherical structures ranged from less than 0.01% to about 7% depending on the culture conditions. Phage particles of morphological type B and spherical bodies were also found free in the medium surrounding the cells. Spherical bodies with discontinuities in their walls, through which phage-like particles sometimes appeared to be escaping, were also found both intra- and extracellularly. The biological significance of these distinctive spherical structures is a matter of conjecture.
Bacteroides fragilis is an anaerobic nonspore-forming gram-negative bacillus. It is a prominent constituent of the human colonic flora, outnumbering coliforms at least 1000 to 1 (3). There is an increasing awareness of the importance of Bacteroides fragilis in infectious processes, and it is now recognized as a significant human pathogen (4). However, relative to the common intestinal aerobes very little is known about the basic biology of this important microorganism. In the course of studies on the genetics, molecular biology, and ultrastructure of certain colonic anaerobes, we observed a unique spherical body associated with phagelike particles in a single strain of Bacteroides fragilis. The present communication provides a morphological description of this structure.
postfixed, dehydrated, and embedded by methods previously described (2). For negative staining, drops of diluted cell suspensions were placed on Teflon blocks. Grids were floated in sequence on drops of cell suspension, fixative (phosphate-buffered 3% glutaraldehyde), 1% ammonium acetate, and 2% uranyl acetate for 60 s each.
MATERIALS AND METHODS Bacteroides fragilis subsp. fragilis (American Type Culture Collection no. 23745) was used in this study. Broth media was prereduced brain heart infusion broth supplemented with yeast extract (0.5%), hemin (0.0005%) and menadione (0.00001%) (Scott Laboratories). Tubes were inoculated under a stream of N2 gas and incubated at 37 C. Agar plates (brain heart infusion agar with 5% sheep blood) were incubated at 37 C in the Gas-Pak anaerobic system (Baltimore
Biological Laboratories). For thin sectioning, cells in broth cultures were pelleted at 8000 x g for 10 min and flooded with fixative (phosphate-buffered 3% glutaraldehyde) or were collected with a loop from the surface of agar plates and transferred in clumps directly to fixative. Cells were fixed for 1 to 2 h. These preparations were 894
RESULTS
The phage-associated spherical bodies (Fig. 1A) were observed in thin sections of Bacteroides fragilis subsp. fragilis (ATCC 23745), both in our laboratory stock strain as well as in a lyophilized culture of the same strain from the American Type Culture Collection. We failed to detect the spherical structure in seven other strains of B. fragilis subsp. fragilis. As many as 20 spherical bodies were counted in a longitudinal thin section of a single intact cell. They were also found in cell ghosts (Fig. IB) or free in the medium (Fig. 1C to E), but were most frequent in intact cells. Serial sectioning verified that the structures under consideration were indeed spherical. The spheres were 300 nm in diameter and comprised an outer multilayered wall 60 nm thick and a central space with heterogenous contents. The contents included objects which corresponded in size, shape, and density to the heads of phage-like particles present in the cell cytoplasm (Fig. 2A) and in the medium (Fig. 2B). These particles were always present in intact spheres and were of two types according to electron density. Spheres contained either pale
.
--.At4
m
Ti7- AL' Iq liLl.
I
r=p ,Ir:;
t, 7 "
II!,
-1
I-% 1.
1.1
I I
t
.Ilk-, iNk
-
F
I
Al
."j -
Is
{i
A
c FIG. 1. (A) Intact cell containing phage-like particles and spherical bodies with multilayered walls. Some of the particles are free, whereas others are sequestered within the spherical structures. One spherical body contains dense phage-like particles; the other three contain pale particles. x100,000. (B) Cell ghost containing spherical bodies and phage-like particles. A tail is seen on the particle at the far left. x52,000. (C) Spherical body free in the medium. x86,000. (D) Free spherical body with a discontinuity in its wall. x86,000. (E) An open spherical body closely apposed to the cell wall of a bacterium. Within the sphere an empty phage capsid is attached to the bacterium by a tail. A second attached tail can be seen. x86,000. 895
896
SILVER ET AL.
J. VIROL.
W..
u
4. ..
*A
.4--
.14 V ,.
.:..A
i.
*B.w~~~-S+.Sx >s ;.-
.
~; ~~~~~C
Li)
FIG. 2. (A) Intact bacterial cell containing spherical structures and large numbers of phage-like particles. x43,000. (B) Phage particle attached by a tail to a bacterial cell. x86,000. (C) Open spherical body within a bacterial cell. x52,000. (D) Negatively stained free phage particle. x 150,000.
or dense particles exclusively, although both pale and dense containing spheres were commonly found in the same cell (Fig. IA). A maximum of' 17 alternating dark and light layers could be resolved in the wall of the spherical body (Fig. IC). Allowing for minor variations caused by differences in focus and plane of sectioning, it appeared that the number of layers and the thickness and relative density of each layer was the same in all spheres. A single significant exception to this was a variation in the density of the innermost layer, which was usually very dense in spheres containing the dense phage-like particles and less dense in those containing pale phage-like particles (Fig. IA). Occasional cells contained incomplete spheres from which the phage-like particles appeared to be escaping (Fig. 2C). Incomplete spheres were also present in the medium; some were empty fragments of' various sizes, whereas others still contained phage-like particles (Fig. ID and IE). That these particles were related to phage was particularly evident in Fig. IE, which shows an incomplete sphere partially enclosing an empty-headed phage attached to the surface of' a bacterium. The morphology of the phage-like particles
(Fig. IE, 2B, and 2D) placed them in Bradley's group B (1). They had long, noncontractile tails, (185 nm long by 15 nm wide) and polyhedral, probably octahedral, heads (50 to 60 nm in diameter). Both empty and f'ull heads were f'ound in the medium, and this leads us to assume that the intracellular pale and dense particles represent capsids devoid of', or full of', nucleic acid, respectively. The spherical bodies were f'irst observed at very low frequencies (usually less than 0.01'.%) in cells harvested from broth cultures 6 h after dilution with fresh medium. The f'requency increased dramatically to approximately 7%' in cells grown for 60 h on the surface of' blood agar
plates.
DISCUSSION We have described an unusual biological system in which a complex spherical structure is formed in conjunction with the spontaneous expression of' what is presumably temperate phage. To our knowledge. the only other comparable system is the production of' refractile bodies in kappa, the bacterial endosymbiont of' killer strains of Paramecium aurelia (6). Like the spherical structures described here, ref'ractile bodies are associated with phage-like parti-
VOL. 15, 1975
PHAGE-ASSOCIATED BODIES IN B. FRAGILIS
cles and vary in frequency with culture conditions (6). Both structures are also lamellar. However, refractile bodies are hollow cylinders formed from a single coiled ribbon of homogeneous composition (5), whereas spheres comprise multiple layers of differing thickness and density, suggesting a more complex composition. The biological significance of phageassociated spheres in a single strain of Bacteroides fragilis subsp. fragilis is still obscure. None of our observations indicate how or when, relative to the assembly of phage particles, the spheres are formed. We do not know if the genetic information for the production of spheres is carried by the phage, by the bacterium, or both. Three possible functions come to mind, none of which can be definitely excluded. The presence of phage-like particles within the spheres raises the possibility that they are involved in phage morphogenesis. On the other hand, Fig. IE and our observations on negatively stained material indicate that extracellular spheres often occur in close apposition to the cell wall of intact bacteria. This suggests that spheres might play some role in phage dissemination, perhaps by retarding inactivation of phage in a hostile extracellular environment. For either of these functions the spheres would probably be products of the phage genome. Another possibility is that the spheres are produced by the bacterial cells as a defense
897
mechanism to retard the spread of phage. However, it is difficult to imagine the bacteria evolving an elaborate and clearly imperfect defense mechanism against an agent to which it is already immune because of lysogeny. Employing standard methods of phage induction such as mitomycin C and UV light, we failed to enhance the proportion of cells expressing spherical bodies and associated phage. Attempts to find strains of Bacteroides fragilis sensitive to the phage have also been unsuccessful. ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of Suni Kloss. This work was supported by Institutional Research Funds, Veterans Administration Hospital, Sepulveda, Calif. LITERATURE CITED 1. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 2. Chase, D. G., and L. Pik6. 1973. Expression of A- and C-type particles in early mouse embryos. J. Natl. Cancer Inst. 51:1971-1975. 3. Gorbach, S. L. 1971. Intestinal microflora. Gastroenterology 60:1110-1129. 4. Gorbach, S. L., and J. G. Bartlett. 1974. Anaerobic infections. N. Engl. J. Med. 290:1177-1184, 1237-1245, 1289-1294. 5. Preer, J. R., Jr., and L. B. Preer. 1967. Virus-like bodies in killer paramecia. Proc. Natl. Acad. Sci. U.S.A. 58:1774-1781. 6. Preer, J. R., Jr., L. B. Preer, and A. Jurand. 1974. Kappa and other endosymbionts in iParamecium aurelia.. Bacteriol. Rev. 38:113-163.
