JOURNAL OF BACTERIOLOGY, Feb. 1975. p. 704-710 Copyright ( 1975 American Society for Microbiology
Vol. 121, No. 2 Printed in U.S.A.
Ultrastructure of Methylosinus trichosporium as Revealed by Freeze Etching TERRY L. WEAVER* AND PATRICK R. DUGAN LaboratorY of Microbiology, Cornell University, Ithaca, New York 14850,* and Department of Microbiology, Ohio State University, Columbus, Ohio 43210 Received for publication 31 October 1974
The methane-oxidizing bacterium Methylosinus trichosporium forms extensive intracytoplasmic membranes that lie near the cell periphery and parallel to it. These membranes enclose cavities within the cytoplasm and exist as flattened, balloon-like vesicles. The internal membranes are passed along to both cells during budding. The bacteria accumulate poly-f-hydroxybutyrate granules that lie in the center of the cells, neither within the internal membrane vesicles nor attached to them. Intercellular bridges result in the formation of chains of bacteria two to four cells in length.
Methylosinus trichosporium was first isolated and described by Whittenbury as a gram-negative, methane-oxidizing bacterium (11). An interesting characteristic of the methane-oxidizing bacteria is the presence of complex systems of intracytoplasmic membranes (1, 7-9, 11). Within the bacteria these membranes are oriented in either of two ways, which have been designated types I and II (1). The type I membranes lie in stacks in the central part of cells, whereas the type II membranes lie near the cell periphery and parallel to it. In thin sections these internal membranes appear similar to the intracytoplasmic membranes of the nitrifying and photosynthetic bacteria (5, 6). The membranes of the methane-oxidizing bacteria have been described variously as tubules (11), leaflets (8), saccules (9), and vesicles (1); however, neither serial sectioning nor freeze etching has been used in these examinations. M. trichosporium was selected for this study because its ultrastructure had not previously been reported, and freeze etching was used to reveal the three-dimensional organization of the intracytoplasmic membranes.
water: KNO3, 1; MgSO4 .7H20, 2 x 10-'; CaCl2, 2 x 10-2; FeSO4 7H20, 10 2; Na2HPO4, 2.3 x 10-l; NaH2PO4, 7 x 10-2; CuSO4 5H20, 5 x 10-6; H3BO4, 10- 5; MnSO4 H20, 7 x 10-6; ZnSO4 7H20, 7 x 10- 5; and MoO3, 10'. Preparation of bacteria for electron microscopy. A drop of bacteria concentrated by centrifugation in CM medium was placed on a gold specimen holder, frozen for 5 s in liquid Freon, and then quickly transferred to liquid nitrogen. The frozen sample was placed on the specimen platform of Balzer's freeze etch apparatus at - 100 C, evacuated to 106 torr, and fractured with a knife cooled to - 196 C. The sample was then etched for 4.5 min, shadowed with platinum, and carbon coated. The carbon replicas were floated on distilled water, cleaned in concentrated H2SO4 and 5% sodium hypochlorite, and recovered on 300-mesh uncoated copper grids. For thin sectioning, cells were fixed for 16 h at 5 C in 1% glutaraldehyde, washed in Veronal acetate buffer, and fixed for 2 h at 21 C in 1% osmium tetroxide. The fixed preparations were suspended in 2% agar blocks, dehydrated through an ethanol series (30 to 100%), and embedded in Epon resin. Thin sections were cut with an LKB ultramicrotome. Preparations were examined with Philips EM-300 electron microscope.
MATERIALS AND METHODS Bacteria and culture conditions. M. trichosporium (Ob3b) was kindly provided by R. Whittenbury, University of Warwick, Coventry, England, and Methylomonas methanica was obtained by streaking enrichments (10) on CM medium plates solidified with washed purified agar (Difco) and incubated under methane and air (1:1). The bacteria were cultured in CM salts on a rotary shaker (2 cycles/s) at 21 C and under an atmosphere of methane and air (1:1). The mineral salts growth medium CM contained the following in grams per liter of distilled
RESULTS Figure 1 shows a longitudinal fracture and Fig. 2 shows a transverse fracture through M. trichosporium. The internal membranes are revealed as closed, flattened circles near the cell periphery. The circles are formed by 9-nm-thick membranes and range from 16 to 300 nm in width, sometimes running the entire length of the cell. The type II morphology of these membranes is compared with a type I bacterium, M. methanica, in Fig. 3. The fracture plane in Fig. 4 shows that these
n
Z*.--
jm
FIG. 1. Longitudinal freeze etching of M. trichosporium showing the intracytoplasmic membranes. Symbols: V, Vesicle; S, direction of shadow (this symbol is also used in subsequent figures).
@lG Vs
FIG. 2. Transverse freeze etching of M. trichosporium showing the intracytoplasmic membranes. Symbol: V, Vesicle. 7 05
706
J. BACTERIOL. WEAVER AND DUGANT
FIG. 3. Longitudinal freeze etching of M. methanica showing the intracytoplasmic membranes. Symbol: IM, Intracytoplasmic membrane.
0.5m _
'
9_V
( PM/
-W
_
'
-.
FIG. 4. Freeze etching of M. trichosporium showing the vesicular structure of the intracytoplasmic membranes. Symbols: V, Vesicles; PM, plasma membrane.
internal membranes exist as flattened, balloonlike vesicles rather than as tubules, sheets, or circles. The pear shape of this 2- to 3-,m bacterium, ranging in width from 0.5 to 1 Aim, can also be seen in one of the cells. Granules ranging in size from 0.2 to 0.25 ,m (Fig. 5) are frequently observed near the centers of the cells
but neither within the membranes nor attached to them. A cell in the process of budding (Fig. 6) suggests that intracytoplasmic membranes are included in the daughter cell during this process. This inclusion of internal membranes in both cells during a division process was also observed in the type I bacterium, M. meth-
VOL. 121, 1975
ULTRASTRUCTURE OF M. TRICHOSPORIUM
707
FIG. 5. Freeze etching of M. trichosporium showing poly-fl-hydroxyb.utyrate granules (PHB).
s
0.5~~~~~~~~~~~~~
FIG. 6. Freeze etching of M. trichosporium in the process of budding showing the distribution of the intracytoplasmic membranes.
anica, used for comparison (Fig. 7). Figure 8 reveals the presence of 30 x 75-nm intercellular bridges that sometimes connect M. trichosporium cells. DISCUSSION The results reveal a type II membrane system for M. trichosporium. This is consistent with a
report by Whittenbury et al. (12), although electron micrographs were not shown. This also correlates with the report by Lawrence and Quayle (4) of hydroxypyruvate reductase activity in cell-free extracts of M. trichosporium. Hydroxypyruvate reductase is involved in the metabolism of serine, which is formed via the transhydroxymethylation of glycine during C1
J. BACTERIOL.
WEAVER AND DUGAN
708
0
0.5 pm FIG. 7. Thin section of M. methanica that is starting to divide showing the distribution of the intracytoplasmic membranes. Symbols: CW, Cell wall; PM, plasma membrane; IM, intracytoplasmic membranes.
Ae ft.
-
p
%."4v
A
s
Io
.
&.#;I. "
""WI
I*."
k- 't N
4 I
A I
k
B
4
I i
A
FIG. 8. Freeze etching of M. trichosporium showing intercellular bridges. Symbol: B, Intercellular bridge.
fixation by type II methane oxidizers. An im- indications of the vesicular nature of the type I portant question clarified by the freeze etch membranes via negative staining of lysed cell preparations is the three-dimensional morphol- preparations, they could not extend the obserogy of the type II intracytoplasmic membranes. vation to any type II organisms. Therefore, the Although Davies and Whittenbury (1) obtained vesicular nature of these type II membranes as
VOL. 121, 1975
ULTRASTRUCTURE OF M. TRICHOSPORIUM
709
FIG. 9. Cut-away model illustrating the proposed morphology of M. trichosporium. Symbols: IM, Intracytoplasmic membranes; PHB, poly-,8-hydroxybutyrate; PM, plasma membrane; OCW, outer cell wall; ICW, inner cell wall.
shown in these micrographs is a point that should be emphasized. In view of the close similarities among the methane-oxidizing bacteria, other methane oxidizers may also be expected to possess this closed vesicular internal membrane structure as opposed to open circles, sheets, or tubules. The cytoplasmic granules shown look like other published micrographs (2) of poly-f,hydroxybutyric acid. This suggestion is strengthened by a positive assay for poly-f3hydroxybutyric acid in M. trichosporium via the method of Law and Slepecky (3). The intercellular bridges observed in this study can also be seen in negatively stained cell suspensions. Functionally, the bridges result in the formation of chains of bacteria usually two to four cells in length. These may be the "holdfast structures" reported by Whittenbury et al. (12); however, they saw attachment at only one end of the cells. Bisection of the intracytoplasmic membrane organelles during division or budding raises the interesting question of whether the membrane structures are self-perpetuating rather than arising de novo after cell division. Figures 6 and 7 suggest that perhaps at least to some extent the membranes are self-perpetuating. Murray and Watson made a similar observation with the internal membranes of Nitrocystis (5). The importance of membranes to living organisms is
well known, and in at least one methane-oxidizing bacterium the internal membranes have been implicated in the methane oxidation process (8). Therefore, perpetuation of internal membranes during a division process would certainly be advantageous to a microorganism with whose physiology the membranes are intimately involved. Based upon observations made in this study, a model for the cellular ultrastructure of M. trichosporium is proposed in Fig. 9. No connections are shown between the cytoplasmic membrane and the intracytoplasmic membranes because these were not observed. However, such connections have been reported with other methane-oxidizing bacteria (1) and also with the nitrifying bacteria (5) and may exist in M. trichosporium under other conditions. In view of the observation that the internal membranes in M. trichosporium can be passed along during budding, it should be possible for subsequent internal membrane development to occur without intervention of the cytoplasmic membrane. ACKNOWLEDGMENT This research was supported by grant A-027-OHIO from the Office of Water Resources Research, Department of the Interior.
LITERATURE CITED 1. Davies, S., and R. Whittenbury. 1970. Fine structure of methane and other hydrocarbon utilizing bacteria. J.
710
WEAVER AND DUGAN Gen. Microbiol. 61:227-232.
J. BACTERIOL.
Bacteriol. 32:118-121.
2. Dunlop, W. F., and A. W. Robards. 1973. Ultrastructural study of poly-$-hydroxybutyrate granules from Bacil-
8. Smith, U., and D. W. Ribbons. 1970. Fine structure of
lus cereus. J. Bacteriol. 114:1271-1280. Law, J. H., and R. S. Slepecky. 1961. Assay of polv-betahvdroxvbutvric acid. J. Bacteriol. 82:33-36. Lawrence, A. J., and J. R. Quayle. 1970. Alternative carbon assimilation pathways in methane utilizing bacteria. J. Gen. Microbiol. 63:371-374. Murray, R. G. E., and S. W. Watson. 1965. Structure of Nitrocvstis oceanus and comparison with Nitrosomonas and Nitrobacter. J. Bacteriol. 89:1594-1609. Oelze, J., and G. Drews. 1972. Membranes of photosynthetic bacteria. Biochim. Biophys. Acta 265:209-239. Proctor, H. M., J. R. Norris, and D. W. Ribbons. 1969. Fine structure of methane utilizing bacteria. J. Appl.
74:116-122. 9. Smith, U., D. W. Ribbons, and D. S. Smith. 1970. The fine structure of Methylococcus capsulatus. Tissue &
3. 4.
5.
6.
7.
Methanomonas methanoxidans. Arch. Mikrobiol.
Cell 2:513-521. 10. Weaver, T. L., and P. R. Dugan. 1972. The eutrophica-
tion implications of interactions between naturally occurring particulates and methane oxidizing bacteria. Water Res. 6:817-828. 11. Whittenbury, R. 1969. Microbial utilization of methane. Process Biochem. 4:51-56. 12. Whittenbury, R., K. C. Phillips, and J. F. Wilkinson. 1970. Enrichment, isolation, and some properties of methane utilizing bacteria. J. Gen. Microbiol. 61:205-218.
Vol. 121, No. 2 Printed in U.S.A.
Ultrastructure of Methylosinus trichosporium as Revealed by Freeze Etching TERRY L. WEAVER* AND PATRICK R. DUGAN LaboratorY of Microbiology, Cornell University, Ithaca, New York 14850,* and Department of Microbiology, Ohio State University, Columbus, Ohio 43210 Received for publication 31 October 1974
The methane-oxidizing bacterium Methylosinus trichosporium forms extensive intracytoplasmic membranes that lie near the cell periphery and parallel to it. These membranes enclose cavities within the cytoplasm and exist as flattened, balloon-like vesicles. The internal membranes are passed along to both cells during budding. The bacteria accumulate poly-f-hydroxybutyrate granules that lie in the center of the cells, neither within the internal membrane vesicles nor attached to them. Intercellular bridges result in the formation of chains of bacteria two to four cells in length.
Methylosinus trichosporium was first isolated and described by Whittenbury as a gram-negative, methane-oxidizing bacterium (11). An interesting characteristic of the methane-oxidizing bacteria is the presence of complex systems of intracytoplasmic membranes (1, 7-9, 11). Within the bacteria these membranes are oriented in either of two ways, which have been designated types I and II (1). The type I membranes lie in stacks in the central part of cells, whereas the type II membranes lie near the cell periphery and parallel to it. In thin sections these internal membranes appear similar to the intracytoplasmic membranes of the nitrifying and photosynthetic bacteria (5, 6). The membranes of the methane-oxidizing bacteria have been described variously as tubules (11), leaflets (8), saccules (9), and vesicles (1); however, neither serial sectioning nor freeze etching has been used in these examinations. M. trichosporium was selected for this study because its ultrastructure had not previously been reported, and freeze etching was used to reveal the three-dimensional organization of the intracytoplasmic membranes.
water: KNO3, 1; MgSO4 .7H20, 2 x 10-'; CaCl2, 2 x 10-2; FeSO4 7H20, 10 2; Na2HPO4, 2.3 x 10-l; NaH2PO4, 7 x 10-2; CuSO4 5H20, 5 x 10-6; H3BO4, 10- 5; MnSO4 H20, 7 x 10-6; ZnSO4 7H20, 7 x 10- 5; and MoO3, 10'. Preparation of bacteria for electron microscopy. A drop of bacteria concentrated by centrifugation in CM medium was placed on a gold specimen holder, frozen for 5 s in liquid Freon, and then quickly transferred to liquid nitrogen. The frozen sample was placed on the specimen platform of Balzer's freeze etch apparatus at - 100 C, evacuated to 106 torr, and fractured with a knife cooled to - 196 C. The sample was then etched for 4.5 min, shadowed with platinum, and carbon coated. The carbon replicas were floated on distilled water, cleaned in concentrated H2SO4 and 5% sodium hypochlorite, and recovered on 300-mesh uncoated copper grids. For thin sectioning, cells were fixed for 16 h at 5 C in 1% glutaraldehyde, washed in Veronal acetate buffer, and fixed for 2 h at 21 C in 1% osmium tetroxide. The fixed preparations were suspended in 2% agar blocks, dehydrated through an ethanol series (30 to 100%), and embedded in Epon resin. Thin sections were cut with an LKB ultramicrotome. Preparations were examined with Philips EM-300 electron microscope.
MATERIALS AND METHODS Bacteria and culture conditions. M. trichosporium (Ob3b) was kindly provided by R. Whittenbury, University of Warwick, Coventry, England, and Methylomonas methanica was obtained by streaking enrichments (10) on CM medium plates solidified with washed purified agar (Difco) and incubated under methane and air (1:1). The bacteria were cultured in CM salts on a rotary shaker (2 cycles/s) at 21 C and under an atmosphere of methane and air (1:1). The mineral salts growth medium CM contained the following in grams per liter of distilled
RESULTS Figure 1 shows a longitudinal fracture and Fig. 2 shows a transverse fracture through M. trichosporium. The internal membranes are revealed as closed, flattened circles near the cell periphery. The circles are formed by 9-nm-thick membranes and range from 16 to 300 nm in width, sometimes running the entire length of the cell. The type II morphology of these membranes is compared with a type I bacterium, M. methanica, in Fig. 3. The fracture plane in Fig. 4 shows that these
n
Z*.--
jm
FIG. 1. Longitudinal freeze etching of M. trichosporium showing the intracytoplasmic membranes. Symbols: V, Vesicle; S, direction of shadow (this symbol is also used in subsequent figures).
@lG Vs
FIG. 2. Transverse freeze etching of M. trichosporium showing the intracytoplasmic membranes. Symbol: V, Vesicle. 7 05
706
J. BACTERIOL. WEAVER AND DUGANT
FIG. 3. Longitudinal freeze etching of M. methanica showing the intracytoplasmic membranes. Symbol: IM, Intracytoplasmic membrane.
0.5m _
'
9_V
( PM/
-W
_
'
-.
FIG. 4. Freeze etching of M. trichosporium showing the vesicular structure of the intracytoplasmic membranes. Symbols: V, Vesicles; PM, plasma membrane.
internal membranes exist as flattened, balloonlike vesicles rather than as tubules, sheets, or circles. The pear shape of this 2- to 3-,m bacterium, ranging in width from 0.5 to 1 Aim, can also be seen in one of the cells. Granules ranging in size from 0.2 to 0.25 ,m (Fig. 5) are frequently observed near the centers of the cells
but neither within the membranes nor attached to them. A cell in the process of budding (Fig. 6) suggests that intracytoplasmic membranes are included in the daughter cell during this process. This inclusion of internal membranes in both cells during a division process was also observed in the type I bacterium, M. meth-
VOL. 121, 1975
ULTRASTRUCTURE OF M. TRICHOSPORIUM
707
FIG. 5. Freeze etching of M. trichosporium showing poly-fl-hydroxyb.utyrate granules (PHB).
s
0.5~~~~~~~~~~~~~
FIG. 6. Freeze etching of M. trichosporium in the process of budding showing the distribution of the intracytoplasmic membranes.
anica, used for comparison (Fig. 7). Figure 8 reveals the presence of 30 x 75-nm intercellular bridges that sometimes connect M. trichosporium cells. DISCUSSION The results reveal a type II membrane system for M. trichosporium. This is consistent with a
report by Whittenbury et al. (12), although electron micrographs were not shown. This also correlates with the report by Lawrence and Quayle (4) of hydroxypyruvate reductase activity in cell-free extracts of M. trichosporium. Hydroxypyruvate reductase is involved in the metabolism of serine, which is formed via the transhydroxymethylation of glycine during C1
J. BACTERIOL.
WEAVER AND DUGAN
708
0
0.5 pm FIG. 7. Thin section of M. methanica that is starting to divide showing the distribution of the intracytoplasmic membranes. Symbols: CW, Cell wall; PM, plasma membrane; IM, intracytoplasmic membranes.
Ae ft.
-
p
%."4v
A
s
Io
.
&.#;I. "
""WI
I*."
k- 't N
4 I
A I
k
B
4
I i
A
FIG. 8. Freeze etching of M. trichosporium showing intercellular bridges. Symbol: B, Intercellular bridge.
fixation by type II methane oxidizers. An im- indications of the vesicular nature of the type I portant question clarified by the freeze etch membranes via negative staining of lysed cell preparations is the three-dimensional morphol- preparations, they could not extend the obserogy of the type II intracytoplasmic membranes. vation to any type II organisms. Therefore, the Although Davies and Whittenbury (1) obtained vesicular nature of these type II membranes as
VOL. 121, 1975
ULTRASTRUCTURE OF M. TRICHOSPORIUM
709
FIG. 9. Cut-away model illustrating the proposed morphology of M. trichosporium. Symbols: IM, Intracytoplasmic membranes; PHB, poly-,8-hydroxybutyrate; PM, plasma membrane; OCW, outer cell wall; ICW, inner cell wall.
shown in these micrographs is a point that should be emphasized. In view of the close similarities among the methane-oxidizing bacteria, other methane oxidizers may also be expected to possess this closed vesicular internal membrane structure as opposed to open circles, sheets, or tubules. The cytoplasmic granules shown look like other published micrographs (2) of poly-f,hydroxybutyric acid. This suggestion is strengthened by a positive assay for poly-f3hydroxybutyric acid in M. trichosporium via the method of Law and Slepecky (3). The intercellular bridges observed in this study can also be seen in negatively stained cell suspensions. Functionally, the bridges result in the formation of chains of bacteria usually two to four cells in length. These may be the "holdfast structures" reported by Whittenbury et al. (12); however, they saw attachment at only one end of the cells. Bisection of the intracytoplasmic membrane organelles during division or budding raises the interesting question of whether the membrane structures are self-perpetuating rather than arising de novo after cell division. Figures 6 and 7 suggest that perhaps at least to some extent the membranes are self-perpetuating. Murray and Watson made a similar observation with the internal membranes of Nitrocystis (5). The importance of membranes to living organisms is
well known, and in at least one methane-oxidizing bacterium the internal membranes have been implicated in the methane oxidation process (8). Therefore, perpetuation of internal membranes during a division process would certainly be advantageous to a microorganism with whose physiology the membranes are intimately involved. Based upon observations made in this study, a model for the cellular ultrastructure of M. trichosporium is proposed in Fig. 9. No connections are shown between the cytoplasmic membrane and the intracytoplasmic membranes because these were not observed. However, such connections have been reported with other methane-oxidizing bacteria (1) and also with the nitrifying bacteria (5) and may exist in M. trichosporium under other conditions. In view of the observation that the internal membranes in M. trichosporium can be passed along during budding, it should be possible for subsequent internal membrane development to occur without intervention of the cytoplasmic membrane. ACKNOWLEDGMENT This research was supported by grant A-027-OHIO from the Office of Water Resources Research, Department of the Interior.
LITERATURE CITED 1. Davies, S., and R. Whittenbury. 1970. Fine structure of methane and other hydrocarbon utilizing bacteria. J.
710
WEAVER AND DUGAN Gen. Microbiol. 61:227-232.
J. BACTERIOL.
Bacteriol. 32:118-121.
2. Dunlop, W. F., and A. W. Robards. 1973. Ultrastructural study of poly-$-hydroxybutyrate granules from Bacil-
8. Smith, U., and D. W. Ribbons. 1970. Fine structure of
lus cereus. J. Bacteriol. 114:1271-1280. Law, J. H., and R. S. Slepecky. 1961. Assay of polv-betahvdroxvbutvric acid. J. Bacteriol. 82:33-36. Lawrence, A. J., and J. R. Quayle. 1970. Alternative carbon assimilation pathways in methane utilizing bacteria. J. Gen. Microbiol. 63:371-374. Murray, R. G. E., and S. W. Watson. 1965. Structure of Nitrocvstis oceanus and comparison with Nitrosomonas and Nitrobacter. J. Bacteriol. 89:1594-1609. Oelze, J., and G. Drews. 1972. Membranes of photosynthetic bacteria. Biochim. Biophys. Acta 265:209-239. Proctor, H. M., J. R. Norris, and D. W. Ribbons. 1969. Fine structure of methane utilizing bacteria. J. Appl.
74:116-122. 9. Smith, U., D. W. Ribbons, and D. S. Smith. 1970. The fine structure of Methylococcus capsulatus. Tissue &
3. 4.
5.
6.
7.
Methanomonas methanoxidans. Arch. Mikrobiol.
Cell 2:513-521. 10. Weaver, T. L., and P. R. Dugan. 1972. The eutrophica-
tion implications of interactions between naturally occurring particulates and methane oxidizing bacteria. Water Res. 6:817-828. 11. Whittenbury, R. 1969. Microbial utilization of methane. Process Biochem. 4:51-56. 12. Whittenbury, R., K. C. Phillips, and J. F. Wilkinson. 1970. Enrichment, isolation, and some properties of methane utilizing bacteria. J. Gen. Microbiol. 61:205-218.
