Cell, Vol. 13, 57-64,
January
1978,
Copyright
0 1978 by MIT
Inhibition of Mycoplasma Cytochalasin B Amit Ghosh, Jack Maniloff* and David A. Gerling Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester, New York 14642
Summary Mycoplasma gallisepticum has subcellular organelles which may function as a primitive “mitoticlike” apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time “mitotic-like” structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle. Introduction The mycoplasmas are a group of procaryotes that do not have cell walls; each cell is bounded by a single lipoprotein “unit” membrane (reviewed by Maniloff and Morowitz, 1972). As the smallest freeliving cells, these organisms are interesting model cellular systems. In particular, the species Mycoplasma gallisepticum has certain novel biological features not found in other mycoplasmas (or in other procaryotes). M. gallisepticum is one of the mycoplasmas with the smallest reported genome size, 4.6 x 1O8 daltons (Bak et al., 1969). Although the cells are small (about 0.5 pm), each has specialized polar subcellular organelles consisting of a terminal bleb structure and an infrableb area between the bleb and the rest of the cell (reviewed by Maniloff and Morowitz, 1972; also see Figure 7 in this paper). A newly formed cell has a single polar bleb structure and a dividing cell has two blebs, each at opposite poles (Morowitz and Maniloff, 1966). Electron micrographs show cytoplasmic areas near some in* To whom
all correspondence
should
be addressed
Cell Division
by
frablebs which appear to be additional blebs, suggesting that new structures may be assembled at preexisting ones (Maniloff and Quinlan, 1973). The cells’ bleb structures have been shown to contain the DNA replication complex, and we have therefore proposed that these organelles may function as a primitive “mitotic-like” apparatus involved in chromosome organization and segregation (Maniloff and Quinlan, 1974). These data led us to consider how, if the blebs do indeed act as a primitive “mitotic-like” apparatus, the organelles could function to segregate daughter chromosomes before cell division. One possibility might be through actin-like proteins. To investigate this, we have studied the effects of cytochalasin B on M. gallisepticum. The cytochalasins are a group of fungal metabolites which have been found to have inhibitory effects on a variety of eucaryotic cell processes. Most studies have utilized cytochalasin B (CB) (reviewed by Pollard and Weihing, 1974; Lin and Spudich, 1974). The effects of CB which have been demonstrated are primarily of two types: inhibition of the uptake of small sugars (such as glucose) and nucleosides (such as thymidine); and inhibition of cellular movements which involve actin or actin-like proteins. Hence the inhibition of a cellular process by CB can be due to one or both effects. Thus far, CB has been shown to be an inhibitor only in eucaryotes; no CB effect was found in studies of the bacteria Escherichia coli (Wessels et al., 1971; Betina, Micekova and Nemec, 1972), Bacillus subtilis (Betina et al., 1972), and strains of Cytophaga and Acinetobacter (Henrichsen, 1972). We report here on studies showing that CB inhibits cell division of M. gallisepticum. This effect is shown not to be caused by inhibition of glucose or thymidine uptake. Biochemical and morphological data suggest that CB blocks at two places in the cell cycle. Results Effect of CB on Cell Growth In addition to M. gallisepticum, CB was also tested on another sterol-requiring mycoplasma, M. capricolum, and a sterol nonrequiring mycoplasma, A. laidlawii. These latter two organisms do not have any subcellular structures, such as found in M. gallisepticum, and were chosen as general representatives of the two groups of mycoplasmas (that is, sterol-requiring and nonrequiring). The effect of CB was studied by measuring colony-forming units (cfu) after 24 hr of incubation in growth medium containing various CB concentrations. The data (Figure 1) show that CB does not affect
Cell
58
growth of M. capricolum and A. laidlawii, does inhibit the growth of M. gallisepticum.
but CB
Effects of Colchicine and Vinblastine Experiments similar to that in Figure 1 were performed using colchicine (up to 100 pg/ml) and vinblastine (up to 10 pg/ml). Neither drug had any effect on growth of the three mycoplasmas used in this study. Kinetics of Growth Inhibition by CB To examine the kinetics of CB action on M. gallisepticum, a logarithmically growing culture was divided into two parts: one was kept as a control, and CB was added to the other part to a final concentration of 100 kg/ml. The cultures were incubated at 37X, and at various times, samples were withdrawn and assayed for cfu. These data are shown in Figure 2; inhibition of cell growth by CB was rapid.
Effect of CB on Sugar Uptake Since one of the reported effects of CB in eucaryotes is the inhibition of sugar uptake, glucose transport was measured in M. gallisepticum. For these studies, exponentially growing cells were treated with 100 pg CB/ml for 3 hr at 37°C; ‘X-amethyl-glucose was added, and glucose uptake was followed. Uptake was also measured in a parallel untreated culture. The results (Figure 3) show that there is no difference in sugar uptake between untreated and CB-treated cells. In both cases, the uptake follows identical complex kinetics, probably reflecting the approach to steady state of the influx and efflux processes. Effect of CB on Macromolecule Synthesis DNA, RNA and protein synthesis were followed by measuring this incorporation of 3H-thymidine, 3Huridine or a 3H-amino acid mixture, respectively, into acid-precipitable material. Both untreated cells and cells treated with 100 pg CB per ml (added at the time of addition of the labeled precursor) were examined. As seen in Figure 4, in CBtreated cells, DNA synthesis levels off after about 2 generation times, while RNA and protein synthesis continue at a somewhat reduced rate. The inhibition in DNA synthesis could be related to the inhibition of cell division, or it could result from a CB-induced inhibition of thymidine transport.
1 ? 0
_
.i 5, C E 5 k 5 .-c IL
0
20
1. Effect
of CB on Mycoplasma
40 CB
Figure
concentration
60
00
-
I
100
0
(ug/ml) Growth
Exponentially growing cultures (about IO6 cfu/ml) of M. gallisepticum (0), M. capricolum (0) and A. laidlawii (A) were incubated with various CB concentrations for 24 hr at 37°C. cfu were assayed before CB addition and after 24 hr of growth.
Figure
2. Kinetics
I 2
I I I 4 6 8 Time (hr) of CB Action
I IO
I
12
on M. Gallisepticum
Growth of untreated cells (0) and cells treated with 100 wg CB per ml (0) was measured as a function of incubation time at 37°C after CB addition. The initial cell titer was 5 x lo5 cfu/ml.
Mycoplasmas 59
and Cytochalasin
B
Effect of CB on Thymidine Uptake Thymidine uptake was followed in experiments similar to those used to measure sugar uptake. For these studies, however, cells were treated for 7 hr with 100 pg CB per ml, to assure that DNA synthesis had stopped, before 3H-thymidine was added. As shown in Figure 5, there is no measurable difference in thymidine uptake in untreated and CB-treated cells. The inhibition of DNA synthesis caused by CB (Figure 4) is therefore not due to an inhibition in thymidine uptake. Cell Growth and DNA Synthesis after CB Removal The fact that CB-inhibited M. gallisepticum cells could form colonies when the samples were diluted and plated (as seen in the experiments shown in Figures 1 and 2) indicated that the CB effect was reversible. To examine this reversibility, after either a 3 or 12 hr treatment with 100 pg CB/ml, a culture was diluted 100 fold to reduce the CB concentration and the number of cfu was assayed as a function of time after CB removal. It was observed
3 m ‘0
x2 h i?
0 -0 0 Figure 3. Glucose septicum
2
4 Uptake
6 8 IO Time (min) in Untreated
12
14
and CB-Treated
I6 M. Galli-
The uptake of ‘%-u-methyl-glucoside was measured in untreated cells (0) and cells treated with 100 pg CB/ml (0). The glucose was added to exponentially growing cells (1 x 10’ cfu/ml) and uptake was measured as a function of time. Since the cell number in the untreated culture increased during the 3 hr in which the treated culture was being incubated with CB, the number of cells in the untreated and treated cultures were different at the time of addition of the labeled glucose. Thus to normalize these data, the results are expressed as cpm/cfu.
Figure 4. Effect of CB Treatment Synthesis in M. gallisepticum
on
DNA,
RN#
and
Protein
For each experiment, an exponentially growing culture was split into two parts: one was kept as a control (0), and CB was added to the other(O) at a final concentration of 100 pglml. 3H-precursors were added to both cultures as follows: (A) V-thymidine, (B) 3Huridine and (C) 3H-amino acid mixture. Label incorporation into acid-insoluble material was followed as a function of time.
I 0
I 2
I 4 Time
I I 6 8 (hr)
I
IO
Cell 60
Figure 6. Growth and after Removal of CB
DNA
Synthesis
of M. gallisepticum
Cells
Cells were treated either (A) and (C) for 12 hr or (B) and (D) for 3 hr with 100 pg CB per ml. (A and B) After 100 fold dilution in medium to reduce the CB concentration, cfu were measured as a function of incubation time at 37°C. (C and D) During CB treatment, a sample of untreated control cells was kept at 4°C (as described for Figure 5). After 100 fold dilution in medium to reduce the CB concentration, 3H-thymidine was added, and incorporation into acid-insoluble material was followed as a function of time. (0) control; (0) CB-treated cells.
Time Figure 5. Thymidine gallisepticum
Uptake
(hr)
in Untreated
and
CB-Treated
M.
An exponentially growing culture was split into two parts: one (0) was kept at 4°C for 6 hr and then at 37°C for 1 hr; the other (0) was treated with CB (100 pg/ml final concentration) at 37°C for 7 hr. This protocol gives an equal number of cells in both cultures at the end of the 7 hr period. 3H-thymidine was then added to both cultures and uptake was followed as a function of time.
(Figures 6A and 6B) that the longer the duration of drug treatment, the greater the lag time before cell growth resumed after CB removal. The cell growth curves in these and in repeated experiments suggested some degree of synchrony in the first cell division following release of CB inhibition. This observation was not investigated further. To see whether after removal of CB, DNA synthesis resumes before or after cell division, ceils were treated for either 3 or 12 hr with 100 pg CB/ml. At these times, each culture was diluted 100 fold to reduce the amount of CB, 3H-thymidine was added and DNA synthesis was measured. In cells treated for 3 hr (Figure 6D), DNA synthesis resumed immediately after CB removal and continued at the rate observed in untreated cells. Cells treated for 12 hr (Figure 6C) had a 2 hr lag in DNA synthesis, after which synthesis resumed at the same rate as in untreated cells. Comparing this with the studies on cell growth (Figures 6A and 6B), it is seen that DNA synthesis resumes before the first cell division following CB removal.
Electron Microscopy of CB-Treated Cells Thin sections of CB-treated M. gallisepticum cells were examined by electron microscopy to see whether any cytological changes could be observed. No differences were seen between untreated and CB-treated cells. Both one-bleb (Figure 7A) and two-bleb cells (Figure 7B) were seen. Since few two-bleb cells were observed due to the low probability of the section plane passing through both blebs, it was not possible to decide whether or not the distribution of cells between one- and two-bleb states was different in untreated and CBtreated cells. To measure the number of one- and two-bleb cells, untreated cells and cells treated for 4 hr with 100 pg CB/ml were negatively stained and examined by electron microscopy. This allowed whole cells to be seen, and one-bleb (Figure 7C) and two-bleb cells (Figure 7D) could be counted. At least 250 cells were counted for each sample. In the untreated culture, the cells were found to be 81% one-bleb and 19% two-bleb. Cells treated with CB were found to be 63% one-bleb and 37% two-bleb. Hence there is about a doubling in two-bleb cells in CB-treated cultures. Untreated and CB-treated cells, prepared by freeze etching, appeared the same (Figures 7E and 7F). In the two samples, no difference could be found in the particle distribution on the membrane fracture faces. Most cells (in both preparations) seemed to have fewer particles on the membrane
Mycoplasmas 61
Figure
and Cytochalasin
7. Electron
Micrographs
(A and B) Thin sections stained with silicotungstate. treated cultures. (E and relative to the number of pg CB/ml. The arrows in
B
of M. gallisepticum
Cells
showing the cell membrane (m), bleb (b), nuclear material (n) and ribosomes (r). (C and D) Cells negatively The one-bleb cells in (A) and (C) are from untreated cultures, and the two-bleb cells in (B) and (D) from CBF) Freeze-etched cells. Fewer particles are seen on the membrane fracture face around the bleb (arrowhead), particles on the membrane face around the rest of the cell. (E) Untreated cells: (F) cells treated for 4 hr with 100 (E) and (F) indicate the direction of shadowing. Bar = 0.25 pm.
fracture face around the bleb than brane around the rest of the cell.
on the
mem-
Uptake of CB 3H-CB, mixed with unlabeled CB, was added to an M. gallisepticum culture at a concentration of 100 pg CB/ml (5 pCi/ml) and uptake was measured. CB uptake reached a plateau by 1 hr (Figure 8A), indicating a saturation of CB binding sites at this time. To obtain a measure of the affinity of these sites for CB, the uptake experiment was repeated, and after 1 hr uptake was stopped by reducing the CB
concentration 100 fold by dilution with growth medium. The loss of CB from binding sites was followed by measuring the label remaining in the cells as a function of time. A gradual loss of CB was found (Figure 8B), and by 2 hr after dilution, only about 20% of the original bound CB remained in the cells. Characterization of a Subcellular Fraction Binding CB The fact that cells lose bound 3H-CB at a slow rate suggested that it might be possible to isolate a subcellular fraction with bound CB. For these stud-
Cell 62
Discussion
Tima ,m,n, Figure
6. Binding
to M. gallisepticum
Cells
(A) 3H-CB, mixed with unlabeled CB, was added to tially growing culture and bound label was measured of time. (6) After 1 hr of 3H-CB binding, a M. culture was diluted 100 fold to reduce exogenous remaining bound CB was measured as a function of
an exponenas a function gallisepticum CB and the time.
ies, 3H-CB was added to an exponentially growing M. gallisepticum culture at a final concentration of 100 pg/ml (5 &i/ml), and the culture was incubated for 8 hr at 37°C to assure saturation of the CB binding sites. The cells were harvested by centrifugation and washed with O..Ol M Tris buffer (pH 7). The cell pellet was resuspended in 2 ml 0.01 M Tris and lysed by freeze-thawing and sonication. In these studies, it was noted that 15-20 cycles of freeze-thawing were necessary to lyse CB-treated cells; lysis was judged by the reduction in turbidity of the cell suspension. This is 4-5 times the number of cycles required to lyse untreated cells, and indicates that CB treatment has made the cells more resistant to freeze-thaw lysis. The reason for this difference was not investigated. The cell lysate was centrifuged at 4000 x g for 4 min to remove unlysed cells. This procedure has been shown to leave lysed cells, membrane vesicles, bleb structures and cytoplasmic material in the supernatant (Maniloff and Quinlan, 1974). The supernatant was removed and centrifuged at 40,000 x g for 30 min to pellet membranes and blebs. The supernatant, which contained about 35% of the 3H-CB, was discarded. The pellet was resuspended in Tris buffer, sonicated to separate the bleb end of the cell from the rest of the cell membrane, and centrifuged through a sucrose step gradient. Two bands could be seen, one on the 55% sucrose shelf and one on the 62% shelf. Material banding on the 55% shelf has been shown to be membrane vesicles (Maniloff and Quinlan, 1974) and was found to contain 35-40% of the 3HCB. Bleb structures have been found to band on the 62% shelf, and this material contained 60 to 65% of the 3H-CB. Hence both cell membranes and bleb structures probably contain CB binding sites.
The inhibition of M. gallisepticum growth described here is the only report, of which we know, of a CB effect on a procaryote. It was shown in these studies that this effect is specific for M. gallisepticum; CB does not inhibit other mycoplasmas (Figure 1). As is discussed below, we believe that CB inhibition is a consequence of the unique subcellular organelles found in M. gallisepticum. Parenthetically, no inhibition was observed with either colchicine or vinblastine. The reported effects of CB on eucaryotic cells are of two types (reviewed by Pollard and Weihing, 1974): inhibition of uptake of small molecules, and inhibition of cellular processes involving actin or actin-like proteins. Since we have been unable to demonstrate a CB inhibition of small molecule uptake, we suggest that the CB inhibition of M. gallisepticum growth is due to the effect of CB on some actin-like protein required at one or more steps of the cell cycle. In agreement with this suggestion are preliminary studies on M. pneumoniae, the only other mycoplasma having a terminal structure (Maniloff and Morowitz, 1972), which have shown that these cells are inhibited by CB (A. Ghosh and J. Maniloff, unpublished data) and that an actin-like protein can be isolated from them (H. C. Neimark, 1976, Abstracts Ann. Meeting Am. Sot. Microbial., p. 61). Considering the data presented here, together with the available data on the analysis of M. gallisepticum subcellular organelles, growth cycle and DNA replication, we propose a model for the action of CB on M. gallisepticum. The morphological events (from Morowitz and Maniloff, 1966) begin with the newly formed daughter cell containing one terminal bleb. During the cell cycle, a second bleb structure is formed, perhaps at the preexisting bleb. Assuming that the fraction of two-bleb cells in a culture represents the fraction of the cell cycle during which a cell is bipolar with two bleb organelles, since about 20% of the cells have two blebs, each cell must be in a two-bleb state during the last 20% of the cell cycle. A constriction leading to cell division occurs at about 2 hr in the cell cycle. Each M. gallisepticum daughter cell receives one genome copy (Ghosh, Das and Maniloff, 1977) which is replicated once per cell cycle (Quinlan and Maniloff, 1973). In a study of synchronously growing M. gallisepticum, it was shown that there is a gap of about 30-40 min in DNA synthesis during cell division (Quinlan and Maniloff, 1973). Hence DNA synthesis is initiated after the start of the cell cycle and terminated before cell division. Since CB immediately stops the increase in cfu, it must block at the cell division step. A second CB
Mycoplasmas 63
and Cytochalasin
B
block earlier in the cell cycle, however, is indicated by the observation that less than half of CB-inhibited ceils reach the two-bleb state. This result suggests that in the presence of CB, M. gallisepti‘cum cells which have begun formation of a second bleb can complete the structure, but cells at an earlier point in the cell cycle cannot initiate formation of a second bleb. In this context, formation of a second bleb may mean either assembly of a bleb structure or migration of a preexisting structure to give the morphological appearance of a two-bleb cell. DNA synthesis continues for 2-3 generation times after CB addition, and it should be noted that more DNA is made than would be expected if synthesis were only completing rounds of replication. Nothing is known about the coupling between DNA synthesis and the formation of bleb structures, but the DNA replication complex is known to be at the bleb (Maniloff and Quinlan, 1974). One possible explanation of the CB effect is that CB has disrupted the assembly of actin-like proteins required at two points of the cell cycle: early in the ceil cycle for the formation of a second bleb, and late in the cycle for constriction and cell division. In agreement with this possibility, preliminary studies have shown that a protein can be isolated from M. gallisepticum with actin-like properties (U. Chaudhuri and J. Maniloff, unpublished data); the protein has a molecular weight about 45,000 daltons, polymerizes in the presence of KCI and depolymerizes with added ATP. Experimental
Procedures
Mycoplaemas and Media The mycoplasmas used in this study were M. gallisepticum strain A5969, M. capricolum strain 14 and Acholeplasma laidlawii strain B [the isolation of these strains has been described by Tourtellotte and Jacobs (1960)]. A. laidlawii and M. capricolum were grown in tryptose broth and assayed as colony-forming units (cfu) on tryptose agar plates, as described previously (Quinlan, Liss and Maniloff, 1972). M. gallisepticum was grown in MBB medium and assayed as cfu on MBB agar plates as described by Ghosh et al. (1977). Drug Treatment The drugs used were cytochalasin B (Aldrich Chemical Co., Milwaukee, Wisconsin), colchicine (Sigma Chemical Co., St. Louis, Missouri), and vinblastine sulfate (Eli Lilly and Co., Indianapolis, Indiana). The latter two were dissolved directly in growth medium. To make CB solutions, 1 mg CB was dissolved in 0.1 ml ethanol and then diluted with medium. Controls for CB treatments were made up with amounts of ethanol equivalent to the corresponding CB samples. In all experiments, the ethanol concentrations were 1% or less, since loss of cell viability was observed only at higher solvent concentrations. Measurement of Macromolecular Synthesis For each experiment (that is, measurement of DNA, RNA or protein synthesis), an appropriate radioisotopically labeled precursor, at a final activity of 50 @Zi/ml, was added to a tube of exponentially growing cells (4.5 ml). Precursor incorporation into macromolecular material was followed by pipetting 50 ~1 culture samples into 1 ml ice cold 10% trichloroacetic acid at various
times. After 30 min in the cold, precipitates filters, washed and assayed for radioactivity (Quinlan et al., 1972).
were collected in toluene-Omnifluor
on
Glucose Uptake Sugar transport was studied by measuring the uptake of ‘+-amethyl-D-glucoside, a nonmetabolizable analogue of glucose, as described by Rottem and Razin (1969). The labeled glucose was added to a final activity of 50 &i/ml to exponentially growing ceils. 50 /II samples were withdrawn after different times and filtered through a 0.22 pm Millipore (Bedford, Massachusetts) filter. The filters containing the cells were washed with 50 ml icecold growth medium, dried and counted as above. Thymidine Uptake These experiments were performed uptake, except that 3H-thymidine tion of IO &i/ml.
similarly was used
to those for glucose at a final concentra-
CB Uptake These experiments were done similarly to those for glucose uptake, except 3H-cytochalasin B (New England Nuclear, Boston, Massachusetts) was used. The 3H-CB was mixed with unlabeled CB and added to a culture to give a final concentration of 100 fig CB/ml and 5 &i/ml. At various times, 1 ml samples were filtered, washed and counted as described for glucose uptake measurements. Density Gradients For the analysis of the cell components which bind CB, cells were incubated with 3H-CB as above for 8 hr. Cells were then disrupted by freeze-thawing and sonication, as described by Maniloff and Quinlan (1974). The lysate was layered over a discontinuous sucrose gradient with 3.5 ml steps of 40, 50, 55 and 62% (w/v) sucrose and spun in a Beckman SW27 rotor for 8 hr at 23,000 rpm at 5°C. This procedure has been shown (Maniloff and Quinlan, 1974) to band membrane vesicles on top of the 55% step and bleb structures on the 62% step. Bands were removed by puncturing the side of the centrifuge tube and assayed for radioactivity, as above. Electron Microscopy For thin-section studies, a culture was mixed with an equal volume of cold 12.5% glutaraldehyde (in 0.1 M cacodylate) and kept at 4°C for 2 hr. The cells were then harvested and washed overnight at 4°C with 0.1 M cacodylate. The pellet was then embedded and stained by the method of Hills and Plaskitt (1968). For freeze fracture studies, fixed cells were centrifuged, and the cell pellet was placed in 40% (v/v) glycerol. The freeze fracture procedure was carried out using a Denton (Cherry Hill, New Jersey) Model DFE-2 Freeze-Etching Unit, as described by Steere (1969). For examination of whole cells, a drop of glutaraldehyde-fixed culture was put on formvar-coated grids and negatively stained with 1% silicotungstic acid (pH 6.8). Grids were examined with a Siemens Elmiskop IA electron microscope with a liquid nitrogen decontamination device and 50 pm objective aperture, operating at 80 kV. Acknowledgments We thank Dr. James Coleman for his suggestions regarding the use of cytochalasin B. This study was supported by a USPHS grant from the National Institute of Allergy and Infectious Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
August
11, 1977;
revised
October
14, 1977
Cell 64
References Bak, L. A., Black, F. T., Christiansen, (1969). Genome size of mycoplasmal 1210.
C. and Freundt. DNA. Nature 224,
E. A. 1209-
Betina, V., Micekova, D. and Nemec, P. (1972). Antimicrobial properties of cytochalasins and their alteration of fungal morphology. J. Gen. Microbial. 71, 343-349. Ghosh, A., Das, J. and ultraviolet light damage Biol. 116, 337-344. Henrichsen, unaffected
Maniloff, J. (1977). Lack of repair of in Mycoplasma gallisepticum. J. Mol.
J. (1972). Gliding and twitching motility of bacteria by cytochalasin 8. Acta Pathol. Stand. B 80, 623-624.
Hills, G. J. and Plaskitt, L. (1968). A protein stain for the electron microscopy of small isometric plant virus particles. J. Ultrastructure Res. 25, 323-329. Lin, S. and Spudich. action of cytochalasin
J. A. (1974). B. J. Supramol.
On the molecular basis Structure 2, 728-736.
Maniloff, J. and Morowitz, H. J. (1972). mycoplasmas. Bacterial. Rev. 36, 263-290.
Cell
Maniloff, J. and Quinlan, D. C. (1973). Biosynthesis lar organization of nucleic acids in Mycoplasma A5969. Ann. NY Acad. Sci. 225, 181-189. Maniloff, membrane coplasma
biology
of
of the
and subcellugdisepticum
J. and Quinlan, D. C. (1974). Partial purification of a associated deoxyribonucleic acid complex from Mygallisepticum. J. Bacterial. 720, 495-501.
Morowitz, H. J. and Maniloff, of Mycoplasma gallisepticum.
J. (1966). Analysis of the life cycle J. Bacterial. 91, 1644-1683.
Pollard, T. D. and Weihing. R. Ft. (1974). Actin cell movement. CRC Crit. Rev. Biochem. 2, i-65.
and myosin
Quinlan, D. C. and Maniloff, J. (1972). Membrane the deoxyribonucleic acid growing point region gallisepticum. J. Bacterial. 772, 1375-1379.
and
association of in Mycoplasma
Quinlan, D. C. and Maniloff, J. (1973). Deoxyribonucleic acid synthesis in synchronously growing Mycoplasma gallisepticum. J. Bacterial. 175, 117-120. Quinlan, medium 6, 179-l
D. C., Liss, A. and Maniloff. J. (1972). Eagle’s basal as a defined medium for mycoplasma studies. Microbios 85.
Rottem, S. and Razin, S. (1969). Sugar ga//isepticum. J. Bacterial. 97, 787-792. Steere, 306-323.
R. L. (1969).
Freeze-etching
Tourtellotte, M. E. and Jacobs, serologic comparison of PPLO Acad. Sci. 79, 521-530.
transport simplified.
R. E. (1960). from various
in Mycoplasma Cryobiology
5,
Physiological and sources. Ann. NY
Wessels, N. K., Spooner, 6. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T. and Yamada, K. M. (1971). Microfilaments in cellular and developmental processes. Science 777, 135-143.
January
1978,
Copyright
0 1978 by MIT
Inhibition of Mycoplasma Cytochalasin B Amit Ghosh, Jack Maniloff* and David A. Gerling Department of Microbiology University of Rochester School of Medicine and Dentistry Rochester, New York 14642
Summary Mycoplasma gallisepticum has subcellular organelles which may function as a primitive “mitoticlike” apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time “mitotic-like” structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle. Introduction The mycoplasmas are a group of procaryotes that do not have cell walls; each cell is bounded by a single lipoprotein “unit” membrane (reviewed by Maniloff and Morowitz, 1972). As the smallest freeliving cells, these organisms are interesting model cellular systems. In particular, the species Mycoplasma gallisepticum has certain novel biological features not found in other mycoplasmas (or in other procaryotes). M. gallisepticum is one of the mycoplasmas with the smallest reported genome size, 4.6 x 1O8 daltons (Bak et al., 1969). Although the cells are small (about 0.5 pm), each has specialized polar subcellular organelles consisting of a terminal bleb structure and an infrableb area between the bleb and the rest of the cell (reviewed by Maniloff and Morowitz, 1972; also see Figure 7 in this paper). A newly formed cell has a single polar bleb structure and a dividing cell has two blebs, each at opposite poles (Morowitz and Maniloff, 1966). Electron micrographs show cytoplasmic areas near some in* To whom
all correspondence
should
be addressed
Cell Division
by
frablebs which appear to be additional blebs, suggesting that new structures may be assembled at preexisting ones (Maniloff and Quinlan, 1973). The cells’ bleb structures have been shown to contain the DNA replication complex, and we have therefore proposed that these organelles may function as a primitive “mitotic-like” apparatus involved in chromosome organization and segregation (Maniloff and Quinlan, 1974). These data led us to consider how, if the blebs do indeed act as a primitive “mitotic-like” apparatus, the organelles could function to segregate daughter chromosomes before cell division. One possibility might be through actin-like proteins. To investigate this, we have studied the effects of cytochalasin B on M. gallisepticum. The cytochalasins are a group of fungal metabolites which have been found to have inhibitory effects on a variety of eucaryotic cell processes. Most studies have utilized cytochalasin B (CB) (reviewed by Pollard and Weihing, 1974; Lin and Spudich, 1974). The effects of CB which have been demonstrated are primarily of two types: inhibition of the uptake of small sugars (such as glucose) and nucleosides (such as thymidine); and inhibition of cellular movements which involve actin or actin-like proteins. Hence the inhibition of a cellular process by CB can be due to one or both effects. Thus far, CB has been shown to be an inhibitor only in eucaryotes; no CB effect was found in studies of the bacteria Escherichia coli (Wessels et al., 1971; Betina, Micekova and Nemec, 1972), Bacillus subtilis (Betina et al., 1972), and strains of Cytophaga and Acinetobacter (Henrichsen, 1972). We report here on studies showing that CB inhibits cell division of M. gallisepticum. This effect is shown not to be caused by inhibition of glucose or thymidine uptake. Biochemical and morphological data suggest that CB blocks at two places in the cell cycle. Results Effect of CB on Cell Growth In addition to M. gallisepticum, CB was also tested on another sterol-requiring mycoplasma, M. capricolum, and a sterol nonrequiring mycoplasma, A. laidlawii. These latter two organisms do not have any subcellular structures, such as found in M. gallisepticum, and were chosen as general representatives of the two groups of mycoplasmas (that is, sterol-requiring and nonrequiring). The effect of CB was studied by measuring colony-forming units (cfu) after 24 hr of incubation in growth medium containing various CB concentrations. The data (Figure 1) show that CB does not affect
Cell
58
growth of M. capricolum and A. laidlawii, does inhibit the growth of M. gallisepticum.
but CB
Effects of Colchicine and Vinblastine Experiments similar to that in Figure 1 were performed using colchicine (up to 100 pg/ml) and vinblastine (up to 10 pg/ml). Neither drug had any effect on growth of the three mycoplasmas used in this study. Kinetics of Growth Inhibition by CB To examine the kinetics of CB action on M. gallisepticum, a logarithmically growing culture was divided into two parts: one was kept as a control, and CB was added to the other part to a final concentration of 100 kg/ml. The cultures were incubated at 37X, and at various times, samples were withdrawn and assayed for cfu. These data are shown in Figure 2; inhibition of cell growth by CB was rapid.
Effect of CB on Sugar Uptake Since one of the reported effects of CB in eucaryotes is the inhibition of sugar uptake, glucose transport was measured in M. gallisepticum. For these studies, exponentially growing cells were treated with 100 pg CB/ml for 3 hr at 37°C; ‘X-amethyl-glucose was added, and glucose uptake was followed. Uptake was also measured in a parallel untreated culture. The results (Figure 3) show that there is no difference in sugar uptake between untreated and CB-treated cells. In both cases, the uptake follows identical complex kinetics, probably reflecting the approach to steady state of the influx and efflux processes. Effect of CB on Macromolecule Synthesis DNA, RNA and protein synthesis were followed by measuring this incorporation of 3H-thymidine, 3Huridine or a 3H-amino acid mixture, respectively, into acid-precipitable material. Both untreated cells and cells treated with 100 pg CB per ml (added at the time of addition of the labeled precursor) were examined. As seen in Figure 4, in CBtreated cells, DNA synthesis levels off after about 2 generation times, while RNA and protein synthesis continue at a somewhat reduced rate. The inhibition in DNA synthesis could be related to the inhibition of cell division, or it could result from a CB-induced inhibition of thymidine transport.
1 ? 0
_
.i 5, C E 5 k 5 .-c IL
0
20
1. Effect
of CB on Mycoplasma
40 CB
Figure
concentration
60
00
-
I
100
0
(ug/ml) Growth
Exponentially growing cultures (about IO6 cfu/ml) of M. gallisepticum (0), M. capricolum (0) and A. laidlawii (A) were incubated with various CB concentrations for 24 hr at 37°C. cfu were assayed before CB addition and after 24 hr of growth.
Figure
2. Kinetics
I 2
I I I 4 6 8 Time (hr) of CB Action
I IO
I
12
on M. Gallisepticum
Growth of untreated cells (0) and cells treated with 100 wg CB per ml (0) was measured as a function of incubation time at 37°C after CB addition. The initial cell titer was 5 x lo5 cfu/ml.
Mycoplasmas 59
and Cytochalasin
B
Effect of CB on Thymidine Uptake Thymidine uptake was followed in experiments similar to those used to measure sugar uptake. For these studies, however, cells were treated for 7 hr with 100 pg CB per ml, to assure that DNA synthesis had stopped, before 3H-thymidine was added. As shown in Figure 5, there is no measurable difference in thymidine uptake in untreated and CB-treated cells. The inhibition of DNA synthesis caused by CB (Figure 4) is therefore not due to an inhibition in thymidine uptake. Cell Growth and DNA Synthesis after CB Removal The fact that CB-inhibited M. gallisepticum cells could form colonies when the samples were diluted and plated (as seen in the experiments shown in Figures 1 and 2) indicated that the CB effect was reversible. To examine this reversibility, after either a 3 or 12 hr treatment with 100 pg CB/ml, a culture was diluted 100 fold to reduce the CB concentration and the number of cfu was assayed as a function of time after CB removal. It was observed
3 m ‘0
x2 h i?
0 -0 0 Figure 3. Glucose septicum
2
4 Uptake
6 8 IO Time (min) in Untreated
12
14
and CB-Treated
I6 M. Galli-
The uptake of ‘%-u-methyl-glucoside was measured in untreated cells (0) and cells treated with 100 pg CB/ml (0). The glucose was added to exponentially growing cells (1 x 10’ cfu/ml) and uptake was measured as a function of time. Since the cell number in the untreated culture increased during the 3 hr in which the treated culture was being incubated with CB, the number of cells in the untreated and treated cultures were different at the time of addition of the labeled glucose. Thus to normalize these data, the results are expressed as cpm/cfu.
Figure 4. Effect of CB Treatment Synthesis in M. gallisepticum
on
DNA,
RN#
and
Protein
For each experiment, an exponentially growing culture was split into two parts: one was kept as a control (0), and CB was added to the other(O) at a final concentration of 100 pglml. 3H-precursors were added to both cultures as follows: (A) V-thymidine, (B) 3Huridine and (C) 3H-amino acid mixture. Label incorporation into acid-insoluble material was followed as a function of time.
I 0
I 2
I 4 Time
I I 6 8 (hr)
I
IO
Cell 60
Figure 6. Growth and after Removal of CB
DNA
Synthesis
of M. gallisepticum
Cells
Cells were treated either (A) and (C) for 12 hr or (B) and (D) for 3 hr with 100 pg CB per ml. (A and B) After 100 fold dilution in medium to reduce the CB concentration, cfu were measured as a function of incubation time at 37°C. (C and D) During CB treatment, a sample of untreated control cells was kept at 4°C (as described for Figure 5). After 100 fold dilution in medium to reduce the CB concentration, 3H-thymidine was added, and incorporation into acid-insoluble material was followed as a function of time. (0) control; (0) CB-treated cells.
Time Figure 5. Thymidine gallisepticum
Uptake
(hr)
in Untreated
and
CB-Treated
M.
An exponentially growing culture was split into two parts: one (0) was kept at 4°C for 6 hr and then at 37°C for 1 hr; the other (0) was treated with CB (100 pg/ml final concentration) at 37°C for 7 hr. This protocol gives an equal number of cells in both cultures at the end of the 7 hr period. 3H-thymidine was then added to both cultures and uptake was followed as a function of time.
(Figures 6A and 6B) that the longer the duration of drug treatment, the greater the lag time before cell growth resumed after CB removal. The cell growth curves in these and in repeated experiments suggested some degree of synchrony in the first cell division following release of CB inhibition. This observation was not investigated further. To see whether after removal of CB, DNA synthesis resumes before or after cell division, ceils were treated for either 3 or 12 hr with 100 pg CB/ml. At these times, each culture was diluted 100 fold to reduce the amount of CB, 3H-thymidine was added and DNA synthesis was measured. In cells treated for 3 hr (Figure 6D), DNA synthesis resumed immediately after CB removal and continued at the rate observed in untreated cells. Cells treated for 12 hr (Figure 6C) had a 2 hr lag in DNA synthesis, after which synthesis resumed at the same rate as in untreated cells. Comparing this with the studies on cell growth (Figures 6A and 6B), it is seen that DNA synthesis resumes before the first cell division following CB removal.
Electron Microscopy of CB-Treated Cells Thin sections of CB-treated M. gallisepticum cells were examined by electron microscopy to see whether any cytological changes could be observed. No differences were seen between untreated and CB-treated cells. Both one-bleb (Figure 7A) and two-bleb cells (Figure 7B) were seen. Since few two-bleb cells were observed due to the low probability of the section plane passing through both blebs, it was not possible to decide whether or not the distribution of cells between one- and two-bleb states was different in untreated and CBtreated cells. To measure the number of one- and two-bleb cells, untreated cells and cells treated for 4 hr with 100 pg CB/ml were negatively stained and examined by electron microscopy. This allowed whole cells to be seen, and one-bleb (Figure 7C) and two-bleb cells (Figure 7D) could be counted. At least 250 cells were counted for each sample. In the untreated culture, the cells were found to be 81% one-bleb and 19% two-bleb. Cells treated with CB were found to be 63% one-bleb and 37% two-bleb. Hence there is about a doubling in two-bleb cells in CB-treated cultures. Untreated and CB-treated cells, prepared by freeze etching, appeared the same (Figures 7E and 7F). In the two samples, no difference could be found in the particle distribution on the membrane fracture faces. Most cells (in both preparations) seemed to have fewer particles on the membrane
Mycoplasmas 61
Figure
and Cytochalasin
7. Electron
Micrographs
(A and B) Thin sections stained with silicotungstate. treated cultures. (E and relative to the number of pg CB/ml. The arrows in
B
of M. gallisepticum
Cells
showing the cell membrane (m), bleb (b), nuclear material (n) and ribosomes (r). (C and D) Cells negatively The one-bleb cells in (A) and (C) are from untreated cultures, and the two-bleb cells in (B) and (D) from CBF) Freeze-etched cells. Fewer particles are seen on the membrane fracture face around the bleb (arrowhead), particles on the membrane face around the rest of the cell. (E) Untreated cells: (F) cells treated for 4 hr with 100 (E) and (F) indicate the direction of shadowing. Bar = 0.25 pm.
fracture face around the bleb than brane around the rest of the cell.
on the
mem-
Uptake of CB 3H-CB, mixed with unlabeled CB, was added to an M. gallisepticum culture at a concentration of 100 pg CB/ml (5 pCi/ml) and uptake was measured. CB uptake reached a plateau by 1 hr (Figure 8A), indicating a saturation of CB binding sites at this time. To obtain a measure of the affinity of these sites for CB, the uptake experiment was repeated, and after 1 hr uptake was stopped by reducing the CB
concentration 100 fold by dilution with growth medium. The loss of CB from binding sites was followed by measuring the label remaining in the cells as a function of time. A gradual loss of CB was found (Figure 8B), and by 2 hr after dilution, only about 20% of the original bound CB remained in the cells. Characterization of a Subcellular Fraction Binding CB The fact that cells lose bound 3H-CB at a slow rate suggested that it might be possible to isolate a subcellular fraction with bound CB. For these stud-
Cell 62
Discussion
Tima ,m,n, Figure
6. Binding
to M. gallisepticum
Cells
(A) 3H-CB, mixed with unlabeled CB, was added to tially growing culture and bound label was measured of time. (6) After 1 hr of 3H-CB binding, a M. culture was diluted 100 fold to reduce exogenous remaining bound CB was measured as a function of
an exponenas a function gallisepticum CB and the time.
ies, 3H-CB was added to an exponentially growing M. gallisepticum culture at a final concentration of 100 pg/ml (5 &i/ml), and the culture was incubated for 8 hr at 37°C to assure saturation of the CB binding sites. The cells were harvested by centrifugation and washed with O..Ol M Tris buffer (pH 7). The cell pellet was resuspended in 2 ml 0.01 M Tris and lysed by freeze-thawing and sonication. In these studies, it was noted that 15-20 cycles of freeze-thawing were necessary to lyse CB-treated cells; lysis was judged by the reduction in turbidity of the cell suspension. This is 4-5 times the number of cycles required to lyse untreated cells, and indicates that CB treatment has made the cells more resistant to freeze-thaw lysis. The reason for this difference was not investigated. The cell lysate was centrifuged at 4000 x g for 4 min to remove unlysed cells. This procedure has been shown to leave lysed cells, membrane vesicles, bleb structures and cytoplasmic material in the supernatant (Maniloff and Quinlan, 1974). The supernatant was removed and centrifuged at 40,000 x g for 30 min to pellet membranes and blebs. The supernatant, which contained about 35% of the 3H-CB, was discarded. The pellet was resuspended in Tris buffer, sonicated to separate the bleb end of the cell from the rest of the cell membrane, and centrifuged through a sucrose step gradient. Two bands could be seen, one on the 55% sucrose shelf and one on the 62% shelf. Material banding on the 55% shelf has been shown to be membrane vesicles (Maniloff and Quinlan, 1974) and was found to contain 35-40% of the 3HCB. Bleb structures have been found to band on the 62% shelf, and this material contained 60 to 65% of the 3H-CB. Hence both cell membranes and bleb structures probably contain CB binding sites.
The inhibition of M. gallisepticum growth described here is the only report, of which we know, of a CB effect on a procaryote. It was shown in these studies that this effect is specific for M. gallisepticum; CB does not inhibit other mycoplasmas (Figure 1). As is discussed below, we believe that CB inhibition is a consequence of the unique subcellular organelles found in M. gallisepticum. Parenthetically, no inhibition was observed with either colchicine or vinblastine. The reported effects of CB on eucaryotic cells are of two types (reviewed by Pollard and Weihing, 1974): inhibition of uptake of small molecules, and inhibition of cellular processes involving actin or actin-like proteins. Since we have been unable to demonstrate a CB inhibition of small molecule uptake, we suggest that the CB inhibition of M. gallisepticum growth is due to the effect of CB on some actin-like protein required at one or more steps of the cell cycle. In agreement with this suggestion are preliminary studies on M. pneumoniae, the only other mycoplasma having a terminal structure (Maniloff and Morowitz, 1972), which have shown that these cells are inhibited by CB (A. Ghosh and J. Maniloff, unpublished data) and that an actin-like protein can be isolated from them (H. C. Neimark, 1976, Abstracts Ann. Meeting Am. Sot. Microbial., p. 61). Considering the data presented here, together with the available data on the analysis of M. gallisepticum subcellular organelles, growth cycle and DNA replication, we propose a model for the action of CB on M. gallisepticum. The morphological events (from Morowitz and Maniloff, 1966) begin with the newly formed daughter cell containing one terminal bleb. During the cell cycle, a second bleb structure is formed, perhaps at the preexisting bleb. Assuming that the fraction of two-bleb cells in a culture represents the fraction of the cell cycle during which a cell is bipolar with two bleb organelles, since about 20% of the cells have two blebs, each cell must be in a two-bleb state during the last 20% of the cell cycle. A constriction leading to cell division occurs at about 2 hr in the cell cycle. Each M. gallisepticum daughter cell receives one genome copy (Ghosh, Das and Maniloff, 1977) which is replicated once per cell cycle (Quinlan and Maniloff, 1973). In a study of synchronously growing M. gallisepticum, it was shown that there is a gap of about 30-40 min in DNA synthesis during cell division (Quinlan and Maniloff, 1973). Hence DNA synthesis is initiated after the start of the cell cycle and terminated before cell division. Since CB immediately stops the increase in cfu, it must block at the cell division step. A second CB
Mycoplasmas 63
and Cytochalasin
B
block earlier in the cell cycle, however, is indicated by the observation that less than half of CB-inhibited ceils reach the two-bleb state. This result suggests that in the presence of CB, M. gallisepti‘cum cells which have begun formation of a second bleb can complete the structure, but cells at an earlier point in the cell cycle cannot initiate formation of a second bleb. In this context, formation of a second bleb may mean either assembly of a bleb structure or migration of a preexisting structure to give the morphological appearance of a two-bleb cell. DNA synthesis continues for 2-3 generation times after CB addition, and it should be noted that more DNA is made than would be expected if synthesis were only completing rounds of replication. Nothing is known about the coupling between DNA synthesis and the formation of bleb structures, but the DNA replication complex is known to be at the bleb (Maniloff and Quinlan, 1974). One possible explanation of the CB effect is that CB has disrupted the assembly of actin-like proteins required at two points of the cell cycle: early in the ceil cycle for the formation of a second bleb, and late in the cycle for constriction and cell division. In agreement with this possibility, preliminary studies have shown that a protein can be isolated from M. gallisepticum with actin-like properties (U. Chaudhuri and J. Maniloff, unpublished data); the protein has a molecular weight about 45,000 daltons, polymerizes in the presence of KCI and depolymerizes with added ATP. Experimental
Procedures
Mycoplaemas and Media The mycoplasmas used in this study were M. gallisepticum strain A5969, M. capricolum strain 14 and Acholeplasma laidlawii strain B [the isolation of these strains has been described by Tourtellotte and Jacobs (1960)]. A. laidlawii and M. capricolum were grown in tryptose broth and assayed as colony-forming units (cfu) on tryptose agar plates, as described previously (Quinlan, Liss and Maniloff, 1972). M. gallisepticum was grown in MBB medium and assayed as cfu on MBB agar plates as described by Ghosh et al. (1977). Drug Treatment The drugs used were cytochalasin B (Aldrich Chemical Co., Milwaukee, Wisconsin), colchicine (Sigma Chemical Co., St. Louis, Missouri), and vinblastine sulfate (Eli Lilly and Co., Indianapolis, Indiana). The latter two were dissolved directly in growth medium. To make CB solutions, 1 mg CB was dissolved in 0.1 ml ethanol and then diluted with medium. Controls for CB treatments were made up with amounts of ethanol equivalent to the corresponding CB samples. In all experiments, the ethanol concentrations were 1% or less, since loss of cell viability was observed only at higher solvent concentrations. Measurement of Macromolecular Synthesis For each experiment (that is, measurement of DNA, RNA or protein synthesis), an appropriate radioisotopically labeled precursor, at a final activity of 50 @Zi/ml, was added to a tube of exponentially growing cells (4.5 ml). Precursor incorporation into macromolecular material was followed by pipetting 50 ~1 culture samples into 1 ml ice cold 10% trichloroacetic acid at various
times. After 30 min in the cold, precipitates filters, washed and assayed for radioactivity (Quinlan et al., 1972).
were collected in toluene-Omnifluor
on
Glucose Uptake Sugar transport was studied by measuring the uptake of ‘+-amethyl-D-glucoside, a nonmetabolizable analogue of glucose, as described by Rottem and Razin (1969). The labeled glucose was added to a final activity of 50 &i/ml to exponentially growing ceils. 50 /II samples were withdrawn after different times and filtered through a 0.22 pm Millipore (Bedford, Massachusetts) filter. The filters containing the cells were washed with 50 ml icecold growth medium, dried and counted as above. Thymidine Uptake These experiments were performed uptake, except that 3H-thymidine tion of IO &i/ml.
similarly was used
to those for glucose at a final concentra-
CB Uptake These experiments were done similarly to those for glucose uptake, except 3H-cytochalasin B (New England Nuclear, Boston, Massachusetts) was used. The 3H-CB was mixed with unlabeled CB and added to a culture to give a final concentration of 100 fig CB/ml and 5 &i/ml. At various times, 1 ml samples were filtered, washed and counted as described for glucose uptake measurements. Density Gradients For the analysis of the cell components which bind CB, cells were incubated with 3H-CB as above for 8 hr. Cells were then disrupted by freeze-thawing and sonication, as described by Maniloff and Quinlan (1974). The lysate was layered over a discontinuous sucrose gradient with 3.5 ml steps of 40, 50, 55 and 62% (w/v) sucrose and spun in a Beckman SW27 rotor for 8 hr at 23,000 rpm at 5°C. This procedure has been shown (Maniloff and Quinlan, 1974) to band membrane vesicles on top of the 55% step and bleb structures on the 62% step. Bands were removed by puncturing the side of the centrifuge tube and assayed for radioactivity, as above. Electron Microscopy For thin-section studies, a culture was mixed with an equal volume of cold 12.5% glutaraldehyde (in 0.1 M cacodylate) and kept at 4°C for 2 hr. The cells were then harvested and washed overnight at 4°C with 0.1 M cacodylate. The pellet was then embedded and stained by the method of Hills and Plaskitt (1968). For freeze fracture studies, fixed cells were centrifuged, and the cell pellet was placed in 40% (v/v) glycerol. The freeze fracture procedure was carried out using a Denton (Cherry Hill, New Jersey) Model DFE-2 Freeze-Etching Unit, as described by Steere (1969). For examination of whole cells, a drop of glutaraldehyde-fixed culture was put on formvar-coated grids and negatively stained with 1% silicotungstic acid (pH 6.8). Grids were examined with a Siemens Elmiskop IA electron microscope with a liquid nitrogen decontamination device and 50 pm objective aperture, operating at 80 kV. Acknowledgments We thank Dr. James Coleman for his suggestions regarding the use of cytochalasin B. This study was supported by a USPHS grant from the National Institute of Allergy and Infectious Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
August
11, 1977;
revised
October
14, 1977
Cell 64
References Bak, L. A., Black, F. T., Christiansen, (1969). Genome size of mycoplasmal 1210.
C. and Freundt. DNA. Nature 224,
E. A. 1209-
Betina, V., Micekova, D. and Nemec, P. (1972). Antimicrobial properties of cytochalasins and their alteration of fungal morphology. J. Gen. Microbial. 71, 343-349. Ghosh, A., Das, J. and ultraviolet light damage Biol. 116, 337-344. Henrichsen, unaffected
Maniloff, J. (1977). Lack of repair of in Mycoplasma gallisepticum. J. Mol.
J. (1972). Gliding and twitching motility of bacteria by cytochalasin 8. Acta Pathol. Stand. B 80, 623-624.
Hills, G. J. and Plaskitt, L. (1968). A protein stain for the electron microscopy of small isometric plant virus particles. J. Ultrastructure Res. 25, 323-329. Lin, S. and Spudich. action of cytochalasin
J. A. (1974). B. J. Supramol.
On the molecular basis Structure 2, 728-736.
Maniloff, J. and Morowitz, H. J. (1972). mycoplasmas. Bacterial. Rev. 36, 263-290.
Cell
Maniloff, J. and Quinlan, D. C. (1973). Biosynthesis lar organization of nucleic acids in Mycoplasma A5969. Ann. NY Acad. Sci. 225, 181-189. Maniloff, membrane coplasma
biology
of
of the
and subcellugdisepticum
J. and Quinlan, D. C. (1974). Partial purification of a associated deoxyribonucleic acid complex from Mygallisepticum. J. Bacterial. 720, 495-501.
Morowitz, H. J. and Maniloff, of Mycoplasma gallisepticum.
J. (1966). Analysis of the life cycle J. Bacterial. 91, 1644-1683.
Pollard, T. D. and Weihing. R. Ft. (1974). Actin cell movement. CRC Crit. Rev. Biochem. 2, i-65.
and myosin
Quinlan, D. C. and Maniloff, J. (1972). Membrane the deoxyribonucleic acid growing point region gallisepticum. J. Bacterial. 772, 1375-1379.
and
association of in Mycoplasma
Quinlan, D. C. and Maniloff, J. (1973). Deoxyribonucleic acid synthesis in synchronously growing Mycoplasma gallisepticum. J. Bacterial. 175, 117-120. Quinlan, medium 6, 179-l
D. C., Liss, A. and Maniloff. J. (1972). Eagle’s basal as a defined medium for mycoplasma studies. Microbios 85.
Rottem, S. and Razin, S. (1969). Sugar ga//isepticum. J. Bacterial. 97, 787-792. Steere, 306-323.
R. L. (1969).
Freeze-etching
Tourtellotte, M. E. and Jacobs, serologic comparison of PPLO Acad. Sci. 79, 521-530.
transport simplified.
R. E. (1960). from various
in Mycoplasma Cryobiology
5,
Physiological and sources. Ann. NY
Wessels, N. K., Spooner, 6. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T. and Yamada, K. M. (1971). Microfilaments in cellular and developmental processes. Science 777, 135-143.
