JOURNAL OF BACTERIOLOGY, Mar. 1977, p. 1495-1501 Copyright © 1977 American Society for Microbiology
Vol. 129, No. 3 Printed in U.S.A.
Motility of Mycoplasma pneumoniae U. RADESTOCK AND W. BREDT1 * Institut fur Medizinische Mikrobiologie, Johannes Gutenberg-Universitat, D-6500 Mainz, Germany
Received for publication 26 July 1976
Cells of Mycoplasma pneumoniae FH gliding on a glass surface in liquid medium were examined by microscopic observation and quantitatively by microcinematography (30 frames per min). Comparisons were made only within the individual experiments. The cells moved in an irregular pattern with numerous narrow bends and circles. They never changed their leading end. The average speed (without pauses) was relatively constant between 0.2 and 0.5 ,um/s. The maximum speed was about 1.5 to 2.0 ,um/s. The movements were interrupted by resting periods of different lengths and frequency. Temperature, viscosity, pH, and the presence of yeast extract in the medium influenced the motility significantly; changes in glucose, calcium ions, and serum content were less effective. The movements were affected by iodoacetate, p-mercuribenzoate, and mitomycin C at inhibitory or subinhibitory concentrations. Sodium fluoride, sodium cyanide, dinitrophenol, chloramphenicol, puromycin, colchicin, and cytochalasin B at minimal inhibitory concentrations did not affect motility. The movements were effectively inhibited by anti-M. pneumoniae antiserum. Studies with absorbed antiserum suggested that the surface components involved in motility are heat labile. The gliding of M. pneumoniae cells required an intact energy metabolism, and the proteins involved seemed to have a low turnover. Motility of bacteria is, in most cases, dependent on the presence of flagella. Relatively few species are known to move by gliding (17, 19). Little is known about the mechanism of gliding and its relation to flagellar motility (17, 19). Among the gliding microorganisms discovered in recent years are three species of mycoplasmas: Mycoplasma pulmonis (1, 21, 22), M. pneumoniae (4, 6, 9), and M. gallisepticum (5). Gliding mycoplasmas deserve special consideration for several reasons. Their genetic content is smaller than that of normal bacteria (5 x 108 versus 1 x 109 to 2 x 109 daltons) (2). Moreover, their membrane is in direct contact with substrate, without an interfering cell wall. Therefore, mycoplasmas may serve as a useful model for studies on the mechanisms of gliding movements. Furthermore, all three motile mycoplasma species are pathogens of the respiratory tract of humans or animals (13, 24). Their ability to move by gliding may therefore be involved in the process of pathogenicity. Little is known about the movements of mycoplasmas. Only M. pneumoniae is a useful model for experimental studies, since its movements are fast and constant even after numerous passages on artificial media. In contrast, M. pulmonis ceases to move after a few pas' Present address: Zentrum fur Hygiene, D-7800 Freiburg, Germany.
sages in vitro (21, 22), and M. gallisepticum shows only slow movements (J. T. Liauw, M.D. thesis, Johannes Gutenberg University, Mainz, Germany, 1976). A few data were obtained from cinematographic studies on M. pneumoniae (10). However, no experimental system existed for the quantitation of movements. Therefore, it was the purpose of this study to develop a reliable method for the quantitation of mycoplasma gliding movements. By this method, information was obtained about the influence of environmental conditions and inhibitory substances on M. pneumoniae motility.
MATERIALS AND METHODS Mycoplasma. M. pneumoniae FH was grown in liquid medium (15), filtered through a polycarbonate filter (0.4 gm, Nucleopore, General Electric Co.), and stored in small portions at -70°C. After thawing, the suspension contained about 107 colonyforming units per ml. A nonmotile mutant of M. pneumoniae, M129P175, with reduced adherence was obtained from W. A. Clyde, Jr., Chapel Hill, N.C. It was grown in the same medium and used only for immunization of rabbits. Media. Liquid and agar media were prepared by the method of Hayflick (15). Penicillin (1,000 U/ml) and thallium acetate (0.05%) were added for most of the experiments. Agamma horse serum (Microbiological Associates) was used instead of horse serum (Flow Laboratories) for all microscopic experiments. For studies on the effect of increased viscosity, vary1495
1496
RADESTOCK AND BREDT
ing amounts of a 10% solution of gelatin in PPLO broth (Difco) were used instead of pure PPLO broth for preparation of the final medium. For the metabolic inhibition test, the medium contained 0.025% 2,3,5-triphenyl-tetrazolium chloride (Serva) and 1% glucose (23). Buffer. Tris(hydroxymethyl)aminomethanebuffered saline (8) was used in several experiments. Viscosity of media. The viscosity of the media used was determined by measuring the flow velocity (in seconds): water, 299; liquid medium, 329; medium with 1% gelatin, 502; medium with 3% gelatin, 984; and medium with 5% gelatin, 1,376. Chemicals. The following substances were used: Freund complete adjuvant (Difco), calcium chloride, magnesium chloride, sodium fluoride, tris (hydroxymethyl)aminomethane (Merck); chloramphenicol (Parke Davis); dimethyl sulfoxide (Roth); and puromycin, cholesterol, colchicin, cytochalasin B, 2,4dinitrophenol, ethylenediaminetetraacetic acid (EDTA), ethylenglycoltetraacetic acid (EGTA), actinomycin D, p-chloromercuribenzoate (PCMB), iodoacetate, and adenosine 5'-triphosphate (Serva). Metabolic inhibition test. The minimal inhibitory concentrations (MIC) of chemicals were determined by a modification of the 2,3,5-triphenyl-tetrazolium chloride reduction inhibition test (23). The test was read when the control wells without the test substance began to show formation of red formazan. The test medium contained 3% gelatin. Mycoplasmocidal test. Twofold dilutions of the test substances were prepared in Eppendorf plastic vials with medium containing 3% gelatin. A 490-,ul amount of each concentration was inoculated with 10 1,u of a preincubated (20 h, 37°C) M. pneumoniae suspension. A vial without test substance served as the control. The vials were incubated at 37°C. At 2-h intervals, a 10-,ul volume was removed from each test tube and immediately diluted in 100 ,ul of PPLO broth. With a calibrated platinum loop, three 1-,ul portions of each dilution were spread on agar media. Colonies were counted after 10 to 12 days of incubation. The concentration at which a distinct decrease occurred after 2 h was considered a mycoplasmocidal concentration (MCC). Antisera. Antisera against M. pneumoniae strains FH and M129P175 were prepared by immunizing rabbits with glass-grown organisms in Freund complete adjuvant (20). The antisera were stored at -20°C. Absorption of antiserum. M. pneumoniae FH was grown in four Roux bottles (100 ml of medium in each bottle), washed, and harvested in 4 ml of buffer. A 2-ml portion of this suspension was heated at 100°C for 1 h; 2 ml remained unheated. A 1-ml portion of each suspension (either heated or native) was centrifuged (7,000 x g), and the pellet was suspended in 400 ul of a 1:5-diluted anti-M. pneumoniae FH antiserum. The suspension was incubated for 1 h at 37°C and centrifuged, and the supernatant was -absorbed once more with the same amount of organisms. UV. The mycoplasmas grown in cover slip chambers were irradiated with ultraviolet light (UV) (254 nm) for 30 to 180 s at a distance of 8 cm (Sylvania lamp type TL 900).
J. BACTERIOL.
Determination of lectin-binding capacity of the medium. Concanavalin A was diluted in gelatin medium in a microtiter plate, and sheep erythrocytes were added. The agglutination titer was read after 2 h, and the lowest agglutinating concentration was considered to be just above the concanavalin A-binding capacity of the medium. Microscopic observation. A frozen suspension of M. pneumoniae was thawed and incubated at 37°C with constant shaking to avoid clumping. After 20 h this suspension was used to inoculate a 3% gelatin medium (1:1,000). The inoculated medium was placed in cover slip chambers (3). After 20 h of incubation, the mycoplasmas grown on cover slips were transferred to new chambers containing the test or control media. The chambers were incubated further at 37°C, and the movements of the cells were examined microscopically after 0.5, 1, 2, 4, 6, and 24 h. During examination, the chambers were kept at 37°C by a Sage Air Curtain incubator. Cinematographic studies. Cover slips with M. pneumoniae were prepared as described above and transferred to chambers containing the test or control medium, respectively. The control medium in these studies always consisted of medium with 3% gelatin (pH 7.2), incubated at 37°C. The two chambers were fastened to metal frames (18) with a wax mixture (Deckglaskitt, Merck) and incubated for 2 h at 37°C. After 2 h, seven series of cinematographic pictures were taken from each preparation. Each series covered a different field of view and lasted for 5 min. The frequency was 30 frames per min, and the exposure time was 0.5 s. The films were analyzed by projecting each frame onto paper, using a projector with single-frame projection (Siemens). The location of each cell was marked on the paper by a point, and the frame number was added. The process was repeated for each frame, resulting in 150 points per cell and series. The distances on the paper were measured, and the real distances in micrometers were calculated. From the distances and intervals obtained, the following data were calculated for each individual cell: total distance, total observation time, average speed, average speed without pauses (corrected speed), maximum speed, total time of movements, total time of resting, and percentage of resting periods. About 2,100 frames were examined for each experiment. Microscopic and photographic equipment. The movements of the cells were examined qualitatively with a Zeiss photomicroscope equipped with a phasecontrast condenser of long working distance (7 mm). The cinematographic studies were performed on a Zeiss inverted microscope (UPL) enclosed in a Plexiglas incubation chamber with electronic temperature regulation (Hessische Feinmechanik, Giessen). A phase-contrast objective with an optical aperture of 1.40 (x63) was used for all quantitative studies. Cinematography was performed with a cinematographic system from Wild, Herbrugg, Switzerland, consisting of a Bolex-Paillard 16-mm camera, variotimer, light meter, and heavy metal stand. Kodak 16-mm Plus-X reversal film was used for all experiments. Statistical test. Results of test and control preparations were examined for significant differences by
MOTILITY OF M. PNEUMONIAE
VOL. 129, 1977
the Wilcoxon test. Data were only compared within the same experiment.
RESULTS General aspects of motility. M. pneumoniae gliding cells always moved in the direction of their tip structure (Fig. 1). Their course was irregular and often circular with no preference for a certain direction. The movements were interrupted by resting periods of different lengths, ranging from 2 to more than 100 s. Under control conditions (medium with 3% gelatin, pH 7.2, 37°C), the cells showed average speeds of 0.2 to 0.5 ,um/s. The corrected speed (resting periods omitted) was higher and showed less variation (mostly between 0.3 and 0.5 ,um/s). The distances covered by the individual cells per 2-s interval were distributed around a mean value (Fig. 2). Occasionally, maximum speeds of 1.5 to 2.0 ,m/s were attained for a short time. The percentage of rest-
/
I
.j
V FIG. 1. M. pneumoniae cells on glass in liquid medium. Phase contrast. Bar represents 2 ,um. Arrow indicates tip structure.
ing periods varied widely among the cells (0 to 80%). Technique of cinematographic measurements. The system for cinematographic recording of M. pneumoniae cell movements produced reliable results under certain conditions. The frequency of 30 frames per min provided a sufficient number of measuring points to follow the often very narrow curves of the moving cells. Lower frequencies resulted in seemingly shorter, and therefore incorrect, distances. The number of cells per field of view was critical: with more than five to seven cells per field, collisions were likely to occur, which disturbed the follow-up of individual cells during analysis. An observation time of more than 5 min increased the chances of individual cells to leave the observation field, thereby reducing the number of cells with comparable observation time. Some of the data obtained from the measurements could be plotted graphically (Fig. 3), and possible differences could be examined statistically. Influence of environmental factors. Changes in the medium viscosity affected the movements significantly (Table 1). Higher viscosity reduced the speed and increased the percentage of resting periods. The latter was caused by more and extended individual pauses. A lower viscosity resulted in higher speed but did not change the percentage of pauses. The maximum speed of the cells was also affected by viscosity. The addition of 2% more gelatin reduced the maximum values about 0.1 ,um/s.
100 90-
(50 J
0
< 40-
1497
80-
70600~ 50CL 40z 3020 L) w 10
I
I 0
0 0
0 * a
000
0
Z30-
LL
O 20LL
E2 10z
w
0./4 5
6
I 7
.8 .9 1.0 1.1 1.2 (Dim)
FIG. 2. Typical distribution of the average speed (without pauses) (micrometers per 2-s interval) during the 300-s observation period. Bars represent three different cells. The number of intervals indicates the number of 2-s periods in which the indicated distance was covered.
o
00
08
*P%.
_
00
0.1 .2 .3 .4 .5 .6 0.1 .2 .3 .4 .5 .6 AVERAGE SPEED (,um/sec) FIG. 3. Results of quantitative experiments. (I) Without significant differences (identical media). (II) With significant differences (test medium, pH 6.5; control medium, pH 7.2). Symbols: 0, cells in test medium; 0, control cells. The average speed is that without pauses (i.e., corrected speed).
1498
RADESTOCK AND BREDT
J. BACTERIOL.
TABLE 1. Environmental factors affecting motility of M. pneumoniaea Resting period . . No. Avg speed (gm/s) anaCorrected speed (,gm/s) Expt condition lyzedM lyzed' Mean Range 2a 2 aS Mean Range Mean Range 1% Gelatin 9 0.41 0.35-0.49 0.01 0.46 0.44-0.51 0.01 11 3-27 5 0.35 0.32-0.37 0.41 0.38-0.45 13 5-15 Controlc
5% Gelatin Control
4 4
0.21 0.35
0.19-0.22 0.28-0.39
0.05
0.30 0.41
0.29-0.30 0.38-0.43
0.05
28 12
25-79 8-27
pH 6.5 Control
7 6
0.31 0.23
0.21-0.39 0.05-0.40
0.01
0.41 0.37
0.34-0.44 0.31-0.44
0.01
22 37
6-53 29-84
pH 7.9 Control
4 9
0.14 0.29
0.08-0.32 0.23-0.37
0.05
0.35 0.42
0.32-0.38 0.38-0.45
0.05
59 30
21-79 14-42
320C Control
6 7
0.27 0.39
0.19-0.34 0.33-0.42
0.01
0.35 0.42
0.27-0.45 0.37-0.45
0.01
23 8
6-56 2-9
390C Control
4 4
0.31 0.38
0.22-0.40 0.37-0.40
NSd
0.42 0.43
0.33-0.50 0.42-0.44
NS
9 24
3-13 2-41
Significance tested by the Wilcoxon text. b Number of cells analyzed (each cell was observed for 300 s). c Medium with 3% gelatin (pH 7.2); incubated at 370C. d NS, Not significant (2a _ 0.05). a
Motility in liquid medium without gelatin could not be studied quantitatively, because the moving cells lost contact with the glass and disappeared in the medium. Another factor affecting movement was the pH of the medium. At a more alkaline pH (7.9), the cells moved with reduced speed (Table 1). The percentage of resting periods was increased by both a higher number of pauses and increased length of the individual pause. Reduction of pH to 6.5 had the opposite effect. The movements were faster and the resting periods were shorter, resulting in a lower percentage of pauses (Table 1). At a lower incubation temperature (32°C), the speed was reduced significantly, and the percentage of pauses was increased (Table 1). This increase was caused by more frequent and prolonged pauses. At a higher temperature (39°C), the speed was not affected, but the percentage of resting periods was increased by more frequent and prolonged pauses. The addition of glucose (0.1 to 1%) did not significantly affect the speed of the cells. The percentage of resting periods was slightly increased by 1% glucose (Table 2). A reduced percentage of serum in the medium (10 or 5%) apparently affected only a minority of the cells. Most of the cells did not show any change in motility after 5 h, but about 10% were visibly damaged and immobilized. A reduction in the percentage of yeast extract (to 5%) affected the movements much more. After 1
TABLE 2. Various environmental factors and their effect on M. pneumoniae motility Expt condition Reduction of serum content (10 or 5%)
Effect on motility Most cells unaffected; signs of damage on '10% of
cells Reduction of yeast extract (5%)
No movements after 1 h
Addition of glucose, 0.1%
No effect Slight increase of pauses
Conditioned medium
No effect
Removal of Ca2+ (10 mM EGTA, 5 mM Mg2+)
No effect
Removal of Ca2+ and Mg2+ (5 mM EDTA)
Cells are rounded
swollen
and
h the cells slowed down or stopped gliding totally (Table 2). The removal of Ca2+ by EGTA did not significantly influence the motility. The addition of a Ca2+ and Mg2+ also had no effect on gliding. Removal of Mg2+ by EDTA resulted in visible, general damage (rounding, swelling) of most of the cells and inhibited movement (Table 2). Conditioned medium (preincubated with M. pneumoniae), cholesterol (5 to 20 ,ug/ml), and adenosine 5'-triphosphate (5 to 20 gg/ml) had no visible effect. Influence of inhibitory agents. The MICs and MCCs of the various substances tested are
VOL. 129, 1977
MOTILITY OF M. PNEUMONIAE
summarized in Table 3. The motility of the cells reacted differently to metabolic inhibitors. The MIC of dinitrophenol, NaCN, and NaF did not affect the movements. Higher concentrations were not tested, because the high MIC suggested a low effectiveness of these substances. Iodoacetate (MIC) significantly reduced the average speed and increased the percentage of pauses (Table 4) by more frequent resting periods. The MIC of PCMB increased the frequency and length of the resting periods and reduced the speed (Table 4). However, the number of cells available for analysis was too low for statistical tests. Concentrations higher than the MIC resulted in rounding and immobilization of the cells after about 1 h. Inhibitors of nucleic acid and protein syntheses showed different effects. Mitomycin C (0.5 MIC) resulted in lower speed and more resting time (Table 4). The frequency of pauses remained unchanged, and their length in-
creased. Actinomycin D and chloramphenicol did not show distinct effects at concentrations below the MCC within 2 to 4 h. Motility was reduced by 10 ,ug of chloramphenicol per ml after 24 h, but some of the cells were still moving. Puromycin at a concentration of 10 ,ug/ml did not affect the gliding movements. UV irradiation of the cells (3 min; dose, 900 ergs/mm2) resulted in slower movements within 6 h. No motile cells were seen after 24 h. X rays in a dose of 3,000 rads had no visible effect. Cytochalasin B at a concentration of 50 ,ug/ ml did not influence the movements. Colchicine (10 ,ug/ml) had no effect. Treatment of the membrane surface with homologous antiserum (Ab) inhibited the movements. Ab at a concentration of 1:400 reduced the motility after 1 h. Higher concentrations (1:50) immobilized the cells totally. The action of Ab was reversible. Transfer of antibodytreated cells (30 min, 1:50) to antibody-free medium restored motility to the normal level after about 2 h. Ab against the nonmotile mutant (M129P175) had the same effect on the cells as antiserum against the homologous strain FH. Absorption of the antiserum (1:50) with native M. pneumoniae removed all motility-inhibiting activity. However, antiserum absorbed with heated M. pneumoniae (100°C) showed full inhibition of movements at the same degree as unabsorbed antiserum. Treatment of the membrane surface with concanavalin A (free concentration, ca. 10 jig/ ml) did not affect the movements.
TABLE 3. MIC and MCC of various substances tested on M. pneumoniae Inhibitor acting on:
MCCa
_10.0 2.5 _10.0 -10.0 0.5
NTb 5.0 NT NT 1.0
0.3 5.0
1.25 10.0
3.0 10.0
25.0 NT
15.6
125.0 NT
Substance tested (Stg/ml) (JLgIml)
Energy metabolism
Dinitrophenol Iodoacetate NaCN NaF PCMB
Nucleic acid synthesis
Actinomycin D Mitomycin C
Protein synthesis
Chloramphenicol Puromycin
Contractile ments
Cytochalasin B Colchicine
ele-
MIC
-'10.0
1499
DISCUSSION Studies on mycoplasma gliding are much more difficult to perform than comparable experiments with other gliding bacteria, e.g.,
a Reduction of colony-forming units after 2 h. bNT, Not tested.
TABLE 4. Inhibitory substances significantly affecting motility of M. pneumoniae at or below MIC levelsa Avg speed
No. anaExptco condition n Expt lyzed b
Mean
(inm/s)
Range 0.17-0.26
(Mm/s)
2a_
Mean
Range
2a_
0
0.01
0.37 0.50
0.34-0.39 } 0.46-0.55 f
0-01
0.20 0.34
0.12-0.28 t NDd 0.28-0.40 f
0.39 0.41
0.38-0.41 I 0.40-0.43
ND
0.35
0.24-0.47
0.47
0.44-0.53
Iodoacetate, 2.5 mM Controlc
6 6
0.23 0.45
0.39-0.52
PCMB, 0.5 mM Control
2 5
Mitomycin C, 2.5 ug/
7
ml
Corrected speed
0.01
Resting periods Mean Range 37 26-52 15 3-16
43 7
31-56 5-31
13
2-28
23
3-54
? 0.01
Control 8 0.52 0.42-0.57 J 0.60 0.58-0.61 J a Significance tested by the Wilcoxon test. b Number of cells analyzed (each cell was observed for 300 S. c Medium with 3% gelatin (pH 7.2); incubated at 37°C. d ND, No statistical calculation done because of small number of cells.
1500 RADESTOCK AND BREDT myxobacteria (12, 17). Several factors are involved. Mycoplasmas are very small cells, they are fastidious, their movements can only be seen in a complex liquid medium, and mutants like those used in studies of myxobacteria (R. P. Burchard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G239, p. 70) do not exist or are rather difficult to select. These factors considerably limit the number of possible experiments, especially those about environmental factors. However, it is possible, to a certain extent, to quantitate the movements, as shown by the data presented. As indicated previously (9, 10), the measurements require a relatively high number of cinematographic pictures, and there are certain variations in the control data of the different experiments. However, the results appear reliable as long as adequate numbers of cells are measured and the data are compared only within the individual experiment. The movements of M. pneumoniae are somewhat different from those of other gliding bacteria, especially myxobacteria. Mycoplasmas do not move in swarms, but as single cells. Their moving pattern consists mainly of circles and narrow bends, and in contrast to myxobacteria they never change their leading end (6, 7). Only the speed of mycoplasmas and myxobacteria is comparable (10, 19). The factors that stimulate mycoplasma movement are unknown. The intense motility in densely grown cultures (9) seems to depend mainly on the lowered pH, as confirmed by the quantitative experiments. Yeast extract seems to contain an unknown substance necessary for constant movement. On the other hand, Ca2+ ions are not required. The failure to obtain significant effects by adding glucose may be due to the large amount of metabolizable substance in the complex medium. No evidence of a motility-stimulating factor was found in conditioned medium. Attempts to find a method for measuring possible chemotactic properties of mycoplasmas have failed so far in our laboratory. The effects of iodoacetate and PCMB on movement indicate a requirement for cell-derived energy. This is in agreement with results obtained by Burchard (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G239, p. 70), who found that PCMB inhibits the gliding of myxobacteria. There was no evidence for thermal energy being the sole cause for the gliding. Proteins participating in the gliding mechanism appear to have a relatively slow turnover. Inhibition of protein synthesis did not affect motility for several hours. This suggests that no substantial amounts of protein (e.g., as a gliding layer) are secreted during movement. Furthermore, de novo membrane synthesis
J. BACTERIOL.
does not seem to play an essential role in the gliding process. Factors effecting the cell division by interfering with deoxyribonucleic acid (DNA) synthesis (mitomycin C, UV) had a delayed effect on motility. However, the movements were inhibited much faster than observed with inhibitors of protein synthesis. Therefore, the effect of UV or mitomycin C cannot be explained by mere exhaustion of preformed substances. The results seem to suggest that intact DNA or DNA synthesis is necessary for the function of the motility mechanism. The data do not permit an explanation for this observation. Inhibitors of contractile elements (cytochalasin B, colchicin) (11, 14, 25) did not affect motility, confirming the results of preliminary experiments on M. pneumoniae (6) and M. gallisepticum (W. Bredt, unpublished data) and observations on other prokaryotes (16). However, this does not necessarily indicate that the motility mechanism is insensitive, since it is not known whether the membrane of prokaryotes is permeable to these substances. The problem of whether actin-like proteins are involved in the motility therefore has to be approached by other methods. Substances located on the membrane surface appear to play an essential role in motility, since treatment with antibodies resulted in fast inhibition of movement. The unimpaired effectiveness of antiserum absorbed with heated mycoplasmas indicates a heat-labile antigenic nature of the structures and substances involved. These are possibly proteins responsible for transport mechanisms and/or attachment. No antigenic difference seems to exist in this respect between the motile strain, FH, and the nonmotile mutant. Similar results have been obtained during studies on the adherence of M. pneumoniae (F. W. Gorski, M.D. thesis, Mainz, in preparation). The surface substances involved in attachment were heat labile and protease sensitive, and their reconstitution could be prevented by chloramphenicol. Whether the same substances are participating in both phenomena remains to be tested. The results obtained in this study provide a basis for further experiments. They show that motility is related to environmental factors. However, they provide only a partial and yet incoherent knowledge about the possible mechanisms. Further studies will hopefully reveal more about structures and mechanisms involved in the transformation of energy into movement. ACKNOWLEDGMENTS The work was supported by grant Br 296/8 of the Deutsche Forschungsgemeinschaft.
VOL. 129, 1977 The skillful technical assistance of Dagmar Donarski and Irmgard Thommes is gratefully acknowledged. We are indebted to W. A. Clyde Jr., Chapel Hill, N.C., for giving us the M. pneumoniae mutant obtained in his laboratory. We would like to thank H. J. Berger, Institute for Statistics and Documentation, Mainz, for statistical help, and J. Bereiter-Hahn, Institute for Cinematographic Cell Research, Frankfurt, for his help in analyzing the pictures. LITERATURE CITED 1. Andrewes, C. H., and F. V. Welch. 1946. A motile organism of the pleuropneumonia group. J. Pathol. Bacteriol. 58:578-580. 2. Bak, A. L., and F. T. Black. 1968. DNA base composition of human T-strain mycoplasmas. Nature (London) 219:1044-1045. 3. Borisy, G. G., and E. W. Taylor. 1967. The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. J. Cell Biol. 34:535-548. 4. Bredt, W. 1968. Growth morphology of Mycoplasma pneumoniae strain FH on glass surface. Proc. Soc. Exp. Biol. Med. 128:339-340. 5. Bredt, W. 1968. Motility and multiplication of Mycoplasma pneumoniae. A phase contrast study. Pathol. Microbiol. 32:321-326. 6. Bredt, W. 1972. Phase contrast studies on living mycoplasmas. Med. Microbiol. Immunol. 157:169. 7. Bredt, W. 1973. Motility of mycoplasmas. Ann. N.Y. Acad. Sci. 225:246-250. 8. Bredt, W. 1974. Structure and motility, p. 47-52. In J. M. Bove and J. F. Duplan (ed.), Mycoplasmas of man, animals, plants and insects. Institut National de la Sante et de la Recherche Medicale, Paris. 9. Bredt, W., and D. Bitter-Suermann. 1975. Interactions between Mycoplasma pneumoniae and guinea pig complement. Infect. Immun. 11:497-504. 10. Bredt, W., K. H. Hofling, and H. H. Heunert. 1970. Mycoplasma pneumoniae (Mycoplasmataceae): Bewegung, Vermehrung, Koloniebildung. Encyclopaedia cinematographica Film E 1633. G. Wolf (ed.),
Gottingen. 11. Bredt, W., K. H. Hofling, H. H. Heunert, and B. Milthaler. 1970. Messungen an beweglichen Zellen von
MOTILITY OF M. PNEUMONIAE 1501 Mycoplasma pneumoniae. Z. Med. Mikrobiol. Immunol. 156:39-43. 12. Burchard, R. P. 1974. Studies on gliding motility in Myxococcus xanthus. Arch. Microbiol. 99:271-280. 13. Freundt, E. A. 1974. Present status of the medical importance of mycoplasmas. Pathol. Microbiol. 40:155-187. 14. Furcht, L. T., and R. E. Scott. 1975. Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells. Exp. Cell Res. 96:271-282. 15. Hayflick, L. 1965. Tissue cultures and mycoplasmas. Tex. Rep. Biol. Med. 23(Suppl. 1):285-303. 16. Henrichsen, J. 1972. Gliding and twitching motility of bacteria unaffected by cytochalasin B. Acta Pathol. Mi'crobiol. Scand. Sect. B 80:623-624. 17. Henrichsen, J. 1972. Bacterial surface translocation: a survey and a classification. Bacteriol. Rev. 36:478503. 18. Heunert, H. H. 1962. Methoden zur Verhinderung von Scharfenschwankungen bei Zeitrafferaufnahmen von Agarkulturen. Res. Film 4:382-387. 19. John, T. L., and E. C. Bovee. 1965. Movement and locomotion of microorganism. Annu. Rev. Microbiol. 19:21-58. 20. Morton, H. E., and R. J. Roberts. 1967. Production of anti-mycoplasma antibodies in rabbits. Proc. Soc. Exp. Biol. Med. 125:538-543. 21. Nelson, J. B. 1960. The behaviour of murine PPLO in HeLa cell cultures. Ann. N.Y. Acad. Sci. 79:450-457. 22. Nelson, J. B., and M. J. Lyons. 1965. Phase-contrast and electron microscopy of murine strains of mycoplasma. J. Bacteriol. 90:1750-1763. 23. Senterfit, L. B., and K. E. Jensen. 1966. Antimetabolic antibodies to Mycoplasma pneumoniae measured by tetrazolium reduction inhibition. Proc. Soc. Exp. Biol. Med. 122:786-790. 24. Sharp, J. T. (ed.). The role of mycoplasmas and L-forms of bacteria in disease. 1970. Charles C Thomas, Springfield, Ill. 25. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. Luduena, E. L. Taylor, J. T. Wrenn, and K. M. Yamada. 1971. Microfilaments in cellular and developmental processes. Science 171:135-143.
Vol. 129, No. 3 Printed in U.S.A.
Motility of Mycoplasma pneumoniae U. RADESTOCK AND W. BREDT1 * Institut fur Medizinische Mikrobiologie, Johannes Gutenberg-Universitat, D-6500 Mainz, Germany
Received for publication 26 July 1976
Cells of Mycoplasma pneumoniae FH gliding on a glass surface in liquid medium were examined by microscopic observation and quantitatively by microcinematography (30 frames per min). Comparisons were made only within the individual experiments. The cells moved in an irregular pattern with numerous narrow bends and circles. They never changed their leading end. The average speed (without pauses) was relatively constant between 0.2 and 0.5 ,um/s. The maximum speed was about 1.5 to 2.0 ,um/s. The movements were interrupted by resting periods of different lengths and frequency. Temperature, viscosity, pH, and the presence of yeast extract in the medium influenced the motility significantly; changes in glucose, calcium ions, and serum content were less effective. The movements were affected by iodoacetate, p-mercuribenzoate, and mitomycin C at inhibitory or subinhibitory concentrations. Sodium fluoride, sodium cyanide, dinitrophenol, chloramphenicol, puromycin, colchicin, and cytochalasin B at minimal inhibitory concentrations did not affect motility. The movements were effectively inhibited by anti-M. pneumoniae antiserum. Studies with absorbed antiserum suggested that the surface components involved in motility are heat labile. The gliding of M. pneumoniae cells required an intact energy metabolism, and the proteins involved seemed to have a low turnover. Motility of bacteria is, in most cases, dependent on the presence of flagella. Relatively few species are known to move by gliding (17, 19). Little is known about the mechanism of gliding and its relation to flagellar motility (17, 19). Among the gliding microorganisms discovered in recent years are three species of mycoplasmas: Mycoplasma pulmonis (1, 21, 22), M. pneumoniae (4, 6, 9), and M. gallisepticum (5). Gliding mycoplasmas deserve special consideration for several reasons. Their genetic content is smaller than that of normal bacteria (5 x 108 versus 1 x 109 to 2 x 109 daltons) (2). Moreover, their membrane is in direct contact with substrate, without an interfering cell wall. Therefore, mycoplasmas may serve as a useful model for studies on the mechanisms of gliding movements. Furthermore, all three motile mycoplasma species are pathogens of the respiratory tract of humans or animals (13, 24). Their ability to move by gliding may therefore be involved in the process of pathogenicity. Little is known about the movements of mycoplasmas. Only M. pneumoniae is a useful model for experimental studies, since its movements are fast and constant even after numerous passages on artificial media. In contrast, M. pulmonis ceases to move after a few pas' Present address: Zentrum fur Hygiene, D-7800 Freiburg, Germany.
sages in vitro (21, 22), and M. gallisepticum shows only slow movements (J. T. Liauw, M.D. thesis, Johannes Gutenberg University, Mainz, Germany, 1976). A few data were obtained from cinematographic studies on M. pneumoniae (10). However, no experimental system existed for the quantitation of movements. Therefore, it was the purpose of this study to develop a reliable method for the quantitation of mycoplasma gliding movements. By this method, information was obtained about the influence of environmental conditions and inhibitory substances on M. pneumoniae motility.
MATERIALS AND METHODS Mycoplasma. M. pneumoniae FH was grown in liquid medium (15), filtered through a polycarbonate filter (0.4 gm, Nucleopore, General Electric Co.), and stored in small portions at -70°C. After thawing, the suspension contained about 107 colonyforming units per ml. A nonmotile mutant of M. pneumoniae, M129P175, with reduced adherence was obtained from W. A. Clyde, Jr., Chapel Hill, N.C. It was grown in the same medium and used only for immunization of rabbits. Media. Liquid and agar media were prepared by the method of Hayflick (15). Penicillin (1,000 U/ml) and thallium acetate (0.05%) were added for most of the experiments. Agamma horse serum (Microbiological Associates) was used instead of horse serum (Flow Laboratories) for all microscopic experiments. For studies on the effect of increased viscosity, vary1495
1496
RADESTOCK AND BREDT
ing amounts of a 10% solution of gelatin in PPLO broth (Difco) were used instead of pure PPLO broth for preparation of the final medium. For the metabolic inhibition test, the medium contained 0.025% 2,3,5-triphenyl-tetrazolium chloride (Serva) and 1% glucose (23). Buffer. Tris(hydroxymethyl)aminomethanebuffered saline (8) was used in several experiments. Viscosity of media. The viscosity of the media used was determined by measuring the flow velocity (in seconds): water, 299; liquid medium, 329; medium with 1% gelatin, 502; medium with 3% gelatin, 984; and medium with 5% gelatin, 1,376. Chemicals. The following substances were used: Freund complete adjuvant (Difco), calcium chloride, magnesium chloride, sodium fluoride, tris (hydroxymethyl)aminomethane (Merck); chloramphenicol (Parke Davis); dimethyl sulfoxide (Roth); and puromycin, cholesterol, colchicin, cytochalasin B, 2,4dinitrophenol, ethylenediaminetetraacetic acid (EDTA), ethylenglycoltetraacetic acid (EGTA), actinomycin D, p-chloromercuribenzoate (PCMB), iodoacetate, and adenosine 5'-triphosphate (Serva). Metabolic inhibition test. The minimal inhibitory concentrations (MIC) of chemicals were determined by a modification of the 2,3,5-triphenyl-tetrazolium chloride reduction inhibition test (23). The test was read when the control wells without the test substance began to show formation of red formazan. The test medium contained 3% gelatin. Mycoplasmocidal test. Twofold dilutions of the test substances were prepared in Eppendorf plastic vials with medium containing 3% gelatin. A 490-,ul amount of each concentration was inoculated with 10 1,u of a preincubated (20 h, 37°C) M. pneumoniae suspension. A vial without test substance served as the control. The vials were incubated at 37°C. At 2-h intervals, a 10-,ul volume was removed from each test tube and immediately diluted in 100 ,ul of PPLO broth. With a calibrated platinum loop, three 1-,ul portions of each dilution were spread on agar media. Colonies were counted after 10 to 12 days of incubation. The concentration at which a distinct decrease occurred after 2 h was considered a mycoplasmocidal concentration (MCC). Antisera. Antisera against M. pneumoniae strains FH and M129P175 were prepared by immunizing rabbits with glass-grown organisms in Freund complete adjuvant (20). The antisera were stored at -20°C. Absorption of antiserum. M. pneumoniae FH was grown in four Roux bottles (100 ml of medium in each bottle), washed, and harvested in 4 ml of buffer. A 2-ml portion of this suspension was heated at 100°C for 1 h; 2 ml remained unheated. A 1-ml portion of each suspension (either heated or native) was centrifuged (7,000 x g), and the pellet was suspended in 400 ul of a 1:5-diluted anti-M. pneumoniae FH antiserum. The suspension was incubated for 1 h at 37°C and centrifuged, and the supernatant was -absorbed once more with the same amount of organisms. UV. The mycoplasmas grown in cover slip chambers were irradiated with ultraviolet light (UV) (254 nm) for 30 to 180 s at a distance of 8 cm (Sylvania lamp type TL 900).
J. BACTERIOL.
Determination of lectin-binding capacity of the medium. Concanavalin A was diluted in gelatin medium in a microtiter plate, and sheep erythrocytes were added. The agglutination titer was read after 2 h, and the lowest agglutinating concentration was considered to be just above the concanavalin A-binding capacity of the medium. Microscopic observation. A frozen suspension of M. pneumoniae was thawed and incubated at 37°C with constant shaking to avoid clumping. After 20 h this suspension was used to inoculate a 3% gelatin medium (1:1,000). The inoculated medium was placed in cover slip chambers (3). After 20 h of incubation, the mycoplasmas grown on cover slips were transferred to new chambers containing the test or control media. The chambers were incubated further at 37°C, and the movements of the cells were examined microscopically after 0.5, 1, 2, 4, 6, and 24 h. During examination, the chambers were kept at 37°C by a Sage Air Curtain incubator. Cinematographic studies. Cover slips with M. pneumoniae were prepared as described above and transferred to chambers containing the test or control medium, respectively. The control medium in these studies always consisted of medium with 3% gelatin (pH 7.2), incubated at 37°C. The two chambers were fastened to metal frames (18) with a wax mixture (Deckglaskitt, Merck) and incubated for 2 h at 37°C. After 2 h, seven series of cinematographic pictures were taken from each preparation. Each series covered a different field of view and lasted for 5 min. The frequency was 30 frames per min, and the exposure time was 0.5 s. The films were analyzed by projecting each frame onto paper, using a projector with single-frame projection (Siemens). The location of each cell was marked on the paper by a point, and the frame number was added. The process was repeated for each frame, resulting in 150 points per cell and series. The distances on the paper were measured, and the real distances in micrometers were calculated. From the distances and intervals obtained, the following data were calculated for each individual cell: total distance, total observation time, average speed, average speed without pauses (corrected speed), maximum speed, total time of movements, total time of resting, and percentage of resting periods. About 2,100 frames were examined for each experiment. Microscopic and photographic equipment. The movements of the cells were examined qualitatively with a Zeiss photomicroscope equipped with a phasecontrast condenser of long working distance (7 mm). The cinematographic studies were performed on a Zeiss inverted microscope (UPL) enclosed in a Plexiglas incubation chamber with electronic temperature regulation (Hessische Feinmechanik, Giessen). A phase-contrast objective with an optical aperture of 1.40 (x63) was used for all quantitative studies. Cinematography was performed with a cinematographic system from Wild, Herbrugg, Switzerland, consisting of a Bolex-Paillard 16-mm camera, variotimer, light meter, and heavy metal stand. Kodak 16-mm Plus-X reversal film was used for all experiments. Statistical test. Results of test and control preparations were examined for significant differences by
MOTILITY OF M. PNEUMONIAE
VOL. 129, 1977
the Wilcoxon test. Data were only compared within the same experiment.
RESULTS General aspects of motility. M. pneumoniae gliding cells always moved in the direction of their tip structure (Fig. 1). Their course was irregular and often circular with no preference for a certain direction. The movements were interrupted by resting periods of different lengths, ranging from 2 to more than 100 s. Under control conditions (medium with 3% gelatin, pH 7.2, 37°C), the cells showed average speeds of 0.2 to 0.5 ,um/s. The corrected speed (resting periods omitted) was higher and showed less variation (mostly between 0.3 and 0.5 ,um/s). The distances covered by the individual cells per 2-s interval were distributed around a mean value (Fig. 2). Occasionally, maximum speeds of 1.5 to 2.0 ,m/s were attained for a short time. The percentage of rest-
/
I
.j
V FIG. 1. M. pneumoniae cells on glass in liquid medium. Phase contrast. Bar represents 2 ,um. Arrow indicates tip structure.
ing periods varied widely among the cells (0 to 80%). Technique of cinematographic measurements. The system for cinematographic recording of M. pneumoniae cell movements produced reliable results under certain conditions. The frequency of 30 frames per min provided a sufficient number of measuring points to follow the often very narrow curves of the moving cells. Lower frequencies resulted in seemingly shorter, and therefore incorrect, distances. The number of cells per field of view was critical: with more than five to seven cells per field, collisions were likely to occur, which disturbed the follow-up of individual cells during analysis. An observation time of more than 5 min increased the chances of individual cells to leave the observation field, thereby reducing the number of cells with comparable observation time. Some of the data obtained from the measurements could be plotted graphically (Fig. 3), and possible differences could be examined statistically. Influence of environmental factors. Changes in the medium viscosity affected the movements significantly (Table 1). Higher viscosity reduced the speed and increased the percentage of resting periods. The latter was caused by more and extended individual pauses. A lower viscosity resulted in higher speed but did not change the percentage of pauses. The maximum speed of the cells was also affected by viscosity. The addition of 2% more gelatin reduced the maximum values about 0.1 ,um/s.
100 90-
(50 J
0
< 40-
1497
80-
70600~ 50CL 40z 3020 L) w 10
I
I 0
0 0
0 * a
000
0
Z30-
LL
O 20LL
E2 10z
w
0./4 5
6
I 7
.8 .9 1.0 1.1 1.2 (Dim)
FIG. 2. Typical distribution of the average speed (without pauses) (micrometers per 2-s interval) during the 300-s observation period. Bars represent three different cells. The number of intervals indicates the number of 2-s periods in which the indicated distance was covered.
o
00
08
*P%.
_
00
0.1 .2 .3 .4 .5 .6 0.1 .2 .3 .4 .5 .6 AVERAGE SPEED (,um/sec) FIG. 3. Results of quantitative experiments. (I) Without significant differences (identical media). (II) With significant differences (test medium, pH 6.5; control medium, pH 7.2). Symbols: 0, cells in test medium; 0, control cells. The average speed is that without pauses (i.e., corrected speed).
1498
RADESTOCK AND BREDT
J. BACTERIOL.
TABLE 1. Environmental factors affecting motility of M. pneumoniaea Resting period . . No. Avg speed (gm/s) anaCorrected speed (,gm/s) Expt condition lyzedM lyzed' Mean Range 2a 2 aS Mean Range Mean Range 1% Gelatin 9 0.41 0.35-0.49 0.01 0.46 0.44-0.51 0.01 11 3-27 5 0.35 0.32-0.37 0.41 0.38-0.45 13 5-15 Controlc
5% Gelatin Control
4 4
0.21 0.35
0.19-0.22 0.28-0.39
0.05
0.30 0.41
0.29-0.30 0.38-0.43
0.05
28 12
25-79 8-27
pH 6.5 Control
7 6
0.31 0.23
0.21-0.39 0.05-0.40
0.01
0.41 0.37
0.34-0.44 0.31-0.44
0.01
22 37
6-53 29-84
pH 7.9 Control
4 9
0.14 0.29
0.08-0.32 0.23-0.37
0.05
0.35 0.42
0.32-0.38 0.38-0.45
0.05
59 30
21-79 14-42
320C Control
6 7
0.27 0.39
0.19-0.34 0.33-0.42
0.01
0.35 0.42
0.27-0.45 0.37-0.45
0.01
23 8
6-56 2-9
390C Control
4 4
0.31 0.38
0.22-0.40 0.37-0.40
NSd
0.42 0.43
0.33-0.50 0.42-0.44
NS
9 24
3-13 2-41
Significance tested by the Wilcoxon text. b Number of cells analyzed (each cell was observed for 300 s). c Medium with 3% gelatin (pH 7.2); incubated at 370C. d NS, Not significant (2a _ 0.05). a
Motility in liquid medium without gelatin could not be studied quantitatively, because the moving cells lost contact with the glass and disappeared in the medium. Another factor affecting movement was the pH of the medium. At a more alkaline pH (7.9), the cells moved with reduced speed (Table 1). The percentage of resting periods was increased by both a higher number of pauses and increased length of the individual pause. Reduction of pH to 6.5 had the opposite effect. The movements were faster and the resting periods were shorter, resulting in a lower percentage of pauses (Table 1). At a lower incubation temperature (32°C), the speed was reduced significantly, and the percentage of pauses was increased (Table 1). This increase was caused by more frequent and prolonged pauses. At a higher temperature (39°C), the speed was not affected, but the percentage of resting periods was increased by more frequent and prolonged pauses. The addition of glucose (0.1 to 1%) did not significantly affect the speed of the cells. The percentage of resting periods was slightly increased by 1% glucose (Table 2). A reduced percentage of serum in the medium (10 or 5%) apparently affected only a minority of the cells. Most of the cells did not show any change in motility after 5 h, but about 10% were visibly damaged and immobilized. A reduction in the percentage of yeast extract (to 5%) affected the movements much more. After 1
TABLE 2. Various environmental factors and their effect on M. pneumoniae motility Expt condition Reduction of serum content (10 or 5%)
Effect on motility Most cells unaffected; signs of damage on '10% of
cells Reduction of yeast extract (5%)
No movements after 1 h
Addition of glucose, 0.1%
No effect Slight increase of pauses
Conditioned medium
No effect
Removal of Ca2+ (10 mM EGTA, 5 mM Mg2+)
No effect
Removal of Ca2+ and Mg2+ (5 mM EDTA)
Cells are rounded
swollen
and
h the cells slowed down or stopped gliding totally (Table 2). The removal of Ca2+ by EGTA did not significantly influence the motility. The addition of a Ca2+ and Mg2+ also had no effect on gliding. Removal of Mg2+ by EDTA resulted in visible, general damage (rounding, swelling) of most of the cells and inhibited movement (Table 2). Conditioned medium (preincubated with M. pneumoniae), cholesterol (5 to 20 ,ug/ml), and adenosine 5'-triphosphate (5 to 20 gg/ml) had no visible effect. Influence of inhibitory agents. The MICs and MCCs of the various substances tested are
VOL. 129, 1977
MOTILITY OF M. PNEUMONIAE
summarized in Table 3. The motility of the cells reacted differently to metabolic inhibitors. The MIC of dinitrophenol, NaCN, and NaF did not affect the movements. Higher concentrations were not tested, because the high MIC suggested a low effectiveness of these substances. Iodoacetate (MIC) significantly reduced the average speed and increased the percentage of pauses (Table 4) by more frequent resting periods. The MIC of PCMB increased the frequency and length of the resting periods and reduced the speed (Table 4). However, the number of cells available for analysis was too low for statistical tests. Concentrations higher than the MIC resulted in rounding and immobilization of the cells after about 1 h. Inhibitors of nucleic acid and protein syntheses showed different effects. Mitomycin C (0.5 MIC) resulted in lower speed and more resting time (Table 4). The frequency of pauses remained unchanged, and their length in-
creased. Actinomycin D and chloramphenicol did not show distinct effects at concentrations below the MCC within 2 to 4 h. Motility was reduced by 10 ,ug of chloramphenicol per ml after 24 h, but some of the cells were still moving. Puromycin at a concentration of 10 ,ug/ml did not affect the gliding movements. UV irradiation of the cells (3 min; dose, 900 ergs/mm2) resulted in slower movements within 6 h. No motile cells were seen after 24 h. X rays in a dose of 3,000 rads had no visible effect. Cytochalasin B at a concentration of 50 ,ug/ ml did not influence the movements. Colchicine (10 ,ug/ml) had no effect. Treatment of the membrane surface with homologous antiserum (Ab) inhibited the movements. Ab at a concentration of 1:400 reduced the motility after 1 h. Higher concentrations (1:50) immobilized the cells totally. The action of Ab was reversible. Transfer of antibodytreated cells (30 min, 1:50) to antibody-free medium restored motility to the normal level after about 2 h. Ab against the nonmotile mutant (M129P175) had the same effect on the cells as antiserum against the homologous strain FH. Absorption of the antiserum (1:50) with native M. pneumoniae removed all motility-inhibiting activity. However, antiserum absorbed with heated M. pneumoniae (100°C) showed full inhibition of movements at the same degree as unabsorbed antiserum. Treatment of the membrane surface with concanavalin A (free concentration, ca. 10 jig/ ml) did not affect the movements.
TABLE 3. MIC and MCC of various substances tested on M. pneumoniae Inhibitor acting on:
MCCa
_10.0 2.5 _10.0 -10.0 0.5
NTb 5.0 NT NT 1.0
0.3 5.0
1.25 10.0
3.0 10.0
25.0 NT
15.6
125.0 NT
Substance tested (Stg/ml) (JLgIml)
Energy metabolism
Dinitrophenol Iodoacetate NaCN NaF PCMB
Nucleic acid synthesis
Actinomycin D Mitomycin C
Protein synthesis
Chloramphenicol Puromycin
Contractile ments
Cytochalasin B Colchicine
ele-
MIC
-'10.0
1499
DISCUSSION Studies on mycoplasma gliding are much more difficult to perform than comparable experiments with other gliding bacteria, e.g.,
a Reduction of colony-forming units after 2 h. bNT, Not tested.
TABLE 4. Inhibitory substances significantly affecting motility of M. pneumoniae at or below MIC levelsa Avg speed
No. anaExptco condition n Expt lyzed b
Mean
(inm/s)
Range 0.17-0.26
(Mm/s)
2a_
Mean
Range
2a_
0
0.01
0.37 0.50
0.34-0.39 } 0.46-0.55 f
0-01
0.20 0.34
0.12-0.28 t NDd 0.28-0.40 f
0.39 0.41
0.38-0.41 I 0.40-0.43
ND
0.35
0.24-0.47
0.47
0.44-0.53
Iodoacetate, 2.5 mM Controlc
6 6
0.23 0.45
0.39-0.52
PCMB, 0.5 mM Control
2 5
Mitomycin C, 2.5 ug/
7
ml
Corrected speed
0.01
Resting periods Mean Range 37 26-52 15 3-16
43 7
31-56 5-31
13
2-28
23
3-54
? 0.01
Control 8 0.52 0.42-0.57 J 0.60 0.58-0.61 J a Significance tested by the Wilcoxon test. b Number of cells analyzed (each cell was observed for 300 S. c Medium with 3% gelatin (pH 7.2); incubated at 37°C. d ND, No statistical calculation done because of small number of cells.
1500 RADESTOCK AND BREDT myxobacteria (12, 17). Several factors are involved. Mycoplasmas are very small cells, they are fastidious, their movements can only be seen in a complex liquid medium, and mutants like those used in studies of myxobacteria (R. P. Burchard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G239, p. 70) do not exist or are rather difficult to select. These factors considerably limit the number of possible experiments, especially those about environmental factors. However, it is possible, to a certain extent, to quantitate the movements, as shown by the data presented. As indicated previously (9, 10), the measurements require a relatively high number of cinematographic pictures, and there are certain variations in the control data of the different experiments. However, the results appear reliable as long as adequate numbers of cells are measured and the data are compared only within the individual experiment. The movements of M. pneumoniae are somewhat different from those of other gliding bacteria, especially myxobacteria. Mycoplasmas do not move in swarms, but as single cells. Their moving pattern consists mainly of circles and narrow bends, and in contrast to myxobacteria they never change their leading end (6, 7). Only the speed of mycoplasmas and myxobacteria is comparable (10, 19). The factors that stimulate mycoplasma movement are unknown. The intense motility in densely grown cultures (9) seems to depend mainly on the lowered pH, as confirmed by the quantitative experiments. Yeast extract seems to contain an unknown substance necessary for constant movement. On the other hand, Ca2+ ions are not required. The failure to obtain significant effects by adding glucose may be due to the large amount of metabolizable substance in the complex medium. No evidence of a motility-stimulating factor was found in conditioned medium. Attempts to find a method for measuring possible chemotactic properties of mycoplasmas have failed so far in our laboratory. The effects of iodoacetate and PCMB on movement indicate a requirement for cell-derived energy. This is in agreement with results obtained by Burchard (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G239, p. 70), who found that PCMB inhibits the gliding of myxobacteria. There was no evidence for thermal energy being the sole cause for the gliding. Proteins participating in the gliding mechanism appear to have a relatively slow turnover. Inhibition of protein synthesis did not affect motility for several hours. This suggests that no substantial amounts of protein (e.g., as a gliding layer) are secreted during movement. Furthermore, de novo membrane synthesis
J. BACTERIOL.
does not seem to play an essential role in the gliding process. Factors effecting the cell division by interfering with deoxyribonucleic acid (DNA) synthesis (mitomycin C, UV) had a delayed effect on motility. However, the movements were inhibited much faster than observed with inhibitors of protein synthesis. Therefore, the effect of UV or mitomycin C cannot be explained by mere exhaustion of preformed substances. The results seem to suggest that intact DNA or DNA synthesis is necessary for the function of the motility mechanism. The data do not permit an explanation for this observation. Inhibitors of contractile elements (cytochalasin B, colchicin) (11, 14, 25) did not affect motility, confirming the results of preliminary experiments on M. pneumoniae (6) and M. gallisepticum (W. Bredt, unpublished data) and observations on other prokaryotes (16). However, this does not necessarily indicate that the motility mechanism is insensitive, since it is not known whether the membrane of prokaryotes is permeable to these substances. The problem of whether actin-like proteins are involved in the motility therefore has to be approached by other methods. Substances located on the membrane surface appear to play an essential role in motility, since treatment with antibodies resulted in fast inhibition of movement. The unimpaired effectiveness of antiserum absorbed with heated mycoplasmas indicates a heat-labile antigenic nature of the structures and substances involved. These are possibly proteins responsible for transport mechanisms and/or attachment. No antigenic difference seems to exist in this respect between the motile strain, FH, and the nonmotile mutant. Similar results have been obtained during studies on the adherence of M. pneumoniae (F. W. Gorski, M.D. thesis, Mainz, in preparation). The surface substances involved in attachment were heat labile and protease sensitive, and their reconstitution could be prevented by chloramphenicol. Whether the same substances are participating in both phenomena remains to be tested. The results obtained in this study provide a basis for further experiments. They show that motility is related to environmental factors. However, they provide only a partial and yet incoherent knowledge about the possible mechanisms. Further studies will hopefully reveal more about structures and mechanisms involved in the transformation of energy into movement. ACKNOWLEDGMENTS The work was supported by grant Br 296/8 of the Deutsche Forschungsgemeinschaft.
VOL. 129, 1977 The skillful technical assistance of Dagmar Donarski and Irmgard Thommes is gratefully acknowledged. We are indebted to W. A. Clyde Jr., Chapel Hill, N.C., for giving us the M. pneumoniae mutant obtained in his laboratory. We would like to thank H. J. Berger, Institute for Statistics and Documentation, Mainz, for statistical help, and J. Bereiter-Hahn, Institute for Cinematographic Cell Research, Frankfurt, for his help in analyzing the pictures. LITERATURE CITED 1. Andrewes, C. H., and F. V. Welch. 1946. A motile organism of the pleuropneumonia group. J. Pathol. Bacteriol. 58:578-580. 2. Bak, A. L., and F. T. Black. 1968. DNA base composition of human T-strain mycoplasmas. Nature (London) 219:1044-1045. 3. Borisy, G. G., and E. W. Taylor. 1967. The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. J. Cell Biol. 34:535-548. 4. Bredt, W. 1968. Growth morphology of Mycoplasma pneumoniae strain FH on glass surface. Proc. Soc. Exp. Biol. Med. 128:339-340. 5. Bredt, W. 1968. Motility and multiplication of Mycoplasma pneumoniae. A phase contrast study. Pathol. Microbiol. 32:321-326. 6. Bredt, W. 1972. Phase contrast studies on living mycoplasmas. Med. Microbiol. Immunol. 157:169. 7. Bredt, W. 1973. Motility of mycoplasmas. Ann. N.Y. Acad. Sci. 225:246-250. 8. Bredt, W. 1974. Structure and motility, p. 47-52. In J. M. Bove and J. F. Duplan (ed.), Mycoplasmas of man, animals, plants and insects. Institut National de la Sante et de la Recherche Medicale, Paris. 9. Bredt, W., and D. Bitter-Suermann. 1975. Interactions between Mycoplasma pneumoniae and guinea pig complement. Infect. Immun. 11:497-504. 10. Bredt, W., K. H. Hofling, and H. H. Heunert. 1970. Mycoplasma pneumoniae (Mycoplasmataceae): Bewegung, Vermehrung, Koloniebildung. Encyclopaedia cinematographica Film E 1633. G. Wolf (ed.),
Gottingen. 11. Bredt, W., K. H. Hofling, H. H. Heunert, and B. Milthaler. 1970. Messungen an beweglichen Zellen von
MOTILITY OF M. PNEUMONIAE 1501 Mycoplasma pneumoniae. Z. Med. Mikrobiol. Immunol. 156:39-43. 12. Burchard, R. P. 1974. Studies on gliding motility in Myxococcus xanthus. Arch. Microbiol. 99:271-280. 13. Freundt, E. A. 1974. Present status of the medical importance of mycoplasmas. Pathol. Microbiol. 40:155-187. 14. Furcht, L. T., and R. E. Scott. 1975. Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells. Exp. Cell Res. 96:271-282. 15. Hayflick, L. 1965. Tissue cultures and mycoplasmas. Tex. Rep. Biol. Med. 23(Suppl. 1):285-303. 16. Henrichsen, J. 1972. Gliding and twitching motility of bacteria unaffected by cytochalasin B. Acta Pathol. Mi'crobiol. Scand. Sect. B 80:623-624. 17. Henrichsen, J. 1972. Bacterial surface translocation: a survey and a classification. Bacteriol. Rev. 36:478503. 18. Heunert, H. H. 1962. Methoden zur Verhinderung von Scharfenschwankungen bei Zeitrafferaufnahmen von Agarkulturen. Res. Film 4:382-387. 19. John, T. L., and E. C. Bovee. 1965. Movement and locomotion of microorganism. Annu. Rev. Microbiol. 19:21-58. 20. Morton, H. E., and R. J. Roberts. 1967. Production of anti-mycoplasma antibodies in rabbits. Proc. Soc. Exp. Biol. Med. 125:538-543. 21. Nelson, J. B. 1960. The behaviour of murine PPLO in HeLa cell cultures. Ann. N.Y. Acad. Sci. 79:450-457. 22. Nelson, J. B., and M. J. Lyons. 1965. Phase-contrast and electron microscopy of murine strains of mycoplasma. J. Bacteriol. 90:1750-1763. 23. Senterfit, L. B., and K. E. Jensen. 1966. Antimetabolic antibodies to Mycoplasma pneumoniae measured by tetrazolium reduction inhibition. Proc. Soc. Exp. Biol. Med. 122:786-790. 24. Sharp, J. T. (ed.). The role of mycoplasmas and L-forms of bacteria in disease. 1970. Charles C Thomas, Springfield, Ill. 25. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. Luduena, E. L. Taylor, J. T. Wrenn, and K. M. Yamada. 1971. Microfilaments in cellular and developmental processes. Science 171:135-143.
