Dec 30, 1985 - This enzyme was found in Acholeplasma laidlawii to be associated with Na+ extrusion (6). As mycoplasmas depend on the external supply of ...
Vol. 167, No. 3
JOURNAL OF BACTERIOLOGY, Sept. 1986, p. 1089-1091
0021-9193/86/091089-03$02.00/0 Copyright © 1986, American Society for Microbiology
Changes in Membrane Lipid Composition of Mycoplasma capricolum Affect the Cell Volume NINO ROMANO,t MITCHELL H. SHIRVAN, AND SHLOMO ROTTEM* Department of Membrane and Ultrastructure Research, The Hebrew University-Hadassah Medical School, Jerusalem 91010, Israel Received 30 December 1985/Accepted 21 May 1986
The cellular water volume of Mycoplasma capricolum was markedly increased by a decrease in the cholesterol-to-phospholipid molar ratio in the membrane. An increase in cell volume was also observed with the increase in the phospholipid cell membrane content obtained by the incorporation of exogenous phosphatidylcholine from the growth medium.
In most bacteria, cell volume is maintained by the rigidity of the cell wall (20). In the absence of a rigid cell wall (13), mycoplasmas are faced with the problem of regulating their cell volume. Electrolytes (mainly Na+ and Cl-) and water diffuse into the cell because of the colloid osmotic effect (20). This influx must be counteracted by the extrusion of ions (and water) by ion pumps (19, 20). The composition and physical state of mycoplasma membrane lipids are important factors in determining the rate of the passive permeability of ions (11, 18), as well as the activity of the membrane-bound Mg2 -ATPase (11). This enzyme was found in Acholeplasma laidlawii to be associated with Na+ extrusion (6). As mycoplasmas depend on the external supply of lipids, controlled changes in membrane lipid composition can be induced (15). In the present study we attempted to determine whether altering membrane lipid composition produces changes in cell volume. Mycoplasma capricolum is well suited for such a study. This organism requires cholesterol for growth but can be adapted to grow with low concentrations of cholesterol (3). Moreover, the phospholipid composition and content of this organism can be modified by the incorporation of exogenous phospholipids from the growth medium (5). M. capricolum. The California kid strain of M. capricolum was grown in Edward basal medium (4) supplemented with 0.5% delipidated bovine serum albumin-palmitic and oleic acid (10 ,ug of each per ml)-cholesterol (2 to 20 ,ug/ml). In some experiments, egg-phosphatidylcholine (egg-PC) (Sigma Chemical Co., St. Louis, Mo.) was added to the growth medium for a final concentration of 5 to 20 ,ug/ml. All lipid ingredients were added to the medium as an ethanolic solution. The final concentration of ethanol in the growth medium did not exceed 0.5%. The medium was treated with 0.1% of a frozen inoculum. The cultures were incubated at 37°C for 12 to 26 h, and growth was followed by measuring the A640 of the culture. Cultures were harvested by centrifugation at 12,000 x g for 20 min. To measure the cell volume, cell pellets were suspended in unsupplemented basal medium and kept at 4°C. Before chemical analyses, the cells were washed twice and suspended in a 0.25 M NaCl solution. Membranes were obtained by osmotic lysis of the organisms (12). *
Cells at a final concentration of 1 to 2 mg of cell protein per ml were incubated in 2.2 ml of the basal medium containing 3H-labeled water (0.62 [tCi/ml; New England Nuclear Corp., Boston, Mass.), ['4Clpolyethylene glycol (molecular weight, 4,000; 0.061 ,uCi/ml; New England Nuclear), and 100 pug of unlabeled polyethylene glycol per ml for 5 min at 37°C. Samples of 1 ml were withdrawn and pipetted onto the surface of 0.5-ml silicone oil {550:556 grade (12:13 [vol/vol]); Dow Corning Corp., Midland, Mich.} in 1.5-ml plastic microfuge tubes and centrifuged in an Eppendorf microfuge for 2 min. Under these conditions, the cells passed through the silicone oil forming a pellet at the bottom of the tube while the aqueous phase remained above the oil. Radioactivity in the cell pellet and aqueous phase were then determined. We used 3H20 to measure the total pellet water and ['4C]polyethylene glycol to measure the intercellular space in the cell pellet. The water space minus the polyethylene glycol space was taken as the intracellular water volume. All intracellular water volume experiments were repeated three to five times with various batches of cells. Although there was some variability (up to 30%) in the water volume measurements among the various experiments, within each experiment the cell water volume was affected by the variables (i.e., age, cholesterol, or phosphatidylcholine [PC] content) in the same manner. The results presented are therefore those of a representative experiment. The cell water volume in such experiments was measured in triplicate, and the results presented are the mean of the three measurements. The variations among the triplicates within the same experiment were not greater than +7%. Protein in cell and membrane preparations was estimated by the method of Lowry et al. (10). The viability of the cell suspensions was measured by the colony-counting technique (2). Lipids were extracted by the method of Bligh and Dyer (1) and analyzed as previously described (16). For electron paramagnetic spin resonance measurements, isolated membranes were labeled with 5-doxylstearate (Syva, Palo Alto, Calif.) and analyzed in a Varian E4 spectrometer as described before (17). The molecular motion is reported as the order parameter (S), which is related to the mean angular deviation of the spin-labeled fatty acid chain from its average orientation in the membrane (4). The maximum value for S is 1.0 for perfect order, while complete disorder yields an S value of 0. Low values of the order parameter are associated with higher freedom of motion. The order parameter was calculated by the method of Gaffney (4), and its repeated
Corresponding author.
t Present address: Instituto d'Igiene dell' Universita di Palermo,
90141 Palermo, Italy.
1089
1090
NOTES
J. BACTERIOL. TABLE 2. Effect of cholesterol incorporation on the cell volume of M. capricolum cellsa
0.6
0.5
E 0
Cholesterol in medium
0.4
(p.g/ml)
0.3
2 5 10 20
Cholesterol Cholesterol Cholesterol-toOrder ol/mg of phospholipid parameter cell protein) ratio (mol/mol) (S)
cr w -J
11 23 40 47
0.22 0.46 0.75 0.94
0.54 0.61 0.70 0.74
~~~~~~Cell water vol RI/mg p1/10' of
protein 4.1 3.6 3.2 3.0
CFU
2.6 2.1 1.5 1.4
aCells were grown to the mid-exponential phase of growth (A6Q0 = 0.2 to 0.3) in a medium containing palmitic and oleic acid (10 ,ug of each per ml) and various concentrations of cholesterol. Lipid, cell water volume, electron paramagnetic spin resonance, and CFU were determined as described in the
0
0.2
text. w
4
0.11
INCUBATION TIME ( h) FIG. 1. Effect of cholesterol and egg-PC on the growth of M. capricolum. Cells were grown in a medium containing 0.5% delipidated bovine serum albumin and palmitic and oleic acids (10 jig of each per ml) with (M, A, *) or without (O, A, 0) 20 ,ug of egg-PC per ml and the following concentrations of cholesterol: O and M, 2 R±g/ml; A and A, 5 Rg/ml; 0 and *, 20 ,ug/ml.
determinations on a given membrane preparation yielded a standard deviation of ±0.01. Membrane lipid composition of M. capricolum can be altered in a controlled manner. This organism requires fatty acids and cholesterol for growth (15) and, aside from its de
novo-synthesized phospholipids (mainly phosphatidylglycerol and diphosphatidylglycerol), can incorporate amounts of exogenous phospholipids added to the growth medium (5). The membrane lipid composition and the content of M. capricolum can therefore be manipulated by the cultivation of cells with various concentrations of cholesterol and by the addition of exogenous egg-PC to the growth medium. Growth curves for M. capricolum cells grown with various cholesterol-PC and egg-PC concentrations are shown in Fig. 1. In the absence of egg-PC, reasonable TABLE 1. Changes in cell volume upon aging of M. capricolum cellsa Age of culture
Cell protein
mg/100 ml of
Cell water vol
FI/ml
of cell
Aio
mg/1011 mg/iJ
time (h)
culture
CFU
protein
CFU
12
0.15 0.24 0.43 0.45 0.48
2.0 3.7 5.4 6.1 7.1
4.4 4.6 4.8 5.4 33.2
2.9 3.0 2.7 3.7 1.1
1.3 1.4 1.3 2.0 3.8
Incubation tincubatio
16 21 24 26
A64
FLI/1010 C.110U
a Cells were grown in a medium containing 0.5% delipidated bovine serum albumin, palmitic and oleic acids (10 ,ug of each per ml), and cholesterol (20 pg/ml). Cell water volume, protein, and CFU were determined as described in
the text.
growth was obtained even with the low cholesterol concentration (2 ,uglml), but the growth was slower. The ability of M. capricolum to grow with very low cholesterol concentrations was reported previously (3). In the presence of egg-PC, however, growth was markedly inhibited at the low concentration of cholesterol but enhanced in high-cholesterolcontaining media. As it is most likely that the egg-PC and cholesterol, added simultaneously as an ethanolic solution to the growth medium, forms cholesterol-egg-PC vesicles, it seems that the growth inhibition and enhancement obtained with M. capricolum are associated with the concept that the ability of lipid vesicles to serve as cholesterol donors is dependent on the cholesterol-to-phospholipid ratio (7). The water volumes of M. capricolum cells harvested at the various growth phases are presented in Table 1. Throughout the exponential phase of growth, the cell water volumes were constant. Almost identical values were obtained with cells harvested and suspended in the growth medium, cells fixed with 0.25% glutaraldehyde before they were harvested, and cells whose water volumes were measured before cultures were harvested (not shown). The cell water volume of stationary-phase cells was markedly higher than that of cells from the exponential phase of growth (Table 1). This manifestation of aging may be associated with alte'rations in the composition and physical properties of the cell membrane (13). Such changes may result in altered permeability or a decrease in transport activities that may lead to cell swelling. In the late-stationary phase, cell water volumes were dramatically decreased, apparently due to cell lysis. Varying the cholesterol and egg-PC concentrations in the growth medium has pronounced effects on the chemical composition of M. capricolum membranes (3, 5). Changes in the cholesterol content of the cell membrane were obtained when cells were grown with various concentrations of cholesterol (Table 2). The cholesterol-to-phospholipid molar ratio in the membranes was increased from 0.22 to 0.94 by an increase of the cholesterol concentration in the growth medium from'2 to 20 ,ug/ml. This increase resulted in a pronounced decrease in membrane fluidity and an increase in the cell water volume. The decrease in membrane fluidity was indicated by the increase in the order parameter of the 5-doxylstearate incorporated into the membranes. When the cells were grown in a medium supplemented with egg-PC (Table 3), the total membrane phospholipid content was increased by over 50% because of the incorporation of the egg-PC into the membrane with a concomitant increase in cell water volume. The cholesterol-to-phospholipid ratio in the PC-rich membranes was almost identical to that in membranes from cells grown without egg-PC, yet the order
NOTES
VOL. 167, 1986 TABLE 3. Effect of PC incorporation on the cell volume of M. capricolum cellsa Phospholipid PCPin in PC content medium incorporated (con tf (,ig/ml) ( of total) Mnmollmgof cell protein)
0 5 10 20
55 68 83 88
2.0 22.0 30.0 36.0
Order
Order
parameter
Cell . water vol
Il/mg of
.,l/1lo
CFU
protein
0.74 0.70 0.64 0.62
2.7 2.8 3.7 3.6
1.3 1.4 1.8 1.9
a Cells were grown in a medium containing 0.5% delipidated bovine serum albumin, palmitic and oleic acids (10 ,ug of each per ml), cholesterol (20 jig/ml), and various Concentrations of PC. The cells were harvested at the mid-exponential phase of growth (A6, = 0.2 to 0.3). Lipids, cell water volume, electron paramagnetic spin resonance, and CFU were determined as described in the text.
parameter of the membrane lipids in the PC-rich membranes was lower, apparently due to the increase in the lipid-toprotein ratio in the membrane (17). Our results show that changes in M. capricolum membrane lipids induced by either varying the cholesterol-tophospholipid molar ratio or increasing the lipid-to-protein ratio in the membrane result in significant changes in the cell water volume. The increase in the cell water volume may be due to changes in the surface area of the cell membrane or associated with changes in the physical state of the membrane lipids. The freedom of motion of membrane lipids was shown to affect ion (and water) influx rates (8, 18), as well as ion pumps (6, 9, 11). The critical reading of the manuscript by David Mannock and the secretarial help of Dawn Oare and Chana Neumann are greatly appreciated. LITERATURE CITED 1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 2. Butler, M., and B. C. J. G. Knight. 1960. The survival of washed suspensions of mycoplasma. J. Gen. Microbiol. 9:379-383. 3. Dahl, J. S., C. E. Dahl, and K. Bloch. 1980. Sterols in membranes: growth characteristics and membrane properties of Mycoplasma capricolum cultured on cholesterol and lanosterol. Biochemistry 19:1467-1472.
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4. Gaffney, B. J. 1975. Fatty acid chain flexibility in the membranes of normal and transformed fibroblasts. Proc. Natl. Acad.
Sci. USA 72:664-668. 5. Gross, Z., S. Rottem, and R. Bittman. 1982. Phospholipid interconversions in Mycoplasma capricolum. Eur. J. Biochem. 122:169-174. 6. Jinks, D.C., J. R. Silvius, and R. N. McElhaney. 1978. Physiological role and membrane lipid modulation of the membranebound (Mg+,Na+)-adenosine triphosphatase activity in Acholeplasma laidlawii. J. Bacteriol. 136:1027-1036. 7. Kahane, I., and S. Razin. 1977. Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to mycoplasma membranes. Biochim. Biophys. Acta 471:32-38. 8. Le Grimellec, C., and G. Leblanc. 1978. Effect of membrane cholesterol on potassium transport in Mycoplasma mycoides var. capri (PG 3). Bi6chim. Biophys. Acta 514:152-163. 9. Le Grimellec, C., and G. Leblanc. 1980. Temperature-dependent relationship between K+ influx, Mg'-ATPase activity, transmembrane potential and membrane lipid composition in mycoplasma. Biochim. Biophys. Acta 599:639-651. 10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 11. McElhaney, R. N. 1982. Effect of membrane lipids on transport and enzymatic activities. Curr. Top. Membr. Transp. 17:317-380. 12. Razin, S. 1963. Osmotic lysis of mycoplasma. J. Gen. Microbiol. 33:471-475. 13. Razin, S. 1978. The mycoplasmas. Microbiol. Rev. 42:414-470. 14. Razin, S., and S. Rottem. 1976. Techniques for the manipulation of mycoplasma membranes, p. 3-26. In A. Maddy (ed.), Biochemical analysis of membranes. Chapman & Hall, Ltd., London. 15. Rottem, S. 1980. Membrane lipids of mycoplasmas. Biochim. Biophys. Acta 604:65-90. 16. Rottem, S., and 0. Markowitz. 1979. Membrane lipids of Mycoplasma gallisepticum: a disaturated phosphatidylcholine and phosphatidylglycerol with an unusual positional distribution of fatty acids. Biochemistry 118:2930-2935. 17. Rottem, S., and A. Samuni. 1972. Effect of proteins on the motion of spin labelled fatty acids in mycoplasma membranes. Biochim. Biophys. Acta 298:32-38. 18. Rottem, S., and A. J. Verklei. 1982. Possible association of segregated lipid domains of Mycoplasma gallisepticum membranes with cell resistance to osmotic lysis. J. Bacteriol. 149:338-345. 19. Wilson, T. H. 1954. Ionic permeability and osmotic swelling of cells. Science 120:104-105. 20. Wilson, T. H., C. Linker, and S. R9ttem. 1984. Volume regulation in Mycoplasma gallisepticum. Isr. J. Med. Sci. 20:800-802.
