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The uptake and modification of exogenous phosphatidylcholine (PC) by several Mycoplasma and Spiroplasma species was investigated. While in most Mycoplasma species and in all Spiroplasma species tested the PC appears to be incorporated unchanged from the growth medium, the PC of M. gallisepticum, M. pulmonis, and M. pneumoniae was disaturated PC, apparently formed by modification of 1-saturated-2-unsaturated PC from the growth medium. The modification of the exogenous PC by M. gallisepticum was inhibited by chloramphenicol under conditions that did not affect de novo synthesis of phosphatidylglycerol. A low activity of an endogenous phospholipase A was detected in native M. gallisepticum membranes. The activity was markedly stimulated by treating the membranes with low concentrations of the nonionic detergents. The PC modification was affected by the fatty acid composition of the exogenous PC species. Diunsaturated, 1-saturated-2-unsaturated, and 1-unsaturated-2-saturated PCs were modified to various extents, whereas the disaturated dipalmitoyl PC (DPPC) was not. Both modified and unmodified PCs were incorporated by the cells, but the unmodified DPPC was incorporated at a lower rate and to a lesser extent. The possibility that the incorporation of DPPC into M. gallisepticum cells is associated with the formation of intracytoplasmic membranes is discussed.

Stopped-flow kinetic measurements of the association of filipin with sterols in intact cells and isolated membranes of Mycoplasma capricolum were used to study the effects of varying the phospholipid in the membrane. The phospholipid composition and content of the membrane were varied by growing cells in an albumin-containing medium with cholesterol, palmitic and oleic acids, and various concentrations of exogenous phospholipids. The exogenous phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidic acid) were incorporated up to levels of approximately 50% of the total membrane phospholipids but had no effect on the distribution of cholesterol between the two halves of the membrane bilayer. The sterol structure was varied by growing the cells with 10/micrograms/mL of either cholesterol, beta-cholestanol, 4,6-cholestadien-3 beta-ol, ergosterol, beta-sitosterol, or stigmasterol. With cholesterol, beta-cholestanol, and 4,6-cholestadien-3 beta-ol, approximately 65% of the sterol was found to be present in the outer half of the lipid bilayer. With ergosterol, beta-sitosterol, and stigmasterol, about 89% of the sterol is localized in the outer half of the membrane bilayer. Thus, the behavior of the alkyl-substituted sterols differs from that of cholesterol. The extent to which a sterol is distributed asymmetrically between the two halves of the bilayer is not related to the extent to which maximum growth is produced. These results suggest that growth-supporting sterols need not be translocated extensively.

Mycoplasma capricolum cells increase their phospholipid content by incorporating exogenous phospholipids from the growth medium. Growing the cells in media with increasing serum concentrations resulted in a massive incorporation of phosphatidylcholine and sphingomyelin (up to about 50% of total phospholipids) into the cell membrane. The incorporation of the exogenous phospholipids had essentially no effect on the rate of cell growth and did not decrease the overall phospholipid biosynthesis of the cells. Thus, the ratio of phospholipid to protein in membranes from cells grown with 5% horse serum was 0.5 (mumol/mg) compared to 0.3 (mumol/mg) in cells grown without serum, and the relative content of charged polar lipids was apparently decreased. The consequence of the incorporation of exogenous phosphatidylcholine was an alteration in the relative amount of the major end-products of the de novo phospholipid biosynthesis; a marked increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol was observed. The possibility that the increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol is part of a control mechanism to maintain a mixture of bilayer and non-bilayer lipids is discussed.

The physical state of the lipids in Mycoplasma capricolum membranes was studied by differential scanning calorimetry. Cells grown in the presence of horse serum incorporated large amounts of cholesterol esters into their membranes. After incubation at a low temperature, the cholesterol ester-containing membranes showed an endotherm characteristic of a cholesterol ester transition from a crystalline state to an isotropic liquid that was identical in membranes both before and after thermal protein denaturation. This transition was not observed in membranes of cells grown in medium in which the horse serum was replaced by bovine albumin, fatty acids, and unesterified cholesterol unless cholesterol esters were added to the growth medium. In membrane preparations obtained from both horse serum-grown cells and from cells grown with bovine albumin plus cholesterol and fatty acids, the free cholesterol content was sufficient to eliminate the bilayer order/disorder transition observed in isolated membrane phospholipids. Our calorimetric studies indicate that the majority of cholesterol esters in M. capricolum membranes is not present in attached serum lipoprotein particles nor is it intimately associated with membrane protein but exists as relatively large droplets of cholesterol ester or as pockets in the membrane. The cholesterol esters in these pockets exist in a liquid-like state at growth temperature and appear to be relatively pure, although the presence of small amounts of other membrane components, especially glycerides, is likely. The existence of a low-temperature endotherm in membrane attributable to glycerides suggests there may be glyceride-rich regions in the membranes.

Plasma membranes of Mycoplasma laidlawii and Mycoplasma gallisepticum were solubilized by several ionic and non-ionic detergents. Solubilization of the membranes by sodium dodecyl sulfate (SDS) separated membrane lipid from protein as demonstrated by polyacrylamide gel electrophoresis of the solubilized material. The solubilized membrane material reaggregated spontaneously on removal of the detergent by dialysis or by Sephadex G-25, and formed vesicles limited by a triplelayered membrane of about the same thickness as the original Mycoplasma membrane. A divalent cation (e.g., Mg2+) was essential for membrane reassembly. The ratio of lipid to protein in membrane reaggregates varied considerably according to the Mg2+ concentration. At a low Mg2+ concentration reaggregates contained a higher percentage of lipid. The present results seem to bear out the suggestion of Engelman et al. [Biochim. Biophys. Acta135, 381 (1967)] that the SDS-solubilized membrane material does not consist of homogeneous lipoprotein subunits but of separate SDS-lipid and SDS-protein complexes. The reassembly of solubilized membrane lipid and protein on removal of the detergent indicates that these components contain sufficient structure-determining information to interact spontaneously in the presence of Mg2+ and produce membraneous structures.

The electron paramagnetic resonance (EPR) spectra of spin labeled fatty acid derivatives (1 (m,n)) in Mycoplasma laidlawii membranes showed a steep temperature dependence. The hyperfine splitting ( 2T m ) of these spectra decreased as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives, demonstrating an increase in molecular motion of the nitroxide radical. The freedom of motion of the spin label I (12.3) in M. laidlawii membranes was higher in membranes containing cis-Δ 9 - octadecenoic acid than in membranes containing the corresponding trans isomer. A high freedom of motion was also observed in Mycoplasma membrane reggregates having a high lipid to protein ratio. Upon increasing the relative amount of protein in the reaggregates a decrease in the freedom of motion of the nitroxide radical was found. The freedom of motion of the spin label I (12,3) in reaggregates formed at a high Mg2+ concentration ( >20 mM ) was similar to that of the native membranes ( 2T m = 62.0 gauss ). Throughout the growth cycle of M. laidlawii a high freedom of motion of spin label 1 (12,3) was found in cell membranes from the early growth phase ( 2T m = 58.0 gauss ). These membranes had a low density ( d = 1.165 ) due a high amount of oleic acid in membrane polar lipids. Decreasing the growth temperature of the cells resulted in an increase in the amount of [14C]oleic acid and a decrease in the amount of [3H]cholesterol incorporated into the cell membrane. The freedom of motion of spin label 1 (12,3) in membranes from M. laidlawii cells grown at 15° was much higher ( 2T m − 58.0 gauss ) than that in membranes from cells growtn at 37° ( 2T m = 62.5 gauss ) when compared at the same temperature.

Stable co-aggregates of solubilized mycoplasma membranes and extraneous proteins (bovine serum albumin and bacterial penicillinase) have been constructed. The penicillinase preparation was derived from the culture supernatant of Bacillus cereus strain 569/H and incorporated in a membrane-like structure formed by solubilization and reaggregation of membranes of Acholeplasma laidlawii (formerly Mycoplasma laidlawaii ). In the presence of methicillin the co-aggregated enzyme showed lower In the presence of methicillin the co-aggregated enzyme showed lower thermostability and considerably higher resistance to inactivation by iodine. Antibodies to the soluble enzyme caused partial inhibition of the activity of the co-aggregated enzyme while antibodies to A. laidlawii membrane proteins had no effect even at concentrations sufficient to coat the co-aggregate. Partial removal of lipids from the co-aggregate restored the original characteristics of the enzyme. The properties of the co-aggregated exopenicillinase were compared with those of the native enzyme and of a membrane-bound variant of penicillinase isolated from the exopenicillinase-producing cells.

1. 1. Serial passages of the sterol-requiring Mycoplasma mycoides var. capri in a serum-free medium supplemented with decreasing concentrations of cholesterol resulted in the adaptation of the organisms to grow with no added cholesterol. The cells of the adapted strain were osmotically more fragile than cells of the native strain and were more permeable to erythritol. 2. 2. The cell membrane of the adapted strain contained low amounts of cholesterol (up to 3% of the total membrane lipid as against 22–25% in the native strain) but its polar lipids were more saturated than those of the native strain, as reflected by the preferential incorporation of [14C]palmitic acid when added to the growth medium together with [14C]oleic acid. 3. 3. The native strain was capable of growing at temperatures as low as 25°C, whereas the adapted strain could not. The lowering of the growth temperature of the native strain resulted in a decrease in the cholesterol content of the membrane (from 24–15% of the total lipid) but had no effect on the fatty acid composition of the membrane polar lipids. On the other hand, aging of the culture increased the ratio of saturated to unsaturated fatty acids in both adapted and native strains, and decreased the cholesterol content of the native strain. 4. 4. Freeze-etching of cells of the native and adapted strains, kept at 37°C prior to freezing, showed a random distribution of particles on the fracture faces of the cell membranes. Keeping the cells at 4°C prior to freezing caused the aggregation of particles on the fracture faces of the adapted strain but had no effect on the distribution of particles in the native strain. 5. 5. Our data support the thesis that cholesterol functions as a regulator of membrane fluidity and that changes in the fatty acid composition of membrane lipids may act to compensate for the lack of cholesterol.

Circular dichroism analyses of Acholeplasma laidlawii membranes solubilized by sodium dodecyl sulfate showed a typical α-helix spectrum. The estimated α-helix content was of the order of 35% calculated from the ellipticity at 208 nm of membranes solubilized by 20 mM SDS. Reaggregation of the solubilized membrane material to a membrane-like structure resulted in a distorted spectrum with low amplitude and red-shifted extrema like that of the native membranes. Throughout the growth cycle the circular dichroism spectra of membrane proteins remained the same despite the marked differences in membrane densities. The optical activity of the membranes was not affected by changing the lipid composition or extraction of over 90% of the lipids, although the latter resulted in marked destabilization of the proteins.

Thin sections of Spiroplasma citri , a mycoplasma-like organism isolated from citrus infected with “Stubborn” disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm 3 . Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 μg/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily.

Spin-labeled fatty acids have been incorporated as structural probes into Acholeplasma laidlawii and Mycoplasma hominis native membranes and into pronase-treated membranes from which up to 75% of the protein had been removed. The electron paramagnetic resonance (EPR) spectra showed the mobility of the spin labels in the native and pronase-digested membranes to be temperature dependent, increasing as the nitroxide radical is moved away from the polar head group of the fatty acid. The changes were more pronounced in A. laidlawii than in M. hominis membranes. The mobility of the spin-labeled fatty acids in the pronase-digested membranes increased with protein digestion which was accompanied by a decrease in the isopycnic density of the membranes. On binding lysozyme or cytochrome c to the pronase-digested membranes, the mobility of the spin-labeled fatty acids decreased with a corresponding increase in membrane density. These changes were almost completely reversed when the soluble proteins were removed by 1 M NaCl. Our findings indicate that membrane proteins, including those bound electrostatically to membrane lipids, influence the physical state of membrane lipids.

Acyl carrier protein (ACP) activity was determined by the malonyl-coenzyme A–CO 2 exchange reaction. It was highest in Acholeplasma laidlawii , lower in A. granularum , and lowest in A. axanthum . The sterol-requiring Mycoplasma species examined showed little or negligible ACP activity. A. laidlawii was capable of utilizing pantetheine or coenzyme A but not β-alanine as precursor for ACP synthesis. Its ACP could thus be labeled by growing the organisms with radioactive coenzyme A. The ACP of A. laidlawii appears to be a soluble cytoplasmic protein, which could be purified about 40-fold by treatment of the cytoplasmic fluid with streptomycin sulfate and chromatography of the supernatant fluid on a Biogel P-10 column. Its molecular weight, determined by polyacrylamide gel electrophoresis, is low (about 10,900) resembling that of Escherichia coli , but it is much more sensitive to heat.

Mycoplasma gallisepticum cells were found to contain two different sugar transport systems, one for d -glucose and α-methyl- d -glucoside (α-MG) and the other for d -mannose and d -fructose. Both systems were noninducible, stereospecific, dependent on temperature and p H, and sensitive to sulfhydryl-blocking reagents. The rate of sugar uptake depended on its external concentration, obeying Michaelis-Menten kinetics. The sugar accumulated in the cells against a concentration gradient, and an energy requirement for accumulation was demonstrated with α-MG. Both transport systems thus meet the criteria of active transport. The exit of α-MG from the cells, like its entry, depended on temperature and was accelerated by energy supplied by the oxidizable d -mannose. d -Glucose accelerated α-MG exit, apparently by an exchange reaction. A method for measuring the intercellular space and intracellular free-water volume of Mycoplasma was devised, and several of its applications are described.

Phospholipase C from Bacillus cereus failed to attack the phospholipids of intact cells or isolated membranes of Mycoplasma hominis, but readily hydrolyzed the extracted phospholipids dispersed in water. Subjection of the membranes to prolonged ultrasonic irradiation, alternate freezing and thawing, or low concentrations of detergents did not render the phospholipids susceptible to the enzyme action. 1. 2. The removal of part of the membrane proteins by proteolysis enzymes enabled phospholipase C to hydrolyze membrane phospholipids. The binding of lysozyme to the protein-depleted membranes interfered with phospholipidase C action. 2. 3. A significant part of the phospholipids of reaggregated M. hominis membranes could be hydrolyzed by phospholipase C, indicating that the reaggregated membranes differ from the native membranes in organization. 3. 4. An endogenous phospholipase A activity, hydrolyzing both acyl ester bonds in phosphatidylcholine, was found in M. hominis membranes. The enzyme(s) was (were) neither activated by Ca2+ nor inhibited by EDTA. It was markedly inhibited by p- chloromercuribenzoate and detergents and was completely destroyed by heating at 70°C for 10 min. 4. 5. It is suggested that the phospholipids in native M. hominis membranes are masked, probably by membrane proteins, and are thus protected from hydrolysis by exogenous phospholipase C. The membrane phospholipids may, however, be degraded by an endogenous, membrane-bound phospholipase A, which can exert its activity even at temperatures as low as −20°C.

Sendai and influenza virions are able to fuse with mycoplasmata. Virus-Mycoplasma fusion was demonstrated by the use of fluorescently labeled intact virions and fluorescence dequenching, as well as by electron microscopy. A high degree of fusion was observed upon incubation of both virions with Mycoplasma gallisepticum or Mycoplasma capricolum. Significantly less virus-cell fusion was observed with Acholeplasma laidlawii, whose membrane contains relatively low amounts of cholesterol. The requirement of cholesterol for allowing virus-Mycoplasma fusion was also demonstrated by showing that a low degree of fusion was obtained with M. capricolum, whose cholesterol content was decreased by modifying its growth medium. Fluorescence dequenching was not observed by incubating unfusogenic virions with mycoplasmata. Sendai virions were rendered nonfusogenic by treatment with trypsin, phenylmethylsulfonyl fluoride, or dithiothreitol, whereas influenza virions were made nonfusogenic by treatment with glutaraldehyde, ammonium hydroxide, high temperatures, or incubation at low pH. Practically no fusion was observed using influenza virions bearing uncleaved hemagglutinin. Trypsinization of influenza virions bearing uncleaved hemagglutinin greatly stimulated their ability to fuse with Mycoplasma cells. Similarly to intact virus particles, also reconstituted virus envelopes, bearing the two viral glycoproteins, fused with M. capricolum. However, membrane vesicles, bearing only the viral binding (HN) or fusion (F) glycoproteins, failed to fuse with mycoplasmata. Fusion between animal enveloped virions and prokaryotic cells was thus demonstrated.