Shmuel Razin

Cholesterol in mycoplasma membranes. Composition, ultrastructure and biological properties of membranes from Mycoplasma mycoides var. capri cells adapted to grow with low cholesterol concentrations.

Abstract 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.

Cholesterol in mycoplasm membranes. Correlation of enzymic and transport activities with physical state of lipids in membranes of Mycoplasma mycoides var. capri adapted to grow with low cholesterol concentrations

Abstract 1. 1. Membranes of a Mycoplasma mycoides var. capri strain adapted to grow with very low concentrations of cholesterol undergo a reversible phase transition detectable by differential-scanning calorimetry and fluorescence measurements. No phase transition could be detected in membranes of the cholesterol-containing native strain. 2. 2. EPR spectrometry, as well as fluorescence measurements, demonstrated that at temperatures above phase transition the membranes of the adapted strain were more fluid than membranes of the native strain, although the former contained a higher percentage of saturated fatty acids. 3. 3. Arrhenius plots of the ATPase activity of the adapted strain membranes showed breaks at temperatures corresponding to those of the phase transition of membrane lipids. The temperature of the break depended on the fatty acid composition of membrane lipids and on the age of the culture. No break could be detected in Arrhenius plots of the ATPase activity of the native strain. 4. 4. No break could be demonstrated in the Arrhenius plot of α-methylgucoside uptake by the adapted strain. Yet the activation energy of the uptake process by the adapted strain was much higher than that of the native strain. On the other hand, activation energies of α-methylglucoside phosphorylation were the same for membranes of both strains. However, Arrhenius plots of α-methylglucoside efflux from cells of the adapted strain showed breaks at temperatures corresponding to those of the lipid phase transition. 5. 5. It is concluded that cholesterol, by preventing the crystallization of membrane lipids maintains them in a state of fluidity essential for the optimal manifestation of several key activities of the membrane.

Detection of mycoplasmas infecting cell cultures by DNA hybridization

SummaryInfection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 105 mycoplasmas.

Highlights of mycoplasma research--an historical perspective.

This brief historical development of the biology of the mycoplasmas begins with their discovery in 1898 to the present. Mycoplasmas are the smallest free-living microorganisms and for years were thought to be viruses because they passed through the usual bacterial filters. They lack a cell wall, are widespread in nature and many are animal, plant and human pathogens. The extensive use of cell cultures in the last fifty years and their frequent contamination with mycoplasmas, together with their possession of the smallest genome of any free-living organism, has drawn enormous attention to these organisms and has revealed considerably more about their biology.

Characterization of the mycoplasma membrane proteins II. Solubilization and enzymic activities of Acholeplasma laidlawii membrane proteins

Abstract Treatment of Acholeplasma laidlawii membranes with EDTA in low-ionic strength media released about 11% of the total membrane protein in a water-soluble form. The released protein fraction had no NADH oxidase, ATPase and p -nitrophenylphosphatase activities. The strongly ionic detergents sodium dodecyl sulfate and cetyltrimethylammonium bromide were more effective in the solubilization of A. laidlawii membranes than the nonionic detergents Triton X-100, Lubrol W or Brij 58. Sodium deoxycholate occupied an intermediate position. The solubilization of the membranes by detergents affected their NADH oxidase, ATPase and p -nitrophenylphosphatase activities in two antagonistic ways: activation and inactivation. The balance of these processes depended on the type and concentration of the detergent used and on the enzymic activity tested. The activation effect was most pronounced with low concentrations of the nonionic detergents and with p -nitrophenylphosphatase activity. Inactivation of the enzymes was most pronounced with sodium dodecyl sulfate and cetyltrimethylammonium bromide. The results of the present study favor the use of nonionic detergents for the solubilization and further fractionation of mycoplasma membrane proteins.

Phospholipid and cholesterol uptake by Mycoplasma cells and membranes.

The ability of growing mycoplasma cells and their isolated membranes to take up exogenous phospholipids was correlated with their ability to take up cholesterol. Horse serum or vesicles made of phosphatidylcholine and cholesterol served as lipid donors. Growing cells of five Mycoplasma species took up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three Acholeplasma species failed to take up any of the exogenous phospholipids, and only incorporated low amounts of free cholesterol and no esterified cholesterol. Hence, the ability of mycoplasmas to take up large quantities of cholesterol appears to be correlated with an ability to take up exogenous phospholipids. Isolated membranes of Mycoplasma capricolum and Acholeplasma laidlawii took up lower amounts of cholesterol than did membranes of growing cells and did not take up phospholipids. Inhibition of M. capricolum growth decreased the ability of the cells to take up exogenous phospholipids and cholesterol. The possibility that the contact between the lipid donors and the membrane involves specific receptors best exposed in actively growing cells is discussed.

Synthesis and turnover of membrane protein and lipid in Mycoplasma laidlawii

Abstract The relatioship of lipid to protein synthesis in Mycoplasma laidlawii membranes was studied in heavy suspensions of the organisms in complex or partially defined media. Under the test conditions used the organisms did not multiply, and the amount of total cell protein remained constant. Lipid synthesis was measured by the incorporation of [3H]oleic acid into membrane lipids, while protein synthesis was measured by the incorporation of l -[14C]phenylalanine into membrane proteins. Though the ability of the cells to synthesize both membrane proteins and lipids declined steeply with age their protein-synthesizing ability was impaired at a much earlier stage. When membrane protein synthesis was arrested by treating the cells with chloramphenicol, the membrane lipid synthesis was not affected, so that the cell membranes formed had a density of 1.158 g/cm3 as against 1.170 g/cm3 for untreated cell membranes. The total membrane proteins labeled with l -[14C]phenylalanine turned over at a relatively high rate, having a half life of approx. 3 h. Turnover of the total membrane lipids labeled with [3H]oleic acid became apparent only after a lag period of several hours after the beginning of the chase. It was thus found that in M. laidlawii membrane lipid synthesis can be uncoupled from the synthesis of the membrane protein, so that the two processes are not necessarily synchronized. The possibility of varying the lipid to protein ratio in biological membranes without affecting their function is discussed.

Selective reaggregation of solubilized mycoplasma-membrane proteins and the kinetics of membrane reformation

Abstract 1. 1. Proteins and lipids of mycoplasma membranes solubilized in sodium dodecyl sulfate reaggregated to membraneous structures when the detergent was diluted by dialysis against a Mg 2+ -containing buffer. The Mg 2+ concentration determined the degree of reaggregation, the lipid-to-protein ratio in the reaggregate and, as shown by electrophoretic and enzymic analyses, the species of membrane proteins aggregated. 2. 2. The reassembly of the solubilized membrane components to membraneous structures proceeded rapidly. After 40 min of dialysis against 20 mM Mg 2+ , the reaggregate already contained membranes of a similar triple-layered structure and thickness as the original ones, but with a smaller number of protein species and a higher lipid-to-protein ratio. 3. 3. On density-gradient analysis reaggregates of Mycoplasma laidlawii membranes were found to be heterogeneous, while at 5 mM Mg 2+ only a “light” lipid-rich band ( d = 1.140) was obtained. At higher Mg 2+ concentrations this was accompanied by one or two heavier bands. The “light” band was transformed into a heavier one when the Mg 2+ concentration was increased. 4. 4. Our data suggest that the reaggregated membranes are assembled by a multi-step process and not by the single-step assembly of lipoprotein subunits. 5. 5. The application of the selective aggregation of solubilized membrane proteins to the fractionation and characterization of membrane enzymes and antigens is suggested and discussed.

Physiology of mycoplasmas.

Publisher Summary The chapter focuses on the physiology of Mycoplasmas. The physiology of mycoplasmas, the smallest organisms capable of autonomous growth, is of special interest in view of their extremely simple structure and limited biochemical activities. Because of the recent improvements in cultivation and identification techniques, quite a number of further mycoplasma species could be established. By now, over 40 species occurring in primates, farm and laboratory animals, and a variety of wild animals have been named. In the absence of a cell wall, the mycoplasmas are so fragile and pliable that their morphology was long disputed. Mycoplasma can grow in filaments; the different strains vary in their ability to do so. This may be due to the differences in their ability to synthesize certain membrane components, when the supply of precursors in the growth medium is limited. The extremely simple ultrastructure found in numerous electron microscope studies of thin mycoplasma sections supports the view that they are the simplest and the most primitive organisms extant. Essentially, the mycoplasma cell is built of only three organelles: the cell membrane, the ribosome, and the characteristic prokaryotic chromosome. Though the unusual physiological properties of the new Thermoplasma acidophilum suggest a rather distant relationship to the animal mycoplasmas, its inclusion in the Mollicutes class seems warranted, and its very existence considerably broadens the range of habitats in which mycoplasma-like organisms are found.

Characterization of the myoplasma membrane proteins: III. Gel filtration and immunological characterization of Acholeplasma laidlawii membrane proteins

Abstract Acholeplasma laidlawii (formerly Mycoplasma laidlawii) membranes solubilized by ionic and nonionic detergents were fractionated on Sephadex G-200 columns containing the detergent used for solubilization. When sodium dodecyl sulfate or sodium deoxycholate were present, membrane lipids were resolved as a single elution peak while membrane proteins formed several reproducible peaks. Gel filtration in the presence of the nonionic detergents Triton X-100, Brij 58 and Lubrol W was usually inferior. The proteins were eluted as two broad peaks, one of which included the void volume. Since the NADH oxidase, ATPase and p- nitrophenylphosphatase activities of the solubilized membranes could be detected in the first peak, containing little or no lipid, it appears that these enzymes do not depend on membrane lipids for activity. Serological activity could be demonstrated in membrane fractions isolated by gel filtration, even when sodium dodecyl sulfate was used. However, the use of deoxycholate enabled the separation of a membrane protein fraction highly enriched in antigens which elicited the production of antibodies inhibiting A. laidlawii metabolism and growth. A high content of antigens located on the outer membrane surface was evidenced by the high agglutination titer of A. laidlawii cells exhibited by antisera to this fraction.