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Amino acid assimilation by different representatives of Acholeplasma genus has been investigated. It was shown that all 7 investigated typical strains Acholeplasma laidlawii PG-8, A. granularum BTS-39, A. modicum PG-49, A. oculi 19-L, A. morum 72-043, A. axanthum S-743, A. hippikon C-1 and 37 phytopathogenic strains isolated from the affected plants were able to assimilate asparagine, glutamine, threonine, histidine and proline. A majority of acholeplasmas were able to metabolize phenylalanine, methionine, glutamate and lysine, rarely isoleucine. Each of the investigated species of acholeplasmas had individual spectrum of assimilated amino acids that can be used as an additional systematic characteristic of mollicutes.

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The lamellar gel to lamellar liquid-crystalline (Lbeta/Lalpha) and lamellar liquid-crystalline to inverted hexagonal (Lalpha/H(II)) phase transitions of a number of phosphatidylethanolamines (PEs) and diacyl-alpha-D-glucosyl-sn-glycerols (alpha-D-GlcDAGs) containing linear saturated, linear unsaturated, branched or alicyclic hydrocarbon chains of various lengths were examined by differential scanning calorimetry and low-angle X-ray diffraction. As reported previously, for each homologous series of PEs or alpha-D-GlcDAGs, the Lbeta/Lalpha phase transition temperatures (Tm) increase and the Lalpha/H(II) phase transition temperatures (Th) decrease with increases in hydrocarbon chain length. The Tm and the especially the Th values for the PEs are higher than those of the corresponding alpha-D-GlcDAGs. For PEs having the same effective hydrocarbon chain length but different chain configurations, the Tm and Th values vary markedly but with an almost constant temperature interval (deltaT(L/NL)) between the two phase transitions. Moreover, although the Tm and Th values of the PEs and alpha-D-GlcDAGs are equally sensitive on the temperature scale to variations in the length and chemical configuration of the hydrocarbon chains, the deltaT(L/NL) values are generally larger in the PEs and vary less with the hydrocarbon chain structure. This suggests that the PE headgroup has a greater ability to counteract variations in the packing properties of different hydrocarbon chain structures than does the alpha-D-GlcDAG headgroup. With decreasing chain length, this ability of the PE headgroup to counteract the hydrocarbon chain packing properties increases, significantly expanding the temperature interval over which the Lalpha phase is stable relative to the corresponding regions in the alpha-D-GlcDAGs. Overall, these findings indicate that the PEs have a smaller propensity to form the H(II) phase than do the alpha-D-GlcDAGs with an identical fatty acid composition. In contrast to our previous report, there is some variation in the d-spacings of these various PEs (and alpha-D-GlcDAGs) in both the Lalpha and H(II) phases when the hydrocarbon chain structure is changed while the effective chain length is kept constant. These hydrocarbon chain structural modifications produce different d-spacings in the Lalpha and H(II) phases, but those changes are consistent between the PEs and alpha-D-GlcDAGs, probably reflecting differences in the hydrocarbon chain packing constraints in these two phases. Overall, our experimental observations can be rationalized to a first approximation by a simple lateral stress model in which the primary bilayer strain results from a mismatch between the actual and optimal headgroup areas and the primary strain in the H(II) phase arises from a simple hydrocarbon chain packing term.

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We have investigated the effect of the interaction of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of model lipid bilayer membranes generated from the total membrane lipids of Acholeplasma laidlawii B and Escherichia coli. The A. laidlawii B membrane lipids consist primarily of neutral glycolipids and anionic phospholipids, while the E. coli inner membrane lipids consist exclusively of zwitterionic and anionic phospholipids. We show that the addition of GS at a lipid-to-peptide molar ratio of 25 strongly promotes the formation of bicontinuous inverted cubic phases in both of these lipid model membranes, predominantly of space group Pn3m. In addition, the presence of GS causes a thinning of the liquid-crystalline bilayer and a reduction in the lattice spacing of the inverted cubic phase which can form in the GS-free membrane lipid extracts at sufficiently high temperatures. This latter finding implies that GS potentiates the formation of an inverted cubic phase by increasing the negative curvature stress in the host lipid bilayer. This effect may be an important aspect of the permeabilization and eventual disruption of the lipid bilayer phase of biological membranes, which appears to be the mechanism by which GS kills bacterial cells and lysis erythrocytes.

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Cytoplasmic fractions from species of the Mollicutes genera Entomoplasma, Mesoplasma, Mycoplasma, and Acholeplasma were assayed for NADH oxidase (NADH ox), ATP- and PPi-dependent phosphofructokinase (PFK), ATP- and PPi-dependent deoxyguanosine kinase (dGUOK), thymidine kinase (TK), TMP kinase (TMPK), glucose-6-phosphate dehydrogenase (G6Pde), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxylase, hypoxanthine-guanine phosphoribosyl transferase, dUTPase, and uracil-DNA glycosylase (UNG) activities. Membrane fractions were also examined for NADH ox activity. These activities were used as indicators of the presence and relative activities of major Mollicutes metabolic and DNA repair pathways. This was the first study to determine the presence of these enzymes in members of the genera Entomoplasma and Mesoplasma. Using the data obtained, we constructed a preliminary scheme for distinguishing genera of the class Mollicutes on the basis of the results of signature functional enzyme assays. This scheme includes phylogenetic relationships deduced from rRNA analyses, but is more informative with respect to metabolic potential. The criteria used include the presence of PPi-dependent PFK, urease, dUTPase, and dGUOK activities. Entomoplasma ellychniae ELCN-1T (T = type strain), Entomoplasma melaleucae M-1T, Mesoplasma seiffertii F7T, Mesoplasma entomophilum TACT, Mesoplasma florum L1T, Mycoplasma fermentans PG18T, and Acholeplasma multilocale PN525T were similar in most respects. NADH ox activity was localized in the cytoplasm of these organisms. These strains had ATP-dependent PFK, MDH, LDH, ATP- and PPi-dependent dGUOK, and UNG activities, but not dUTPase or G6Pde activities. In contrast, Acholeplasma equifetale C112T, Acholeplasma oculi 19LT, Acholeplasma hippikon C1T, Acholeplasma modicum PG49T, and Acholeplasma morum 72-043T had membrane-localized NADH ox activity, PPi-dependent PFK, G6Pde, and dUTPase activities, and significantly lower MDH and LDH activities and exhibited a faster rate with PPi than with ATP in the dGUOK reaction. All of the members of the Mollicutes tested had hypoxanthine-guanine phosphoribosyl transferase, phosphoenolpyruvate carboxylase, and (except for Mesoplasma entomophilum TAC(T)) UNG activities. All of the Acholeplasma strains except Acholeplasma multilocale PN525T had TK, TMPK, and UNG activities. Mesoplasma entomophilum TAC(T) was distinguished by having no detectable dUTPase, UNG, TK, and TMPK activities, indicating that there is a severe restriction in or an absence of a synthetic route to dTTP. Our data also suggest that A. multilocale PN525T is a member of an unrecognized metabolic subgroup of the genus Acholeplasma or is not an Acholeplasma strain.

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We investigated 22 mycoplasma and acholeplasma species for their ability to reduce tetrazolium salts by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The test results were evaluated visually, as well as spectrophotometrically, by using an enzyme-linked immunosorbent assay reader. Our results were very similar to the results obtained when the tetrazolium salt reduction assay described by Aluotto et al. was used. However, the MTT reduction assay appeared to be better because it is faster, more objective and sensitive, easier to evaluate, and less expensive; in addition, it allows quantitative determinations. By using regression analysis a linear correlation between formazan production and the number of colony-forming units was demonstrated for all of the species investigated, indicating that the MTT assay can also be used for growth, toxicity, or chemosensitivity tests for the mycoplasma species that are capable of reducing tetrazolium salts.

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Four species in the order Mycoplasmatales, Mycoplasma capricolum, Mycoplasma hominis, Mycoplasma arginini, and Acholeplasma laidlawii, were compared for their ability to accumulate radiolabeled amino acids and polyamines. The use of a novel high-molecular-weight (HMW) medium, from which molecules of less than 12,000 molecular weight had been removed by extensive dialysis, allowed us to discern significant differences among the species in their relative accumulations of [3H]methionine and [3H]leucine and of [3H]spermidine and [3H]putrescine. Accumulation of radiolabeled amino acids in control low-molecular-weight (LMW) medium was small (0.2 to 2% of the label), and the species did not differ in their proportional accumulations of methionine and leucine. Accumulation of methionine was significantly enhanced (5- to 12-fold) in all species in HMW medium. In contrast, leucine accumulation was enhanced sevenfold for A. laidlawii but only twofold for M. hominis and M. capricolum in HMW medium. The nonglycolytic species, M. hominis and M. arginini, accumulated radiolabeled putrescine and spermidine in both media, whereas the glycolytic species, M. capricolum and A. laidlawii, accumulated only radiolabeled spermidine. The ability to accumulate putrescine appeared to be a differential characteristic for nonfermentative, arginine-utilizing mycoplasmas. HMW medium was much more effective than LMW medium for use in radiolabeling M. capricolum proteins with [35S]methionine.

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The physical state of the membrane lipids in the plasma membranes of intact, live Acholeplasma laidlawii B cells was probed by Fourier-transform infrared spectroscopy and compared with that in isolated membranes. Infrared spectra of live A. laidlawii B cells, enriched biosynthetically in the presence of avidin, with saturated deuterated and unsaturated non-deuterated fatty acids have been recorded at a variety of temperatures. The results indicate that within the temperature range of the gel to liquid-crystal phase transition, the live cells are able to keep the 'fluidity' of their plasma membranes at a considerably higher value compared to that in the isolated membranes at the same temperature. While this is a generally valid observation, the degree by which live and isolated membranes differ in their liquid-crystal-phase content at a given temperature depends on the nature of the exogenous fatty acid and the temperature of growth.

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Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.

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Further analysis of three sterol-nonrequiring Mollicutes (strains PS-1, TAC, and YJS) isolated from gut fluids of insects confirms their similarity to Acholeplasma. They are serologically distinct from acholeplasmas of vertebrates and several other sterol nonrequiring Mollicutes isolated from plant surfaces. The PS-1 strain had a DNA G + C content of 31 mol % and a genome size of 1,030 megadaltons (MDa). Optimum temperature is in the range of 23 to 30 C. Thirty-two new nonhelical Mollicutes isolated from a much wider range of insect hosts were examined for acholeplasmas. Twenty-five of the insect isolates were grown consistently in serum-free broth, with or without Tween 80 supplements. Two of the acholeplasmas were serologically related to Acholeplasma florum, 13 strains were serologically identical to the TAC isolate reported earlier, and 10 of the putative acholeplasmas could not be identified with current reference antisera. Seven of the new nonhelical insect isolates appeared to be sterol-requiring Mollicutes. One sterol-requiring isolate (ELCN-1) was recovered from the hemolymph of a firefly, and is the first report of nonhelical Mollicutes in the insect hemocoel. Two of the seven sterol-requiring Mollicutes, which were nonhelical in earlier passages in broth, later reverted to typically helical spiroplasmas. Confirmation of sterol-requiring, nonhelical Mollicutes in insects would provide an important ecological finding that insects constitute an important reservoir for both acholeplasmas and mycoplasmas.

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PPAV is a type of Mollicutes that was isolated in SP4 medium from seeds of apples affected by proliferation, a disease in which mycoplasma-like organisms (MLOs) are involved. However, PPAV is probably not the etiological MLO agent for at least two reasons: 1) optimal temperature growth is 43 C, and 2) in spite of numerous isolation attempts over several years, no second PPAV culture could be obtained. PPAV is surrounded by a single cytoplasmic membrane and forms typical fried egg-shaped colonies on solid medium. The organism grows in simplified mycoplasma media, such as BSR. In growth inhibition, it shows no serological relationships with any other mycoplasmas or acholeplasmas, including those cultured from the surfaces of plants. The absence of relatedness of PPAV to other Mollicutes was confirmed by DNA hybridization studies. The genome of PPAV is close to 10(9) daltons and contains 25.2 mol % G + C. Its genome size is similar to that of Acholeplasma spp. and Spiroplasma spp. It has, however, a clearcut sterol requirement and therefore cannot be an acholeplasma. Neither is it a spiroplasma since, though filamentous in BSR medium, it has never shown signs of helicity. Hence, PPAV is a taxonomical paradox.

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Cultures of the Mollicutes (mycoplasma) Acholeplasma laidlawii B, Acholeplasma morum, Mycoplasma bovis, Mycoplasma arginini, Mycoplasma fermentans and Mycoplasma gallisepticum, representing four metabolic groups, were sampled at intervals over a 40 to 50 h period and assayed for the numbers of c.f.u., changes in pH and glucose concentration, and concentrations of ATP, ADP, AMP, lactate and pyruvate. The adenylate energy charge (ECA), the mean generation time, and the number of nmol of ATP (mg dry weight)-1 were calculated for cultures in the mid-exponential growth phase. The maximum cell concentrations ranged from 0.2 X 10(10) to 5.0 X 10(10) c.f.u. ml-1. Doubling times ranged from 0.34 to 3.29 h. The fermentative, nonarginine-requiring A. laidlawii B, A. morum, and M. gallisepticum, as well as the fermentative, arginine-requiring M. fermentans, utilized glucose and produced lactate and pyruvate. The non-fermentative, non-arginine-requiring M. bovis neither utilized glucose nor produced lactate or pyruvate. The non-fermentative, arginine-requiring M. arginini utilized glucose, but did not produce lactate or pyruvate. At mid-exponential growth phase, the average ECA of A. laidlawii B was 0.90, a value similar to that reported for Spiroplasma citri and other bacteria. In contrast, the average ECA of A. morum and the four Mycoplasma species was 0.70. In A. laidlawii B at mid-exponential growth phase, ATP accounted for 97% of the total adenylate nucleotide pool.(ABSTRACT TRUNCATED AT 250 WORDS)

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The metabolism of the Mollicutes Acholeplasma and Mycoplasma may be characterized as restricted, for example, by virtue of the apparent absence of cytochrome pigments. Some Mollicutes have lowered ECA values during their logarithmic growth phase, which we speculate may be related to insufficient substrate phosphorylation or insufficient ATP synthesis linked to glycolysis. We found that PEP is carboxylated by preparations of A. laidlawii, but not by other Mollicutes; thus in this organism oxaloacetate from PEP may be a link to other pathways. We found phosphoribosylpyrophosphate in A. laidlawii, which suggests that ribosylation of purines and pyrimidines occurs in Mollicutes other than M. mycoides.

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Mycoplasma offer several unique advantages for investigating the mechanism controlling transfer and uptake of exogenous cholesterol and phospholipids by biomembranes, as their plasma membrane interacts directly with exogenous lipid donors and their endogenous lipid synthesis is restricted. Growing cells of five species of Mycoplasma were found to take up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three species of Acholeplasma failed to take up any of the exogenous phospholipids and incorporated only low amounts of free cholesterol and no esterified cholesterol. It is hypothesized that Mycoplasma species have receptors for serum lipoproteins and phospholipid-cholesterol vesicles that facilitate the transfer of cholesterol and phospholipids to the growing cell membrane. Our finding that gentle trypsin treatment of growing Mycoplasma capricolum cells decreased their cholesterol uptake ability by about 50% but did not affect cholesterol uptake by growing Acholeplasma laidlawii cells appears to support the existence of protein receptors for lipoproteins on the surface of Mycoplasma but not on Acholeplasma species. Digestion of membrane phospholipids by phospholipase A2 decreased the cholesterol-binding capacity of isolated A. laidlawii and M. capricolum membranes, roughly in proportion to the amount of phospholipid digested. The total removal of phosphatidylglycerol and diphosphatidylglycerol from A. laidlawii membranes by phospholipase A2 only decreased but did not abolish cholesterol uptake, an indication that glycolipids also participate in cholesterol uptake.

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Biochemical and serological properties of mycoplasmas isolated from the blood, feces and parenchymatous organs of monkeys have been studied to determine their species. It was established that the isolated strains belong to the family Acholeplasmatoceae. The study of their biochemical properties in different tests has revealed the presence of 5 biochemically heterogeneous groups. Their serological properties suggest that 13 out of 45 strains are identical to the reference strain of A. laidlawii A, and all other strains have been classified as new Acholeplasma species which have never been isolated from monkeys before.

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Mycoplasma virus type 2 was shown to adsorb specifically to intact cells, membranes, and lipoglycan of Acholeplasma laidlawii strain JA1 but not to these components of Acholeplasma oculi. The oligosaccharide chain of the lipoglycan defined the specificity of the receptor site since deacylation not only did not reduce adsorption but increased it threefold. Actual adsorption of virus to lipoglycan was demonstrated by sucrose density gradient separation of the virus-lipoglycan complex. A strain of A. laidlawii, JA1r, resistant to infection with mycoplasma virus type 2, was incapable of adsorbing the virus and was devoid of lipoglycan.

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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.

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Aerobic reduction of tellurite by five Acholeplasma species and two Mycoplasma species was investigated by light and electron microscopy. Among the Acholeplasma species, colonies of A. laidlawii and A. oculi exhibited a heavy, macroscopically visible reduction of tellurite, whereas the reaction of A. axanthum was weaker. A. granularum and A. modicum did not reduce the substrate under the experimental conditions employed. The two subspecies of M. mycoides also reacted with tellurite, as did also the investigated strain of M. bovigenitalium although to a lesser extent. Ultrastructurally, reduction sites were localized to the cytoplasmic membrane in the three tellurite positive Acholeplasma species and apparently to the cytoplasm of M. mycoides subsp. mycoides. Reduction sites could not be demonstrated in M. mycoides subsp. capri and in M. bovigenitalium. The results support previous evidence obtained by biochemical methods which indicates membrane localization of redox enzymes in Acholeplasmas.

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Methods are described for the rapid detection of beta-D-glucosidase and phosphatase in mycoplasma cultures using fluorogenic 4-methylumbelliferone substrates. These methods were applied to a selection of mycoplasma cultures and were compared with the conventionally used tests for these enzymes. Results were similar by both methods, but the fluorogenic tests could be read after 1 h, whereas the conventional tests took several days.

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Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity of Acholoplasma laidlawii is confined to the cell membrane. The enzyme could not be released from the membrane by either low- or high-ionic-strength solutions, with or without ethylenediaminetetraacetic acid, nor solubilized by detergents. The enzyme has a general specificity for long-chain saturated and unsaturated fatty acid thioesters. The preferred substrates among the saturated fatty acyl derivatives are the myristyl and palmityl derivatives. Arrhenius plots of thioesterase activities in A. laidlawii membranes enriched with elaidic or palmitic acids showed discontinuities at 12 and 18 degrees C, respectively. The possible regulatory significance of the thioesterase activity for the fatty acid synthetase and the possibllity that the activity of the enzyme is controlled by the physical state of membrane lipids are discussed.

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