[Effect of heat shock on cells of phytopathogenic mycoplasma Acholeplasma laidlawii PG-8A].

Heat shock caused a more active formation of the "dormant" forms (minibodies), as well as increased production of extracellular membrane vesicles by Acholeplasma laidlawii PG-8A cells. Raise of the amount of the minibodies that have increased resistance to biogenic and abiogenic stress factors and pathogenicity may lead to more successful persistence of mycoplasmas in their hosts. Increased production of the extracellular membrane vesicles containing virulence factors by Acholeplasma laidlawii cells during stress may be an additional burden for the infected organism. It has been recently revealed that the vesicles of A. laidlawii contain appreciable quantities of small heat shock protein IbpA (Hsp20). In this paper, using immune-electron microscopy, have shown that at elevated temperature IbpA is associated with A. laidlawii minibodies. Perhaps, IbpA contributes to increased resistance and pathogenicity of the minibodies, keeping their proteins and polypeptides, including protein virulence factors in the folding-competent state.

Extracellular vesicles derived from Acholeplasma laidlawii PG8.

Extracellular vesicle production is believed to be a ubiquitous process in bacteria, but the data on such a process in Mollicutes are absent. We report the isolation of ultramicroforms - extracellular vesicles from supernatants of Acholeplasma laidlawii PG8 (ubiquitous mycoplasma; the main contaminant of cell culture). Considering sizes, morphology, and ultrastructural organization, the ultramicroforms of A. laidlawii PG8 are similar to membrane vesicles of Gram-positive and Gram-negative bacteria. We demonstrate that A. laidlawii PG8 vesicles contain genetic material and proteins, and are mutagenic to lymphocytes of human peripheral blood. We show that Mycoplasma gallisepticum S6, the other mycoplasma, also produce similar structures, which suggests that shedding of the vesicles might be the common phenomenon in Mollicutes. We found that the action of stress conditions results in the intensive formation of ultramicroforms in mycoplasmas. The role of vesicular formation in mycoplasmas remains to be studied.

[Elaboration of the in vitro model system to study the interaction of phytopathogenic mollicutes with plant cells].

The model system based on the sugar beet calluses infected by mycoplasms (mollicutes) was elaborated, and changes in the callus cells morphology under the effect of these microorganisms were also studied. The calluses of sugar beet 3K51 cultivated on the Gamborg medium were infected by phytopathogenic mollicute Acholaplasma laidlawii var. granulum str.118. Under the effect of mollicute infection one could observe changes in the cell morphology of sugar beet calluses: the plant cells were transformed from round to lengthened, the intensity of polyploids forming was increased, their grouping and their total destruction were observed. Data of electron microscopy confirm the presence of the mollicute in the sugar beet calluses: acholeplasma cells were localized between and within undifferentiated plant cells.

Adaptation to altered growth temperatures in Acholeplasma laidlawii B: Fourier transform infrared studies of acyl chain conformational order in live cells.

Acholeplasma laidlawii B cells, highly enriched in saturated C14 and C15 fatty acids, have been grown at several temperatures. Conformational order in the acyl chains of live cell membranes has been monitored with Fourier-transform infrared (FT-IR) spectroscopy. Coupled CH2 wagging progressions, characteristic of the all-trans conformation, have been used to quantitatively monitor the extent of trans-gauche isomerization. A simple model relating the changes in the progression intensity to the introduction of gauche rotamers into the acyl chains suggest that at the growth temperature (25 or 37 degrees C for the C14-enriched cells, 30 or 37 degrees C for the C15-enriched cells) the cell membrane contains about 1.5 gauche bonds per acyl chain. The thermotropic response of the CH2 stretching frequencies near 2850 cm-1 reveals that the gel-liquid-crystal phase transition is shifted toward lower temperatures when the growth temperature is reduced. In addition, at any given temperature, the cells grown at lower temperature are more conformationally disordered than their counterparts grown at higher temperature. This behavior is consistent with the quantitative results from the CH2 wagging measurements. The data reveal that A. laidlawii B cells can control the overall conformational state of their membranes and that the observed degree of disorder (1.5 +/- 0.3 gauche bonds/acyl chain), when achievable, provides optimum cell viability. The ability of this microorganism to control the degree of membrane disorder delineates one possible pathway for homeoviscous adaptation.

Phase transitions of Acholeplasma laidlawii membranes. The involvement of Mg(2+)-ATPase in the C transition.

Highly sensitive differential scanning calorimetry has been employed to study the phase transitions of A. laidlawii membrane. The DSC curves obtained show five distinct transitions between 20 and 80 degrees C which contain a reversible lipid thermotropic transition at about 37 degrees C and four irreversible denaturation transitions of the membrane proteins occurred at about 44 degrees C, 52 degrees C, 62 degrees C, and 67 degrees C, respectively. Total enthalpy of the thermal denaturation of membrane proteins is 3.4 +/- 0.5 cal/g. Further study of A. laidlawii membrane preparations by means of thermal gel analysis and enzyme activity measurements at various temperatures provided information that the third peak (C transition) of the DSC curve involved primarily with Mg(2+)-ATPase on A. laidlawii membranes.

[The isolation of ribosomes from Acholeplasma and the study of their physicochemical characteristics]. / Poluchenie ribosom akholeplazm i issledovanie ikh fiziko-khimicheskikh kharakteristik.

Ribosomes are produced from Acholeplasma laidlawii PG-8 and A. laidlawii var. granulum str. 118. It is proved as possible to use the standard method of differential centrifugation for isolation of mollicute ribosomes. The physico-chemical properties of acholesplasma ribosomes are studied. The protein content for A. laidlawii PG-8 amounted to 39.6%, rRNA content--60.4% and for A. laidlawii var. granulum--39.1 and 60.8%, respectively. The RNA: protein ratio equals 1.5:1, the ratio of optic density indicates at 260 and 280 nm--2, that is peculiar to treated preparations of ribosomes. Sedimentation coefficient of acholeplasma ribosomes is 70S. The produced preparations of acholeplasma ribosomes can be used subsequently for creating the system of translation in vitro to study the biosynthesis processes of mollicute protein.

[Tubular structures in Mycoplasma gallisepticum and the localization of a tubulin-like protein]. / Tubuliarnye struktury Mycoplasma gallisepticum i lokalizatsiia tubulinopodobnogo belka.

In all the strains of M. gallisepticum investigated, a protein with apparent molecular weight 40 kDa was revealed by immunoblotting with polyclonal anti-calf brain tubulin antibodies and monoclonal anti-chicken alpha-tubulin antibodies. In other 8 investigated Mycoplasma species no positive reactions with the same antibodies were found. The M. Gallisepticum cells were examined under electron microscope on fine serial sections and on some sections going at different angles to the long cell axis. Undermembrane system of tubules was revealed and the intracellular pattern of the tubular structures were reconstructed. The immunoelectron microscopic data suggest that tubulin-like protein may be included into the structures.

Comparison of glycoproteins from ten human Mycoplasma species.

Multiple bands of glycoprotein, rare in procaryotes, were detected in ten human Mycoplasma species by staining with periodic acid-Schiff (PAS) reagent after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A major contaminant formed in Hayflick medium (H medium), corresponding to an apparent molecular weight of about 80 kD, was eliminated by using the organisms grown in PPLO broth supplemented with PPLO serum fraction (P medium), except that M. genitalium and M. pneumoniae were grown in H medium as monolayers on the glass surface. The comparison of glycoproteins among ten human Mycoplasma species indicated that their profiles were shown to be species-specific. However, those of M. buccale and M. faucium were very similar, and M. pneumoniae and M. genitalium seemed to be related.

Studies on the purified Na+,Mg(2+)-ATPase from Acholeplasma laidlawii B membranes: a differential scanning calorimetric study of the protein-phospholipid interactions.

The purified Na+,Mg2(+)-ATPase from the Acholeplasma laidlawii B plasma membrane was reconstituted with dimyristoyl phosphatidylcholine and the lipid thermotropic phase behavior of the proteoliposomes formed was investigated by differential scanning calorimetry. The effect of this ATPase on the host lipid phase transition is markedly dependent on the amount of protein incorporated. At low protein/lipid ratios, the presence of increasing quantities of ATPase in the proteoliposomes increases the temperature and enthalpy while decreasing the cooperativity of the dimyristoyl phosphatidylcholine gel to liquid-crystalline phase transition. At higher protein/lipid ratios, the incorporation of increasing amounts of this enzyme does not further alter the temperature and cooperativity of the phospholipid chain-melting transition, but progressively and markedly decreases the transition enthalpy. Plots of lipid phase transition enthalpy versus protein concentration suggest that at the higher protein/lipid ratios each ATPase molecule removes approximately 1000 dimyristoyl phosphatidylcholine molecules from participation in the cooperative gel to liquid-crystalline phase transition of the bulk lipid phase. These results indicate that this integral transmembrane protein interacts in a complex, concentration-dependent manner with its host phospholipid and that such interactions involve both hydrophobic interactions with the lipid bilayer core and electrostatic interactions with the lipid polar head groups at the bilayer surface.

The effect of variations in growth temperature, fatty acid composition and cholesterol content on the lipid polar head-group composition of Acholeplasma laidlawii B membranes.

We have systematically investigated the effect of variations in growth temperature, fatty acid composition and cholesterol content on the membrane lipid polar headgroup composition of Acholeplasma laidlawii B. Two important lipid compositional parameters have been determined from such an analysis. The first parameter studied was the ratio of the two major neutral glycolipids of this organism, monoglucosyldiacylglycerol (MGDG) and diglucosyldiacylglycerol (DGDG). As the former lipid prefers to exist in a reversed hexagonal phase at higher temperatures, with unsaturated fatty acyl chains or in the presence of cholesterol, the ratio of these two lipids reflects the phase state preference of the total A. laidlawii membrane lipids. Although we find that the MGDG/DGDG ratio is reduced in response to an increase in fatty acid unsaturation, increases in growth temperature or cholesterol content reduce this ratio only in cells enriched in a saturated but not an unsaturated fatty acid. The second parameter studied was the ratio of these neutral glycolipids to the only phosphatide in the A. laidlawii membrane, phosphatidylglycerol (PG); this parameter reflects the relative balance of uncharged and charged lipids in the membrane of this organism. We find that the MGDG + DGDG/PG ratio is lowest in cells enriched in the saturated fatty acid even though these cells already have the highest lipid bilayer surface charge density. Moreover, this ratio is not consistently related to growth temperature or changes in cholesterol levels, as expected. We therefore conclude that A. laidlawii strain B, apparently unlike strain A, does not possess coherent regulatory mechanisms for maintaining either the phase preference or the surface charge density of its membrane lipid constant in response to variations in growth temperature, fatty acid composition or cholesterol content.

Purification of membrane proteins in SDS and subsequent renaturation.

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.

Isolation and characterization of a novel monoacylated glucopyranosyl neutral lipid from the plasma membrane of Acholeplasma laidlawii B.

The level of a normally minor component of the membrane polar lipids of Acholeplasma laidlawii B was significantly increased when the glucose supplement in the growth medium was reduced. Under such glucose-limiting conditions the proportion of this component was found to be dependent upon the fatty acid supplement and could approach 55-60% of the total polar lipids when palmitic acid was used to supplement the growth medium. A number of physical measurements, along with specific chemical and enzymic degradation studies followed by a careful analysis of the degradation products, enabled us to tentatively identify this lipid as a polyprenyl-alpha-D-glucoside with a long-chain fatty acid esterified to the 2-hydroxyl group of the sugar moiety. This lipid exhibited some unusual thermotropic phase properties and our observations suggest that it may not be easily miscible with the other membrane lipid components. The structure and physical properties of this unusual glycolipid also suggest that it may be capable of forming non-bilayer phases under physiologically relevant conditions.

Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy.

The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde-fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction. The cytoplasmic membrane of sectioned Acholeplasma-cells dehydrated and infiltrated as described above appeared more diffuse than that of cells fixed with glutaraldehyde and osmium tetroxide in epoxy resin at room temperature. Glutaraldehyde-fixed erythrocyte ghosts retained 85% of their phospholipid content when dehydrated with ethanol between 277 and 223 K and infiltrated with methacrylate at 223 K. Spinach chloroplasts and thylakoid vesicles retained 61% and 35%, respectively, of their chlorophyll content.

Ultrastructural changes in mollicutes induced by the peptide antibiotic herbicolin A.

Electron microscopy of negatively stained mycoplasma, ureaplasma, and acholeplasma cells showed ultrastructural changes after 10 min of treatment of the organisms with the peptide antibiotic herbicolin A in concentrations ranging from 10 micrograms/ml for Mycoplasma capricolum to 600 micrograms/ml for Ureaplasma urealyticum. The morphological changes were shown to be reversible at low concentrations of the antibiotic but irreversible at high concentrations.

Fluorine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. A direct comparison of the effects of cis and trans cyclopropane ring and double-bond substituents on orientational order.

The hydrocarbon chain orientational order parameters of membranes of Acholeplasma laidlawii B, enriched with large quantities of fatty acids containing either a cis or a trans cyclopropane ring or a cis or trans double bond, plus small quantities of one of an isomeric series of monofluoropalmitic acids, were determined via fluorine-19 nuclear magnetic resonance spectroscopy over a range of temperatures spanning the corresponding gel to liquid-crystalline phase transitions (determined via differential scanning calorimetry). Membrane orientational order profiles in the liquid-crystalline state were generally similar, regardless of the particular fatty acid structure present, showing a region of relatively constant order preceding a region of progressively decreasing order toward the methyl terminus of the acyl chain. In the gel state, the order profiles in the presence of either a trans cyclopropane ring or trans double-bond substituent were similar and were characterized by a pronounced head to tail gradient of order at temperatures just below the lipid phase transition, while at temperatures far below the lipid phase transition this gradient was less pronounced, all chain positions showing a more uniformly high degree of orientational ordering. In the gel state, the order profiles in the presence of either a cis cyclopropane ring or a cis double-bond substituent were also similar but were highly unusual in that order first increased and only then subsequently decreased toward the acyl chain methyl terminus. In addition, the substituents in the cis configuration, whether a cyclopropane ring or a double bond, were overall more disordered in the gel state than the corresponding substituents in the trans configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Infrared spectroscopic study of the gel to liquid-crystal phase transition in live Acholeplasma laidlawii cells.

The temperature dependences of the infrared spectra of deuterium-labeled plasma membranes of live Acholeplasma laidlawii B cells and of the isolated plasma membranes demonstrate that the profiles of the gel to liquid-crystal phase transitions are very different. At temperatures within the range of the phase transition, the live mycoplasma is able to keep the "fluidity" of its plasma membrane at a much higher value than that of the isolated plasma membrane at the same temperature. The difference is particularly pronounced at and around the temperature of growth. Live Acholeplasma laidlawii, grown at 37 degrees C on a fatty acid depleted medium supplemented with myristic acid (C14:0), pentadecanoic acid (C15:0), or palmitic acid (C16:0), are highly "fluid"; i.e., at the temperature of growth, the fractional population of the liquid-crystalline phase is 95-100% at 37 degrees C, whereas in the case of the isolated plasma membranes the fractional population of the liquid-crystalline phase at 37 degrees C is only 58% (C14:0), 36% (C15:0), or 38% (C16:0).

The effect of mycoplasmas and acholeplasmas of equine origin on organ cultures of chicken-embryo trachea.

Chicken-embryo tracheal organ cultures were inoculated with equine strains of Mycoplasma arginini, M. equigenitalium, 2 strains of M. subdolum, Acholeplasma laidlawii and 3 strains of A. oculi. All strains established and multiplied in the explant cultures, but only M. subdolum and A. oculi produced a cytopathic effect on ciliated epithelial cells, causing sloughing of cells and cilia after 6 days. There was a correlation between ciliostasis and increase in titre of both M. subdolum and A. oculi and this relationship was not observed with M. equigenitalium and A. laidlawii. All the strains of acholeplasma multiplied to some extent in organ culture media, but reached higher titres in the presence of explants. Cells infected with the M. subdolum strain showed sloughing of cilia, vacuolization, and increase in size of mitochondria, followed by disorganization of epithelium and marked destruction of subcellular organelles. Mycoplasmas were closely attached to the epithelial surface of the tracheal explant 8 days after infection.

Fracturing of melamine-embedded cells and tissues: a new technique for studying cell membranes.

A new technique is presented for studying cell membranes by scanning electron microscopy. It is based on the observation that cells and tissues, embedded in a water-compatible melamine resin, are as hard as glass and consequently can be fractured with ease. Fracture faces so exposed are either sputter-coated for studying the surface topography or re-embedded for thin-sectioning, or both. Scanning electron microscopy shows that the fracture faces of a variety of tissues reveal cell membranes and associated structures with remarkable detail. Re-embedding and thin-sectioning of fractured frog retina, mycoplasma and red blood cells indicate that membranes become divided into their exo- and protoplasmic leaflets during fracturing. In this respect, the results reported here must be compared with conventional freeze-fracture techniques.