[Experimental estimation of proteome size for cells and human plasma].

Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70,000 proteoforms, and plasma--1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)--40,000, E. coli--6200, P. furiosus--3400. In hepatocytes, the amount of proteoforms was the same as in HepG2--70,000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.

Extracellular membrane vesicles and phytopathogenicity of Acholeplasma laidlawii PG8.

For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses) in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing) to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria--invasivity, infectivity--and toxigenicity--and to favor to bacterial phytopathogenicity.

Nutritional effects of culture media on mycoplasma cell size and removal by filtration.

Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 microm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 microm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 microm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 microm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 microm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 microm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum.

[Molecular weight and subunit composition of extracellular fructosobisphosphatase of Acholeplasma laidlawii var. granulum strain 118].

Molecular weight of extracellular fructosobisphosphatase of Acholeplasma laidlawii var. granulum strain 118--an agent of pale-green dwarf of cereals has been determined. This enzyme is the basic factor of pathogenicity of this organism, and, maybe, of all phytoplasmas, owing to realization of the enzyme noncontrolled function in the plant organism, its habit acquires the disease symptoms characteristic of "yellows". It has been established that in the native condition the extracellular fructosobisphosphatase of Acholeplasma laidlawii var. granulum st. 118 has molecular weight 230 000 +/- 5 000 Da, and in denaturating ones--56 600 Da, i.e., the enzyme in the native condition consists of four equal subunits. The subunit composition is peculiar to fructosobisphosphatase of other microorganisms already described in literature; fructosobisphosphatase of the mollicute as to its parameters both in native and denaturated conditions occupies the intermediate place among them.

Effect of independent variations in fatty acid structure and chain length on lipid polar headgroup composition in Acholeplasma laidlawii B membranes: regulation of lamellar/nonlamellar phase propensity.

We have studied the biosynthetic regulation of the membrane lipid polar headgroup distribution in Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin to test whether this organism has the ability to coherently regulate the lamellar/nonlamellar phase propensity of its membrane lipids. The addition of various single normal growth-supporting exogenous fatty acids to such cell cultures produces fatty acid-homogeneous cells in which the hydrocarbon chain length and structure of the fatty acyl chains of the membrane lipids can be independently varied. Moreover, in analyzing our results, we consider the fact that the individual membrane lipid classes of this organism can form either normal micellar, lamellar, or reversed cubic or hexagonal phases in isolation (Lewis, R. N. A. H., and McElhaney, R. N. (1995) Biochemistry 34, 13818-13824). When A. laidlawii cells are highly enriched in one of a homologous series of methyl isobranched, methyl anteisobranched, or omega-cyclohexyl fatty acids, neither the ratio of normal micellar/lamellar nor of inverted cubic or hexagonal/lamellar phase-forming lipids are coherently regulated, and in fact in the former case, the changes in lipid polar headgroup composition observed are generally in a direction opposite to that required to maintain the overall lamellar/nonlamellar phase preference of the total membrane lipids constant when hydrocarbon chain length is varied. Similarly, when lipid hydrocarbon structure is varied at a constant effective chain length, a similar lack of coherent regulation of membrane lipid polar headgroup distribution is also observed, although in this case a weak overall trend in the expected direction occurs. We also confirm our previous finding (Foht, P. J., Tran, Q. M., Lewis, R. N. A. H., and McElhaney, R. N. (1995) Biochemistry 34, 13811-13817) that the ratio of inverted phase-forming monoglucosyl diacylglycerol to the lamellar phase-forming glycolipid diglucosyl diacylglycerol, previously used to estimate membrane lipid phase preference in A. laidlawii A and B, is not by itself a reliable indicator of the overall lamellar/nonlamellar phase propensity of the total membrane lipids of these organisms. Our results indicate that A. laidlawii B lacks a coherent mechanism to biosynthetically regulate the polar headgroup distribution of its membrane lipids to maintain the micellar/lamellar/inverted phase propensity constant in the face of induced variations in either the chain length or the structure of its lipid hydrocarbon chains. Finally, we suggest that the lack of a coherent regulatory mechanism to regulate the overall phase-forming propensity of the total membrane lipids of this organism under these circumstances may result in part from its inability to optimize all of the biologically relevant physical properties of its membrane lipid bilayer simultaneously.

Membrane lipid composition and cell size of Acholeplasma laidlawii strain A are strongly influenced by lipid acyl chain length.

The small, cell-wall-less prokaryote Acholeplasma laidlawii strain A-EF22 could grow with membrane lipids having an average acyl chain length Cn varying over 14.5- almost 20 carbons by exogenous supplementation with selected fatty acids. For 16 < Cn < 18, the cells grew with lipids containing 100% (mol/100 mol) monounsaturated acyl chains, whereas for Cn < 16 and Cn > 18, cell growth only occurred with gradually lower fractions of unsaturated chains. Cn was actively increased and decreased by chain elongation or de novo fatty acid synthesis upon incorporation of short-chain and long-chain fatty acids, respectively. The membrane lipid composition was strongly affected by the acyl chain length and unsaturation, and the metabolic responses are readily explained as a regulation mechanism based on the established phase equilibria of the individual lipids in the A. laidlawii membrane. Monoglucosyldiacylglycerol (Glc-acyl2-Gro) was the dominating lipid with short chains but the fraction of this lipid decreased with increasing Cn, correlating with the decreasing lamellar to nonlamellar phase transition temperatures for this lipid. The fractions of diglucosyldiacylglycerol (Glc2-acyl2Gro) and phosphatidylglycerol (PtdGro), forming lamellar phases only, increased with increasing Cn over the entire chain-length interval. A weaker correlation was usually observed between the relative amount of a lipid and the extent of chain unsaturation; however, the fractions of Glc2-acyl2Gro and PtdGro increased clearly with an increasing degree of unsaturation. Moreover, the synthesis of the nonbilayer-forming lipids acyl2Gro and monoacyl-Glc-acyl2Gro was strongly stimulated by a high degree of chain saturation. Concomitantly, the phase equilibria of Glc-acyl2Gro are shifted towards lamellar phases at the growth temperature. The fraction of the three potentially nonbilayer-forming lipids varied over 10-80% (mol/100 mol) total lipids as a function of the acyl chain composition. The combined molar fractions of the three phospholipids increased strongly with chain unsaturation. However, the fraction of phosphate moieties in the different lipids was constant over the entire chain-length interval. It is concluded that the regulation of the membrane lipid composition aims at maintaining similar phase equilibria and surface charge densities of the lipid bilayer. The size of A. laidlawii cells was changed in a systematic manner and correlated qualitatively with the packing properties of the lipids. Cell diameters were increased by an increase in acyl chain length and saturation, and was affected by additives such an n-dodecane and acyl2Gro.

Fusion of mycoplasmas: the formation of cell hybrids.

Poly(ethylene glycol) (PEG 8000) can induce cell-cell fusion of Mycoplasma capricolum cells, and it can promote the formation of intergeneric hybrids of various Mycoplasma, Acholeplasma and Spiroplasma species. The extent of fusion was quantitatively evaluated by following the dequenching of octadecylrhodamine fluorescent label incorporated into donor cell membranes after their incubation with recipient cells. The results of dequenching experiments were confirmed by electron microscopy, as well as by angle light-scattering measurements. Fusion appeared to require the presence of Mg2+, but was completely inhibited by either 0.1% glutaraldehyde or 100 microM chlorpromazine, and was partially suppressed by proteolytic enzymes, carbonyl cyanide-m-chlorophenylhydrazone, or thiol reagents.

Plasmid transformation and replica filter plating of Acholeplasma laidlawii.

The restriction deficient mutant 8195 of Acholeplasma laidlawii strain JA1 was transformed by the promiscuous streptococcal plasmid vector pNZ18 at a frequency of 4 x 10(-4)/cfu. The plasmid was maintained without structural rearrangements but was lost in the absence of a selection pressure, i.e. kanamycin or neomycin. Transformed primary colonies were easily recognized due to a different colony morphology. Replica filter plating, previously not obtained with mycoplasmas, was achieved using pNZ18 as a marker by incubating the replica filters with the cell side down on the new agar plates. These findings should greatly facilitate the genetic and functional analysis of A. laidlawii.

[Dependence of membrane protein turnover in Mycoplasma cells on the age of the culture]. / Zavisimost' obnovleniia membrannykh belkov kletok mikoplazm ot vozrasta kul'tury.

To understand the molecular mechanisms of damages appearing in biological membranes in the process of cellular aging, changes in the rate of catabolic processes in Mycoplasma cells have been studied. This study has revealed that the aging of Acholeplasma laidlawii culture is accompanied by a decrease in the activity of such catabolic enzymes as DNA-ase, RNA-ase, cathepsin D and beta-glucosidase. A considerable increase in the duration of the half-life of membrane proteins has been registered, which is indicative of a decrease in their turnover rate. The electrophoretic separation of membrane proteins has revealed essential changes in their properties. Such decline in the functional activity of the plasma membrane of Mycoplasma cells at the stationary phase is probably due to the inactivation of membrane enzymes and to the decreased rate of their turnover.

Evidence for the bacterial origin of Acholeplasma laidlawii A.

L-forms from a Corynebacterium were induced in hyperosmolar media and gradually adapted to normal osmolarity over a period of two years. During this adaptation several morphologic L-form variants derived from the L-form cultures and were serologically identified as Acholeplasma laidlawii A. The possibility that the bacterial and L-form cultures were contaminated with acholeplasmas was carefully investigated; this was determined not to be the case. Membrane protein gel electrophoresis patterns of these L-form variants were identical to the ATCC A. laidlawii strain PG-8. Acholeplasma phage MVL-1 displayed no affinity for these L-form variants. Phage MVL-2 showed low affinity, but after virus enhancement in the specific host, high plaquing efficiency ensued. DNA hybridization experiments showed a high level of nucleotide sequence homology (greater than 90%) between the L-form-derived variants and PG-8. The homology between the diphtheroid L-forms and the PG-8 strain was 16.4% with te50 values of 86%; this indicates strong base pairing homology. These findings suggest that the L-form variants are acholeplasmas and that they are biologically and genetically related to the Corynebacterium L-forms.

[Comparative study of transformation and fusion induced by polyethylene glycol in Acholeplasma laidlawii B]. / Etude comparative de la transformation et de la fusion induite par les polyéthyléleneglycols chez Acholeplasma laidlawii B.

This study showed in Acholeplasma laidlawii the possibility of transformation and fusion of protoplasts after induction by polyethylene glycol. Recombination in a genome of the resistance to chloramphenicol and neomycin has been obtained. Minimal inhibitory concentration of these antibiotics towards the recombined strain are identical to those measured with the parental strains. The most suitable experimental conditions have been studied. It appeared that the fusion, which is cared out more easily than the transformation led to a higher frequency of recombination.

The effects of ionizing radiation on the peroxide content of a pure polyunsaturated lipid dispersion and of lipids and membranes derived from Acholeplasma laidlawii.

Dispersions of a pure unsaturated phospholipid, dilinoleoylphosphatidyl choline, formed conjugated diene hydroperoxides when irradiated in air with 7 MeV electrons (150 Gy and 300 Gy). Peroxide formation was optimized when the dispersions were irradiated in air at 37 degrees C at a dose rate of 5 Gy/min. No significant loss of linoleic acid from the irradiated phospholipid dispersions was observed after doses of 150 or 300 Gy. Small amounts of thiobarbituric acid-reactive material were formed in irradiated unsaturated phospholipid dispersions. However, lipids or membranes isolated from 48 hour cultures of Acholeplasma laidlawii grown in media supplemented with either linoleic or linolenic acid did not appear to be peroxidized by irradiation under the same conditions.

[Instability of host-virus association in Acholeplasma laidlawii infected by a mycoplasma virus of the Gourlay group L1]. / Instabilité de la liaison hôte-virus lors de l'infection d'acholeplasma laidlawii par un mycoplasmavirus du groupe L1 de gourlay.

Since L 1 Mycoplasmaviruses have been described by Gourlay for Acholeplasma laidlawii, two hypotheses tried to explain the relationships between virus and host, the first by a "gradual lysis", the second on the basis of a "reduced growth" of infected acholeplasmas. Neither has implied that susceptible cells, free of virus, can be obtained from infected populations, the cells being supposed to die according to the first and to be all infected but still slowly dividing according to the second hypothesis. However, we have shown, that uninfected susceptible cells do survive within infected acholeplasms. So, in the present work, we investigated the possibility that the susceptible cells may arise from a secondary dissociation between virus and host. Testing 51 clones for infectivity after isolation from two different A. laidlawii lines initially infected with two different strains of L 1 virus, we obtained 11 infected clones. The clones were studied for stability of association between virus and host. Six were shown not to be vertically stable during a serial reisolation procedure much longer than the usual cloning ("hypercloning"). Of the remaining 5 vertically stable lines, three were shown to be horizontally unstable when cloning was performed according to a multiple simultaneous technique. This indicates, that the association between virus and host cannot be considered stable and the virus is apparently of a pseudolysogenic type. Moreover, cell cloning is convenient to separate some cells from others, but may not be appropriate to isolate cells from viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid polymers--synthesis and spectral characteristics.

A new approach has been developed for the study of model and natural biomembranes. This involves the cross-linking of diacetylene groups after ultraviolet irradiation. For the study of model biomembranes, pure phospholipids (phosphatidylcholines) have been synthesized containing diacetylene groups in each acyl chain. The physical properties of these lipids have been examined and the conditions under which they polymerise have been determined. Polymerisation occurs when the lipid is in a crystalline phase, either compressed in KBr, dispersed in water (liposomes) or deposited on a suitable support (multilayers). The resultant polymer contains a conjugated backbone and is coloured. The visible spectrum of the phospholipid polymer is sensitive to its environment. Preliminary experiments show that similar polymerisation can be induced in Acholeplasma laidlawii cells grown on diacetylenic fatty acid.

Giant cell formation in Acholeplasma laidlawii, strain JA1.

Striking alterations, which looked like blebs, were observed in colonies of Acholeplasma laidlawii, strain JA 1. "Craters" were seen on the surface of these colonies by scanning electron microscopy. In addition, giant cells of acholeplasma, up to 14 microns in diameter, i.e. approximately 20 times the size of a normal A. laidlawii cell, were visible in the colonies. The large forms were surrounded by a unit membrane. After infection with group 1 and group 2 mycoplasma-viruses, the proportion of altered colonies and the number of large cells within these colonies increased. A strain of A. laidlawii, which was not susceptible to infection with the three known acholeplasma-viruses did not exhibit comparable morphological changes. The development of the giant cells can be explained either by cell fusion or by a lag of cell division behind genome replication. Blebs and "craters" may result from the destruction of mycoplasma organisms within the complex structure of the colony. There is also suggestive evidence that strain JA 1 carries a virus in some cryptic form.

Scanning electron microscopy of Acholeplasma colonies on agar.

The colony formation and morphology of Acholeplasma laidlawii and of an Acholeplasma species was studied by scanning electron microscopy. In the colonies of both Acholeplasma spp. large irregularly shaped cells, spherical cells, chains of beads, and long filaments with small bulbous distensions were seen. The membrane of some of the large cells seemed to be perforated, giving the cell a pitted appearance.

The planar distributions of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids.

We have studied the influence of changes in lipid organization on the planar distribution of two classes of membrane proteins: integral proteins which have amino groups exposed to labelling at the membrane surface by the biotinavidin-ferritin procedure, and those proteins which penetrate the lipid bilayer sufficiently to be seen as intramembranous particles by freeze-fracture electron-microscopy. When the membranes are examined at temperatures below the lipid phase transition, the first class is dispersed and the second patched. At temperatures in the middle of the transition range, both classes are patched. At temperatures just above the phase transition the first class is dispersed and the second patched, and at temperatures well above the transition both classes are dispersed. Freeze-etch studies of avidin-ferritin-labeled membranes confirmed that the distribution seen by the labeling and the freeze-fracture techniques coexist in single membranes. Thus, there exist two distinct classes of membrane proteins with differential organizational responses to the lipid state.

Molecular order in Acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids.

The first application of deuterium magentic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37 degrees C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.