[EFFECT OF MYCOPLASMA INFECTION TO FATTY ACID COMPOSITION OF CALLUS CULTURE SUGAR BEET].

It was studied the effect of Acholeplasma laidlawii var. granulum str. 118 to fatty acid composition of sugar beet calluses. It was established that acting of acholeplasma results to changes in the quantitative content of the individual fatty acids and in the qualitative composition of fatty acids in the lipids of calluses. The changing of the fatty acid composition of calluses lipids of sugar beet infected by A. laidlawii vargranulum str. 118 is observed as nonspecific response to biotic stress.

Adaptation of mycoplasmas to antimicrobial agents: Acholeplasma laidlawii extracellular vesicles mediate the export of ciprofloxacin and a mutant gene related to the antibiotic target.

This study demonstrated that extracellular membrane vesicles are involved with the development of resistance to fluoroquinolones by mycoplasmas (class Mollicutes). This study assessed the differences in susceptibility to ciprofloxacin among strains of Acholeplasma laidlawii PG8. The mechanisms of mycoplasma resistance to antibiotics may be associated with a mutation in a gene related to the target of quinolones, which could modulate the vesiculation level. A. laidlawii extracellular vesicles mediated the export of the nucleotide sequences of the antibiotic target gene as well as the traffic of ciprofloxacin. These results may facilitate the development of effective approaches to control mycoplasma infections, as well as the contamination of cell cultures and vaccine preparations.

Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis.

For the first time, we studied the phytopathogenicity toward Oryza sativa L. of unadapted and adapted to unfavorable environment (starvation) cells of Acholeplasma laidlawii PG8--ubiquitous mycoplasma found in the soil, waste waters, tissues of the highest eukaryotes and being the basic contaminant of cell cultures and a causative agent of phytomycoplasmoses. The features of morphology, ultrastructural organization and proteomes of unadapted and adapted cells of the mycoplasma and infected plants were presented. Using 2D-DIGE and MS, 43 proteins of O. sativa L. that were differentially expressed in the leaves of plants cultivated in media with A. laidlawii PG8 were identified. The qualitative and quantitative responses of the plant proteome toward adapted and unadapted mycoplasma cells differed. That may be explained by differences in the virulence of the corresponding bacterial cells. Using 2D-DIGE and MS, 82 proteins that were differentially expressed in adapted and unadapted mycoplasma cells were detected. In adapted cells of the mycoplasma, in comparison with unadapted ones, a significant increase in the expression of PNPase--a global regulator of virulence in phytopathogenic bacteria occurred; there was also decreased expression of 40 proteins including 14 involved in bacterial virulence and the expression of 31 proteins including 5 involved in virulence was not detected. We propose that differences in the phytopathogenicity of adapted and unadapted A. laidlawii PG8 cells may be related to features of their proteomes and membrane vesicles.

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.

The growth and long term survival of Acholeplasma laidlawii in media products used in biopharmaceutical manufacturing.

Acholeplasma laidlawii is a potential contaminant of bovine serum and has also been found as a contaminant in serum free cell culture media products. Anecdotal evidence of A. laidlawii contamination of tryptone soya broth circulated for a number of years before it was acknowledged that the organism could contaminate microbiological broth powders. The occasional occurrence of A. laidlawii in broth powders and possibly in powdered components of cell culture media as part of the normal bioburden poses a serious threat to routine pharmaceutical and biopharmaceutical operations where filtration is the sterilisation method of choice. Absence of visual evidence of contamination cannot be relied upon as there is variation with both organism strain and media product in the ability to produce turbidity. Strains of A. laidlawii which have been isolated from broth powders are not significantly different in temperature or media preferences from other strains. A. laidlawii is capable of growing to high titre at refrigeration and ambient temperatures in unsupplemented bacteriological sterility media or serum free cell culture media and can survive for prolonged periods in these products.

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.

[Adaptive reactions of mycoplasmas in vitro: "viable but unculturable forms" and nanocells of Acholeplasma laidlawii].

The adaptation of Acholeplasma laidlawii to conditions unfavorable for growth has been found to be accompanied by cell transformation into special morphological structures known as ultramicroforms (nanocells). The ratio of the cells of the two morphological types in the population depended on the growth conditions. Nanocells retained viability for a long time under conditions unfavorable for growth and showed resistance to stressors. Reduction in the cell size occurred due to unequal division, which involved the loss of cytoplasmic material. A. laidlawii ultramicroforms (nanocells) were able to restore proliferative activity and to revert to their initial vegetative form; they measured less than 0.2 microm and are the smallest cells known at present. Nanocells formed in vitro under exposure to abiogenic stressors may correspond to the A. laidlawii minibodies observed in infected plants upon exposure to biogenic stressors. The transformation of A. laidlawii cells into ultramicroforms was accompanied by condensation of the nucleoid, a change in the polypeptide spectrum, and a change in the availability of rRNA operons for in vitro amplification. All these changes are indicative of reorganization of the genetic and metabolic systems of mycoplasmas.

Alternative to fluorescence assays to monitor fusion between Acholeplasma laidlawii cells and liposomes.

AIMS: To develop a new technique as an alternative to the fluorescence assays and electron microscopy for the purpose of monitoring the cell-liposome fusion. METHODS AND RESULTS: Acholeplasma laidlawii whole cells did not oxidize Glucose-6-phosphate (G6P) or Fructose-1,6 diphosphate (F1,6DP) as free (unentrapped) substrates, at concentrations 47 and >270 mM, respectively. Lysed A. laidlawii cells oxidized G6P and F1,6DP at lower concentration of 0.8 and 15 mM, respectively. When these substrates were entrapped inside liposomes, at a final concentration of 1.5 mM, and interacted with A. laidlawii whole cells, in an oxygen electrode chamber, an increase in oxygen uptake was evident. This interaction does not have any effect on cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental system described here is advantageous over classical fluorescence assays in determining the fate of liposome-entrapped material and raises the possibility of studying the kinetics of metabolic substrates, which are normally excluded from the cell by the cell membrane.

Mycoplasma stimulates HIV-1 expression from acutely- and dormantly-infected promonocyte/monoblastoid cell lines.

Treatment of a myelo-monocyte cell line, J22HL-60, dormantly infected with human immunodeficiency virus type 1 (HIV-1) with heat-inactivated extracts of Acholeplasma (A) laidlawii (250 micrograms/ml) enhanced virus production more than 45-fold as assessed by p24 viral core antigen assay. When treated with a suboptimal dose of TPA or TNF-alpha, Acholeplasma extracts further augmented virus production in J22HL-60 cells. H7, an inhibitor of protein kinase C(PKC), almost completely abrogated HIV-1-inducing ability of Acholeplasma extracts in the cells. A. laidlawii and several other mycoplasmas also enhanced acute infection of U937 cells as shown by increased virus-positive cells and augmentation of HIV-1 production in the culture supernatant independent of their pathogenicity to humans.

Ageing of procaryotes. Acholeplasma laidlawii as an object for cell ageing studies: a brief note.

It was established that A. laidlawii cell viability in the stationary phase of culture development decreases according to the Gompertz law. It was shown that the "stationary" ageing of A. laidlawii cells is accompanied by the lower intensity of phosphorus-containing compounds biosynthesis, decreased rate of membrane proteins synthesis and degradation and also by accumulation of DNA single-strand breaks. The biochemical changes found in ageing A. laidlawii cells are similar to ageing manifestations of resting eucaryotic cells. According to the authors opinion, the received data show that the A. laidlawii cell culture may be used as an object for investigation of the cell ageing mechanisms.

Inactivation of the vascular permeability-increasing activity of bradykinin by mycoplasmas.

Mycoplasma pneumoniae, M. genitalium, M. fermentans, M. hominis, M. salivarium, M. orale, Ureaplasma urealyticum and Acholeplasma laidlawii inactivated the vascular permeability-increasing activity of bradykinin when the mixture of bradykinin and mycoplasma cells was injected after incubation at 37 degrees C for 1 h. Cell components responsible for inactivation of the activity of bradykinin were found to be arginine-specific aminopeptidase and carboxypeptidase.

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.

Animal viruses are able to fuse with prokaryotic cells. Fusion between Sendai or influenza virions and Mycoplasma.

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.

Membrane potential, lipid regulation and adenylate energy charge in acyl chain modified Acholeplasma laidlawii.

In Acholeplasma laidlawii variations induced in the transmembrane electrical potential have been shown to affect the membrane lipid composition. Particularly the molar ratio between the predominant glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol, decreases upon hyperpolarization and increases upon depolarization (Clementz et al. (1986) Biochemistry 25, 823-830). Upon variation of the degree of membrane fatty acyl chain unsaturation, known to affect the passive permeability for a number of small molecules, there was no significant correlation between acyl chain composition and the magnitude of the electrical potential. Hyperpolarization by valinomycin decreased the glucolipid ratio for all kinds of membranes, but the size of the decrease was not correlated to the acyl chain composition. However, a clear relationship, independent of acyl chain composition, was found between the extent of hyperpolarization and the size of the decrease in the glucolipid ratio. The adenylate energy charge value (Ec) of the cells was affected by the acyl chain composition, although not exclusively by the proportion of unsaturation. Furthermore, a larger hyperpolarization upon valinomycin addition was accompanied by a stronger reduction in Ec.

Acholeplasma laidlawii cells acutely and chronically infected with group 1 acholeplasmavirus.

Interactions between group 1 acholeplasmaviruses and their host cells were studied. Acutely infected, chronically infected and uninfected cultures of Acholeplasma laidlawii strain JA1 were compared by their growth in broth and on agar, by the sensitivities of the uninfected and chronically infected cells to representatives of each of the three groups of acholeplasmaviruses, and by their SDS-PAGE polypeptide profiles. Acutely infected cells resembled uninfected cells by these criteria, except for the fact that progeny virus was being released. Two types of chronically infected cells were found:rapid growers (the same doubling time as uninfected cells) and slow growers. The latter resembled uninfected cells, except for their slower growth and low-level release of virus, and the former was resistant to group 1 viruses and had a unique polypeptide profile. These biological characterizations help to establish the non-lytic, non-cytocidal cycle of the group 1 acholeplasmaviruses.

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.

Effects of acyl chain composition on production of mycoplasmavirus MV-L2 by Acholeplasma laidlawii.

Membrane-enveloped viruses obtain their lipid envelopes from the host cells. Studies with animal viruses and different cell lines have shown that the composition of the virus envelope affects infectivity. In this study, Acholeplasma laidlawii strain JA1 and the membrane-enveloped virus MV-L2 were used. In this system, it is possible to vary the "stiffness" of the host cytoplasmic membrane within a very wide range by adding different fatty acids to the growth medium. Viruses with different envelopes were produced from these hosts. Eleven hosts and 11 viruses with different fatty acyl chain composition were tested in all combinations. The acyl chain composition and presence of cholesterol had a marked influence on the plaque-forming ability of L2. Virus adsorption rates were also dependent on acyl chain composition, but no apparent correlation with plaque-forming ability could be seen. L2 adsorbed equally well to a virus-resistant strain (A EF22).

Calorimetric and spectroscopic studies of lipid hydrocarbon chain order in the Acholeplasma laidlawii membrane.

The thermotropic phase behavior of Acholeplasma laidlawii B membranes, whose lipids were biosynthetically highly enriched with one of a wide variety of different types of fatty acyl groups, was studied by differential scanning calorimetry. The membrane-lipid-hydrocarbon chain orientational order of these membranes, in both the gel and liquid-crystalline states, was also investigated by 19F-nuclear magnetic resonance spectroscopy, using biosynthetically incorporated monofluoropalmitic acid probes. The gel to liquid-crystalline phase transition midpoint temperature, Tm, for a series of membranes containing different types of fatty acyl groups, but with a similar number of carbon atoms, decreases in the order: linear saturated greater than methyl isobranched greater than methyl anteisobranched greater than or equal to trans-monounsaturated greater than cis-monounsaturated. At comparable reduced temperatures below their respective Tm values, the degree of hydrocarbon chain order correlates strongly and positively with Tm, indicating that Tm is determined primarily by the efficiency of chain packing in the gel state. At comparable "reduced" temperatures above Tm, the degree of hydrocarbon chain orientational order is moderately and inversely correlated with Tm. At any one absolute temperature in the fluid state, however, orientational order does correlate weakly and inversely with Tm, due to a small inherent decrease in order with temperature. Thus, to a first approximation, membrane lipid order in the liquid-crystalline state is directly proportional, and membrane lipid fluidity inversely proportional, to the gel to liquid-crystalline phase transition temperature. However, the magnitude of this effect lessens with decreasing proximity to the Tm. Finally, evidence is presented that the presence of membrane protein has little if any effect on the average orientational order of the membrane lipid hydrocarbon chains in either gel or liquid-crystalline states.