[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 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.

[Effect of wheat germ agglutinin on acholeplasma--the pathogen of phytomycoplasmosis].

Introduction of wheat germ agglutinin (WGA) to cultural medium of pale green dwarf agent of wheat Acholeplasma laidlawii var. granulum str. 118 gives rise to pleiotropic responses of acholeplasma: activation of growth process, an increase of common protein in comparison with control, a decrease of hemagglutinating activity which results in a decrease of the adhesion properties of pathogen.

[Comparative investigation of fructosobisphosphatases of Bacillus subtilis and pale-green dwarfism pathogens of cereals in the reaction of double diffusion in gel according to Ouchterlony].

Serological properties of fructosobisphosphatases (FBPases) of Bacillus subtilis 668 and PGD agent of cereals--the mollicute Acholplasma laidlawii var. granulum st. 118 (Alg 118) were studied in a comparative aspect with the help of the reaction of double diffusion in gel according to Ouchterlony. It was established for each of microorganisms that their extracellular and intracellular enzymes are similar in serologic respect, and each of them is composed of two antigens, one of them being identical in the both microorganisms, while the other displays only partial identity, since it reacts with antibodies in heterological systems with formation of a precipitation line looking as a "spur". That indicates to the fact that antisera to those enzymes contain antibodies both to general determinants of antigens which are compared (FBPases here), and to the determinant absent in one of them. Basing on the investigation results it is concluded that FBPase of B. subtilis is rather similar than identical, in serological aspect, to the enzyme Alg 118 of the same name.

Acholeplasma laidlawii PG8 culture adapted to unfavorable growth conditions shows an expressed phytopathogenicity.

Mycoplasmas are the smallest, self-replicating, prokaryotic organisms with avid biochemical potential and spreading in higher eukaryotes in nature. In this study, Acholeplasma laidlawii PG8 cells were cultivated on a deficient medium for 480 days resulting in a mycoplasma culture that was adapted in vitro to unfavorable growth conditions. Cells that survive this condition had decreased sizes (about 0.2 microm) and increased phytopathogenicity. This resulted in more frequent appearance of various morphological alterations when plants of vinca (Vinca minor L.) were infected by adapted mycoplasma cells. The increasing pathogenicity was accompanied by changes in genome expression in these adapted cells. Further studies are needed to explore the exact mechanisms that permit adaptation to unfavorable growth conditions and changes in phytopathogenic potential.

Taxonomic status of the agent of cereals pale-green dwarf and proposition of founding in the class of Mollicutes, of the order III Acholeplasmatales, family I Acholeplasmataceae, genus II Pluraplasma gen. nov., and its first species Pluraplasma granulum sp. nov.

The paper includes the data concerning the taxonomic status of the agent of cereals pale-green dwarf (ACPGD) which has been defined as the phytopathogenic variant of the mollicute Acholeplasma laidlawii and called A. laidlawii var. granulum. Since besides phytopathogenicity ACPGD has such fundamental differences from A.laidlawii as: a very large genome of 2200 thousand pairs of nucleotides (t.p.n.) to 2310 t.p.n. that practically equals a sum of genomes of A. laidlawii (1600 t.p.n.) and phytoplasmas (710 t.p.n.); two forms of DNA-dependent RNA-polymerase (one form functions in A. laidlawii); a capacity to form extracellular fructosobisphos-phatase which looks like its hypothetical phylogenetic precursor a bacteria Bacillus subtilis; availability of numerous fermentative activities which are absent in acholeplasmas; peculiar relation to sterols availability in nutrient media that is not characteristic of all the known acholeplasmas; extremely rich, as to quantity and quality, composition of antigens to react almost homologously with antibodies to representatives of Acholeplasma genus and separate species of Mycoplasma genus; great similarity (above 88 %) of sequences of 16S rRNA of ACPGD and representatives of Phytoplasma genus and other properties described in the paper, so it is concluded, that proceeding from its characteristics, ACPGD cannot be referred to either of the existing genera of the Mollicules class, because according to all its features this mollicute is a transition form of the microorganism, it is the hitherto unknown chain between these genera of mollicutes and sporiferous bacteria, ACPGD is a probable representative of mollicute precursor with genome of about 1600 t.p.n. and, first of all, of genera Acholeplasma and Phytoplasma which arised as a result of the evolutionary splitting of its genome. On this basis, it is recommended to found for ACPGD in the Mollicutes class, order III Acholeplasmatales, family I Acholeplasmataceae, a new genus II Pluraplasma gen. nov. and its first species Pluraplasma granulum sp. nov., the strain 118 being its typical representative.

[On the nature of the pathogen of pale-green dwarfism in cereals].

The paper presents more precise data concerning optimal temperature demands to growth of white-yellow dwarfness of cereals (WYDC) identified before as Acholeplasma laidlawii var. granulum, its relation to sterols and genome properties was determined using pulse-electrophoresis. It was established that the agent strains 84 and 118, characterized by phytopathogenicity, grew most intensively at 32 degrees C; they behaved as mesoplasmas but, as it had been found, they were capable to synthesis of carotenoids and displayed close serologic affinity for A. laidlawii PG8. That is, the above strains are typical acholeplasmas capable to live in leafhoppers which carry a disease and in cereals plants and cause a disease with typical symptoms of "yellows" in the latter. Molecular weight of the strain 84 genome was 2200 t.p.n. (GC = 33 mol %); in strains 118 it was 2310 t.p.n. (GC = 34.2 mol %). Allowing for the fact that molecular weight of genome of A. laidlavii var. granulum is almost by 1/3 (1600 + 710 t.p.n.) more than that of A. laidlawii PG8 genome, the authors think that the agent of WYDC is the evolution precursor (or one of precursors) which initiated the Acholeplasma and Phytoplasma genera as a results of splitting of their genomes.

[The effect of the Mycoplasma contamination of 2 kidney cell sublines from the kangaroo rat on their karyotypic structure]. / Vliianie mikoplazmennoi kontaminatsii dvukh sublinii kletok pochki kengurovoi krysy na kariotipicheskuiu strukturu.

The karyotypic variability has been investigated for two cell sublines of Rat kangaroo kidney cultured for 40-160 days after contamination with Acholeplasma laidlawii, strain PG-8. The contaminated cultures did not differ from non-contaminated ones in cell distribution for chromosome number. The majority of cells of subline NBL-3-11 with modal number of chromosomes displayed the main structural variant of the karyotype (SVK)--2+2+2+2+2+1; in subline NBL-3-17 the main SVK being 3+3+3+3+3+2. A comparison of intact cultures of these sublines in cell distribution for chromosome number show just the opposite direction of aneuploidy processes: cell heterogeneity for chromosome number decreased in NBL-3-11 and increased in NBL-3-17. The quantity of chromosomal aberrations, primarily chromosomal breaks, increases within 40-160 days of cultivation of contaminated cells of subline NBL-3-11. The number of chromosomal aberrations, mainly at the expense of dicentrics due to telomeric associations, increases after 40 days of cultivation of subline NBL-3-17 contaminated cells. During a long-term cultivation (110 days) of subline NBL-3-17 intact cells, there is an increase in the number of chromosomal aberrations, mainly dicentrics, whereas the extent of chromosomal breaks appears much less. The present results and other additional experimental data make it possible to suppose that the increase in chromosomal instability seen in subline NBL-3-17 at a long-term cultivation may be characteristic of this culture, in distinction to subline NBL-3-11. The most frequent breaks were seen in chromosomes 1, 2 and X of intact and contaminated cells in both the sublines. Chromosomes 1, 2 and 4 are mainly involved in dicentric formations by q (long) arms. The role of dicentrics in cell adaptation to in vitro conditions is discussed.

[The effect of mycoplasmal contamination of cultures of skin fibroblasts from the Indian muntjac and of the subsequent decontamination of the cultures using ciprofloxacin on the karyotypic structure of the cell line]. / Vliianie kontaminatsii mikoplazmoi kul'tur fibroblastov kozhi indiiskogo muntzhaka i posleduiushchei dekontaminatsii kul'tur s pomoshch'iu tsiprofloksatsina na kariotipicheskuiu strukturu kletochnoi linii.

The karyotypic variability of Indian muntjac skin fibroblast cell line, cultured for 95-168 days after contamination with Acholeplasma laidlawii strain PG-8, has been investigated. The contaminated cultures differ from noncontaminated ones in cell distribution for chromosome number. The noncontaminated cultures have modal number of chromosomes equal to 7 with the main structure variant of the karyotype (SVK) 2+2+1+1+1. In the contaminated cultures the cell number with 7 chromosomes and the main SVK 2+2+1+1+1 decreased, whereas the cell number with 6 chromosomes increased along with the main SVK 2+2+1+1 resulting from the loss of chromosome Y1. The treatment of cells with ciprofloxacin for mycoplasma decontamination did not restore the normal cell distribution for chromosome number. The frequency of chromosomal aberrations, mainly dicentrics, due to telomeric associations, increased after 95-168 days of cultivation of contaminated cells. Chromosomes 1 and 2 and their combination are mainly involved in dicentric formations. The treatment of contaminated cells with ciprofloxacin restores the initial frequency of chromosomal aberrations. Chromosomes with altered structures have not been demonstrated. It has been shown that cells became mycoplasma-free after 15 days of treatment with ciprofloxacin. The role of aneuploidy and dicentrics in cell adaptation to culture conditions is discussed.

[Soluble and semisoluble lectins from mollicutes and their possible functions]. / Rozchynni ta napivrozchynni lektyny molikutiv i ïkh mozhlyvi funktsiï.

When studying mollicute lectins it was established that Acholeplasma laidlawii PG-8 synthesizes two half-soluble lectins one of which is specific to N-acetyl-D-glucosamine, and the other--to D-glucosamine.HCl; phytopathogenic strain A. laidlawii var. granulum 118 produced 4 lectins one of which is soluble and specific in respect to fructose-1.6-diphosphate, the rest three lectins are half-soluble and specific to one of the sugars--D-galactosamine.HCl, rafinose and D-glucosamine.HCl. In Mycoplasma pneumoniae FH all the four lectins found in the culture liquid have been classified as half-soluble, specific to one of carbohydrates--D-galactosamine.HCl, talose, N-acetyl-neuramine acid and D-glucose; M. capricolum Cal. Kid. synthesizes four lectins; two of them being defined as soluble (one of the lectins is, respectively, specific to talose and D-glucosamine.HCl, two others, as half-soluble, specific to one of sugars--rafinose or D-glucose. The results obtained permit a conclusion to be made that the half-soluble lectins of mollicutes, on the one hand, are the factors of adhesion on the corresponding organs of macroorganism and, on the other hand take part in the transport of substances from without into the microorganism cell. Soluble lectins determine pathogenicity of mollicutes and form with half-soluble lectins a single chain to providing the mycoplasma cells with nutrients and to protect them from the action of the macroorganism immune system.

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.

Isolation and pathogenicity of mycoplasmas from geese.

Several mycoplasma isolation trials were performed on infertile goose eggs and goose embryos which died during incubation, as well as on geese of different ages. A total of 43 out of 110 goose eggs proved to be contaminated by mycoplasmas. Upon autopsy of birds which laid positive eggs, lesions were observed in the airsacs. Mycoplasmas could be isolated from their air sacs and oviduct. Four out of 15 strains examined biochemically and serologically with antisera prepared against all known avian mycoplasma species were identified as Acholeplasma laidlawii and A. axanthum, respectively. Two strains proved to be glucose-positive and arginine-negative and 9 were glucose-negative but arginine-positive. Some strains caused 50-80% mortality among embryos inoculated intra-yolk-sac at 12 days. In goslings inoculated at the age of 3 days with these strains, we observed fibrinous airsacculitis and peritonitis. By inoculating laying geese with one of the strains, we demonstrated decreasing egg production, increasing early-embryo mortality and egg transmission of mycoplasmas.