[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.

[Mycoplasma heat shock proteins and their genes].

Mycoplasmas (class Mollicutes) are the most simply organized prokaryotic organisms capable to self-reproduction. They are considered as a model of "minimal" cell. Systems preserved by mycoplasmas in their reductive evolution may play a fundamental role in viability of any cell. Information on the genes encoding heat shock proteins (HSP) in completely sequenced mycoplasma genomes was summarized and systematized. An attempt was made to explain the presence or absence in mycoplasmas of important bacterial chaperones and proteases. These HSP are necessary not only for cell stress resistance, but for protein homeostasis under normal conditions. The mechanisms of regulation of transcription of corresponding genes are considered. Properties and functions of the most completely characterized mycoplasmal HSP are discussed: DnaK, DnaJ-like, GroEL/GroES, ClpB, and small heat shock proteins (sHSP).

[Localization of the division protein FtsZ in mycoplasma cells Mycoplasma hominis].

Localization of the protein FtsZ in Mycoplasma hominis cells was determined. Ultra thin sections were treated by rabbit polyclonal antibodies against FtsZ M. hominis: a conjugate of protein A with colloidal gold particles was used instead of secondary antibodies. Considerable polymorphism of cells was seen on electron microscopy pictures of M. hominis cells, which is typical for mycoplasmas. Among a wide variety of cell shapes we distinguished dumbbell-shaped dividing cells, and the cells connected with each other with the aid of thin membrane tubules (former constrictions). Dominants distribution of the label in the constriction area of dividing M. hominis cells and in the area of the thin membrane tubules was observed. We revealed the cross septum in the mycoplasma cells for the first time, as well as the gold labeling of this structure. Furthermore, in some rounded and oval cells colloidal gold particles labeled the whole plasma membrane in ring-shaped manner. Probably, the label in these cases marks a submembrane contractile ring (Z-ring). The facts mentioned above confirm that FtsZ of M. hominis plays an active role in the mycoplasma cytokinesis. In a series of cases spiral-like distribution of gold particles was observed. Probably, FtsZ protofilaments in M. hominis cells can form spiral structures similar to Z-spirals of Bacillus subtilis and Escherichia coli. Its presence in mycoplasma cells may be considered as an important argument in favour of model of Z-ring assembling through reorganization of Z-spirals. FtsZ also may participate in maintenance of mycoplasma cell shape (membrane localization).

[The heat shock protein of alpha-crystallin type from mycoplasma (Acholeplasma laidlawii)].

A considerable increase in several heat shock proteins (HSPs) amount in Acholeplasma laidlawii cells has been revealed after temperature rising of liquid culture; and the quantity of small HSP, named p17, was increased in a hundred of times. The p17 protein was isolated and identified as HSP of alpha-crystallin type (alpha-HSP). It became possible as a result of sequencing of 15 amino acids from N-terminal of the p17 polypeptide chain, followed by revealing of a corresponding open reading frame (ORF) in a completely sequenced genome of A. laidlawii PG 8A. Computer-based search for homologous ORFs in all 17 genomes of Mycoplasmataceae family (the mycoplasmas themselves) that had been completely sequenced to date, gives negative result. But among the representatives of Mollicutes (mycoplasma) class, the genes coding alpha-HSPs were found in two Phytoplasma species (Phytoplasmataceae family) and the acholeplasma examined (Acholeplasmataceae family). It supposed that presence or absence of alpha-HSPs in microorganisms might be connected with the fact that representatives of Acholeplasmataceae and Phytoplasmataceae families inhabit principally in plant tissues in contrast to majority of Mycoplasmataceae family, that inhabit animal and human tissues, i. e. use ecological niches with relatively constant temperature.

[FtsZ and the division of bacterial cell].

In this review we have tried to describe proteins and supermolecular structures which take part in the division of bacterial cell. The principal cell division protein of the most of prokaryotes is FtsZ, a homologue of eukaryotic tubulin. FtsZ just as tubulin is capable to bind and hydrolyze GTP. The division of bacterial cell begins with forming of so called divisome. The basis of such divisome is a contractile ring (Z ring); the ring encircles the cell about midcell. Z ring consists of a bundle of laterally bound protofilaments, which have been formed as a result of FtsZ polymerization. Z ring is rigidly bounded to cytozolic side of inner membrane with participation of FtsA, ZipA, FtsW and many other cell division proteins of divisome. The ring directs the process of cytokinesis transmitting power of constriction to membrane. Primary structures of members of the family of prokaryotic FtsZs differ from eukaryotic tubulines significantly except the region, where the site of GTP binding is placed. There is high degree of homology between structures of these proteins in the region. FtsZ is one of the most conservative proteins in prokaryotes, but ftsZ genes have not been found in completely sequenced genomes of several species of microorganisms. There are 2 species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea among them. So these organisms divide without FtsZ. There are many open reading frames which encode proteins with unknown functions in genomes of U. parvum and M. mobile. The comparison of primary structures of these hypothetical proteins with structures of cell division proteins did not allow researchers to find similar proteins among them. We suppose that the process of cell division of these organisms should recruit proteins with function similar to FtsZ and having homologous with FtsZ or other cell division proteins spatial structures.

[Cloning and expression of the Mycoplasma hominis ftsZ for a cell division protein]. / Klonirovanie i ékspressiia gena, kodiruiushchego belok deleniia mikoplazmy (ftsZ, Mycoplasma hominis).

A Mycoplasma hominis chromosomal fragment containing the full-length ftsZ gene was cloned and sequenced. Natural expression of this gene was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) with total RNA. The M. hominis FtsZ protein was shown to differ substantially from its counterparts of two other Mycoplasma species, M. genitalium and M. pneumoniae. The possibility of M. hominis ftsZ expression in Escherichia coli was demonstrated with several bacterial strains. The M. hominis FtsZ protein was isolated from E. coli cells transformed with recombinant plasmids carrying the M. hominis ftsZ gene. Complementation between the E. coli and M. hominis FtsZ proteins was observed in transformants.

[Interaction of Mycoplasma with immune system of animals and humans]. / Vzaimodeistvie mikoplazm s immnnoi sistemoi zhivotnykh i cheloveka.

Mycoplasmal infections of animals and human belong to latent or chronic ones, being commonly accompanied with immunodeficiency symptoms. The following topics are discussed in this review: 1) a direct influence of mycoplasmas on the immune system cells with an interference and compression of the functional activity of these; 2) influence of mycoplasmas on the immune system via cytokine induction; 3) mitogenes and mitogenic action of mycoplasmas; 4) the role of mycoplasmas in the development of autoimmune processes. Basic host immune response to mycoplasma is presented in diagrams.

[Cloning and expression of the gene for the mycoplasma key division protein FtsZ in Escherichia coli]. / Klonirovanie i ekspressiia gena kliuchevogo belka deleniia FtsZ mikoplazmy v Escherichia coli.

Gene ftsZ responsible for division of bacterial cells was revealed in most prokaryote groups. A 520-bp fragment of the ftsZ gene was amplified on the template of A. laidlawii DNA using degenerate primers. This fragment was sequenced and served as a hybridization probe for cloning of the full-sized copy of the A. laidlawii ftsZ gene. The amplified fragment was cloned in a pGEX3X vector and expressed in E. coli cells. Polyclonal antibodies derived from the chimeric polypeptide containing a fragment of A. laidlawii FtsZ protein interacted only with the A. laidlawii protein with molecular mass of 40 kDa. Comparison of nucleotide sequences of the ftsZ-gene region of A. laidlawii and other bacterial species showed that they were highly homologous in A. laidlawii, E. coli, and Bac. subtilis, while low homology was revealed between the A. laidlawii sequence and those of the members of the genus Mycoplasma. Analysis of the ftsZ-gene nucleotide sequences is suggested as a means to study the evolutionary relatedness of prokaryotes.

[Cloning universal probes for detecting mycoplasma contamination of cell cultures]. / Kolnirovanie universal'nykh zondov dlia vyiavleniia mikoplazmennogo zarazheniia kletochnykh kul'tur.

To obtain the universal polynucleotide hybridization probes for testing mycoplasmal contaminations in cell cultures, we have cloned several DNA fragments from the srRNA gene of Acholeplasma laidlawii. Before cloning, in order to exclude cross-hybridization of these probes with eukaryotic rRNA, the thermodynamic parameters of duplex formation between DNA complementary to mycoplasmal rRNA and eukaryotic rRNA had been studied. Using a set of computer methods, the region which forms weak heteroduplexes with eukaryotic srRNA was revealed. This region occupies positions 250 to 550 position of the mycoplasmal srRNA. Three different DNA fragments which include the region were generated in PCR, cloned in pUC18, and their hybridization characteristics were evaluated. In appropriate hybridization conditions the probes hybridize with all mycoplasmal RNAs studied without cross-hybridization with eukaryotic ribosomal RNA and DNA, and allow one to detect virtually any mycoplasmas (or any prokaryote) in cell cultures. Blot-hybridization of universal probes with mycoplasmal DNA digested by BsuRI allows one to identify the different species of mycoplasmas.

[Diagnosis of mycoplasma infections using directed amplification]. / Diagnostika mikoplazmennykh zarazhenii s pomoshch'iu napravlennoi amplifikatsii.

Test systems for rapid detection of mycoplasmas in biological samples have been elaborated on the base of the polymerase chain reaction (PCR). Amplification of the conservative rDNA sequences was used for testing of cell cultures for mycoplasmal contamination. Mycoplasma pneumoniae detection system has been developed based on amplification of the species-specific DNA sequences. Inversions of some repeated sequences in the Mycoplasma pneumoniae genome make it possible to run the PCR with a single primer. The revealed spacer length polymorphism for 16S-23S rDNA operons can be used in the mycoplasmas identification.

[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.

[Molecular biological differences between strains of Mycoplasma gallisepticum]. / Molekuliarno-biologicheskie razlichiia mezhdu shtammami Mycoplasma gallisepticum.

Differences in virulence of two Mycoplasma gallisepticum strains, S6 and A5969, are confirmed in experiments with chickens. Macromolecular discrepancies detected between these two strains are concerning the genomic size, electrophoretic spectra of DNA and proteins. Cross immunoblotting data with polyclonal and monoclonal antibodies reveal major immunogens of protein nature in both the strains. Homologous proteins with different electrophoretic mobility are detected in other four M. gallisepticum strains. A possible participation of these proteins of M. gallisepticum in adhesion to the host cells is discussed.

[A family of repetitive nucleotide sequences (Sau 3A family) in the genome of 2 forms of the sockeye salmon Oncorhynchus nerka]. / Semeistvo povtoriaiushchikhsia posledovatel'nostei nukleotidov (Sau 3A-semeistvo) v genomakh dvukh form tikhookeanskogo lososia Oncorhynchus nerka.

A family of DNA sequences (Sau3A repeat sequences) has been revealed in genomes of two forms of O. nerka. A DNA fragment belonging to this "family" has been cloned in bacterial plasmid. Its copy number (in genomes of normal (570 +/- 33) and dwarf (66 +/- 12) forms) has been counted. The structural organization of sequences of Sau3A family in genomes of males and females of normal form is not the same. The organization of Sau3A sequences is the same in genomes of dwarf males and dwarf females of normal form, but the quantity of Sau3A sequences copies is smaller. It is supposed that the DNA sequences of Sau3A family bear the sex determination of dwarf form of O. nerka. The fragment of rRNA gene of nerka has been cloned and the number of rRNA gene copies in genomes of normal (200 +/- 62) and dwarf (1690 +/- 24) forms has been defined.

[Detection and identification of Mycoplasma infections by DNA hybridization]. / Obnaruzhenie i identifikatsiia mikoplazmennykh zarazhenii metodom DNK-gibridizatsii.

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.

[The Mycoplasma genome]. / Genom mikoplazm.

A review is presented of the available data on the nature of chromosomal and extrachromosomal DNA of Mollicutes (mycoplasmas)--the smallest and simplest procaryotic organisms.

[Mycoplasmas]. / Mikoplazmy.

Structural peculiarities of the mycoplasmas--the smallest prokaryotic organisms--are reviewed, in addition to their complicated relationships with the eukaryotic cells and with the whole organisms of plants and animals.

[Palindromic and repetitive sequences in initiation sites of DNA replication in Tetrahymena]. / Palindromnye i povtoriaiushchiesia posledovatel'nosti nukleotidov v uchastkakh initsiatsii sinteza DNK u infuzorii Tetrahymena.

DNA replication was induced in Tetrahymena pyriformis cells at the stationary phase of culture growth by a series of heat shocks. The selective nature of this DNA synthesis was established. DNA was uniformly labelled with [3H]thymidine during the exponential phase of culture growth and with [14C]thymidine during the stationary phase. The renaturation kinetics of DNA samples was investigated by measuring the relations of 14C/3H radioactivity in the renatured and denatured DNA fractions after their separation on hydroxyapatite. The results were analysed by means of a computer program based on the experimentally found renaturation curve of T. pyriformis macronuclear DNA. Under conditions described palindromic and repetitive (which are repeated about 1000 times) nucleotide sequences are intensively replicated. It is assumed that synchronous activation of replicons occurs as a consequence of induction. It may well be that replication starts in the central parts of comparatively long palindromes, which are identical or very similar in their nucleotide sequences.