[Biological cost of rifampicin resistance in Helicobacter pylori].

The frequence of mutations in the rifampicin resistant (RIF(r)) clones of microorganisms after adaption to ofloxacin and metronidazole was investigated to estimate the biological cost of H. pylori rifampicin (RIF) resistance. Mutations in rpoB gene responsible for RIF resistance of H. pylori were shown to have biological cost and be compensated by additional mutations in the microorganism genome. Comparison of the mutation frequency in the presence of metroniazole demonstrated that the acquired resistance to RIF resulted in changing of the adaptative capacity of the RIF(r) clones of H. pylori to metronidazole. Thus, a significant increase of the mutation frequency (> 700 times) in one of the RIF(r) clones and a broad spectrum of the mutations responsible for resistance to metronidazole vs. the H. pylori initial strain 26695 were observed. The findings could be evident of the fact that the adaptation to RIF changed the properties of the cell on one hand in such a way that its mutation capacity increased and that the target selection on the other hand revealed hypermutable cells, likely usual for the bacterial population.

[Population identification of Helicobacter pilory isolates from Russia].

Using multilocus sequence typing (MLST), 22 Helicobacter pylori isolates from Russia have been characterized. All of the Russian strains were assigned to a single population, hpEurope.

Comparative analysis of transcription profiles of Helicobacter pylori clinical isolates.

The transcription profiles of four Helicobacter pylori clinical isolates (two cag-negative and two cag-positive) were compared in stationary growth phase using a cDNA-macroarray. The correlation coefficient value between total transcription profiles of clinical isolates H. pylori varied from 0.70 to 0.83. For 44 groups of genes (total number 66) belonging to various functional classes of H. pylori, the correlation coefficient value between these isolates exceeded 0.7, and for 14 groups the value exceeded 0.9. These groups included genes encoding components involved in cell division, adaptations to atypical conditions, electron transport, salvage of nucleosides and nucleotides, glycolysis/gluconeogenesis, folding and stabilization of proteins, translation factors, anaerobic metabolism, and amino acids and amine metabolism. Expression of 52 genes significantly differed between H. pylori clinical isolates. Some of these genes determine microorganism virulence. They include: cytotoxin-associated gene (cagA), genes encoding neutrophil-activating protein (napA), major flagellar protein (flaA), and vacuolizing cytotoxin (vacA), some genes encoding outer membrane proteins (omp), urease alpha and beta subunits (ureA and ureB), and some regulatory proteins, and genes encoding stress-related proteins, such as the chaperone and heat shock protein genes (groEL and dnaK).

[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.

Characterization of the Mycoplasma hominis ftsZ gene and its sequence variability in mycoplasma clinical isolates.

We cloned and sequenced Mycoplasma hominis chromosomal fragment containing ftsZ gene. The wild-type expression of the gene was shown at RNA level by reverse transcription followed by PCR amplification. We revealed that M. hominis FtsZ had a comparatively low similarity to proteins of Mycoplasma genitalium and Mycoplasma pneumoniae. After full ftsZ gene sequencing for 14 clinical isolates of M. hominis, single-nucleotide substitutions were found in 21 positions, 6 of them being common for almost all isolates. This ftsZ gene polymorphism may be used for subtyping of M. hominis in clinical samples. Expression of the M. hominis ftsZ gene in different Escherichia coli strains was also demonstrated, and M. hominis FtsZ protein was purified from E. coli cells transformed with recombinant expression plasmid. Complementation between the M. hominis FtsZ and E. coli FtsZ could be shown. The comparison of FtsZ protein structures may also be used for investigation of bacterial phylogenetic relationships.

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes.

Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H+ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes: the 14-3-3 content in the membranes increased 2- to 3-fold, while the H+-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H+-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H+ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins.

Role of the 14-3-3 proteins in the regulation of H+-ATPase activity in the plasma membrane of suspension-cultured sugar beet cells under cold stress.

All higher plants possess highly specific binding sites for fusicoccin, a metabolite of the fungus Fusicoccum amygdali Del. These sites are harboured in the plasma membranes and formed by a 14-3-3 protein dimer associated with the C-terminal autoinhibitory domain of H+-ATPase. We considered the fusicoccin binding to plasma membranes to be an indicator of complexation between the 14-3-3 dimer and H+-ATPase, we assessed the effect of cold stress on the interaction of these proteins in suspension-cultured sugar beet cells and protoplasts derived from these cells. In both objects, upon lowering the temperature to 0-4 degrees C, a portion of the cytoplasmic 14-3-3 proteins became associated with the plasma membrane, which showed an increasing amount of ATPase/14-3-3 complexes and enhanced ATPase activity. Association between ATPase and 14-3-3 resulted in a several-fold rise in the H+ efflux from protoplasts and intact cells. We suppose that regulation of the H+ pumping under changing external conditions may be based on the interaction between H+-ATPase and the 14-3-3 proteins.

Plant cell pH-static circuit mediated by fusicoccin-binding proteins.

On sugar beet protoplasts that carry two types of fusicoccin-binding sites, a pH downshift in a physiological range (7.0-6.6) markedly enhanced the efficiency of fusicoccin (FC) binding, mainly owing to increased avidity of low-affinity FC-binding sites. This may allow the FC-binding proteins to act as pH-sensitive modulators of cell activity, for instance, via plasma membrane H+-ATPase or potassium channels.