[Repeats in bacterial genomes: evolutionary considerations].

Contemporary data on repeated nucleotide sequences (repeats) in bacterial genomes are discussed. Different classifications and distribution of the repeats in the genomes of bacteria belonging to different species are reviewed. Comparative data on the density of repeats in the genomes of pathogenic and non-pathogenic bacteria, as well as in the genes encoding different functions of microbial cell (genes of stress response, genes controlling DNA metabolism and repair, etc.), are discussed. The role of the repeats in the uptake and loss of genetic material by bacterial genomes is discussed. It is suggested that repeats are responsible not only for the functioning of bacterial genome at the given moment of time, but also for the structures of the implicit genomes, which may appear in the future due to evolutionary significant genomic rearrangements.

[A comparative study of the role of the yadA, invA, and psaA genes in the pathogenicity of Yersinia pseudotuberculosis].

The roles of yadA, invA, and psaA genes introduced into the genetic background of the Y. pseudotuberculosis strain possessing the large p VM82 plasmid in virulence and invasion capacity were studied. Isogenic single mutants as well as double and multiple mutants of these genes were constructed and used. LD50 was used as a measure of virulence and the estimation of the ability to invade mammalian cells and the effect of infection on the weight changes of infected mice were used as additional indicators of pathogenicity. It was shown that the YadA had a major effect on the bacterial virulence when compared with the effects of PsaA and InvA. InvA appears to mediate the main pathway of the cellular invasion. YadA is responsible for the weight loss after infection of mice with sublethal doses of Y. pseudotuberculosis. The effects of YadA on virulence and of InvA on bacterial invasion were independent of the expression of the other genes studied. To our knowledge, this study showed for the first time the direct involvement of YadA in the virulence of Y. pseudotuberculosis in mice. Further pathomorphological studies are required to reveal the differences in the pathogenesis of pseudotuberculosis caused by yadA mutants or yadA+ bacteria of Y. pseudotuberculosis.

[Molecular genetic methods of typing of the Bartonellae].

The primer systems for the PCR detection of four house-keeping genes of bartonellae in clinical material were developed and tested. The tactics of the species RFLP typing was also developed and tested. The scheme of the species RFLP typing of bartonellae was tested using as an example two strains for the first time isolated in Russia from patients with endocarditis and fever of uncertain origin. The results of the typing were supported by sequencing of the amplicons obtained. According to the sequencing the isolates were attributed to the sub species Bartonella vinsonii, subsp. arupensis. The necessity of molecular epidemiological analysis of bartonelloses in Russia was substantiated.

[Wild small mammals are the reservoir hosts of the Bartonella genus bacteria in the south of Moscow region].

A total of 103 blood samples collected from wild small mammals captured in the Prioksko-Terrasny Reserve on the south of Moscow region were studied to determine the bartonellae prevalence. The examined species were the yellow-necked mice Apodemus flavicollis (35 samples), the European wood mouse Apodemus uralensis (10 samples), the bank vole Clethrionomys glareolus (51 samples), the house mouse Mus musculus (3 samples), the common vole Microtus arvalis (2 samples), and the shrew Sorex araneus (2 samples). Initially, we obtained 76 bacterial Bartonella-like isolates after plating onto the surface of the solid nutrient media. 66 of them were PCR-positive at least for three of four targets, gltA, ftsZ, ribC and 16S RNA. Thus, the percentage of the infection in the studied community was 64%. Subsequent RFLP assay showed that obtained isolates belonged to the Bartonella grahamii and/or B. taylorii species. In 7 cases we found both bartonellae species in one animal. These data were confirmed by direct sequencing of four ftsZ, four ribC and two gltA amplicons. According to our data, there is no any marked host specificity for these bartonellae species. Now we have laid the bartonellae strain collection consisting of 31 isolates. To our knowledge, this is the first investigation of the bartonellae prevalence in wild small mammals performed in Russia. The comparison of our data with those obtained by European researchers and issues of coinfection by different bartonellae species and host specificity are discussed.

Expression of the plague plasminogen activator in Yersinia pseudotuberculosis and Escherichia coli.

Enteropathogenic yersiniae (Yersinia pseudotuberculosis and Yersinia enterocolitica) typically cause chronic disease as opposed to the closely related Yersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage by Y. pestis of a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared approximately 70-kb low-calcium-response, or Lcr, plasmids (pCD in Y. pestis and pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (alpha-Pla) and slightly smaller (beta-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only alpha-Pla accumulates in Escherichia coli LE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble alpha and beta forms possessing biological activity. This process also converted cell-bound alpha-Pla to beta-Pla and smaller forms in Y. pestis KIM/pPCP1 and Y. pseudotuberculosis PB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops in Y. pseudotuberculosis PB1/+/pPCP1 occurred more slowly than that in Y. pestis but was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP by Y. pseudotuberculosis did not significantly influence virulence in mice.

Passive immunity to yersiniae mediated by anti-recombinant V antigen and protein A-V antigen fusion peptide.

LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, has been implicated as a regulator of the low-calcium response, virulence factor, and protective antigen. In this study, lcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yielding major peptides of 36, 35, 34, and 32 to 29 kDa. Rabbit gamma globulin raised against this mixture of cleavage products provided significant protection against 10 minimum lethal doses of Y. pestis (P < 0.01) and Y. pseudotuberculosis (P < 0.02). To both stabilize V antigen and facilitate its purification, plasmid pPAV13 was constructed so as to encode a fusion of lcrV and the structural gene for protein A (i.e., all but the first 67 N-terminal amino acids of V antigen plus the signal sequence and immunoglobulin G-binding domains but not the cell wall-associated region of protein A). The resulting fusion peptide, termed PAV, could be purified to homogeneity in one step by immunoglobulin G affinity chromatography and was stable thereafter. Rabbit polyclonal gamma globulin directed against PAV provided excellent passive immunity against 10 minimum lethal doses of Y. pestis (P < 0.005) and Y. pseudotuberculosis (P < 0.005) but was ineffective against Y. enterocolitica. Protection failed after absorption with excess PAV, cloned whole V antigen, or a large (31.5-kDa) truncated derivative of the latter but was retained (P < 0.005) upon similar absorption with a smaller (19.3-kDa) truncated variant, indicating that at least one protective epitope resides internally between amino acids 168 and 275.

Identification of Rickettsia prowazekii using the polymerase chain reaction.

Polymerase chain reaction (PCR) was used to identify Rickettsia prowazekii, the etiologic agent of epidemic typhus. For the PCR, Thermus thermophilus thermostable DNA polymerase was applied with buffer containing a relatively low Mg2+ concentration (1.5-2 mM with dNTP's at 250 microM each). A primer pair used to amplify a 448-base-pair (bp) fragment of R. prowazekii genome was synthesized on the basis of the DNA sequence of gene rpa14/16, coding for a precursor of the mature polypeptides of molecular weight (Mr) 14,000 and/or 16,000 (16kD) from R. prowazekii strain E. For determining the specificity of the primer pair, purified genomic DNAs of 16 rickettsial and 10 other bacterial strains were used.

The difference in the lcrV sequences between Y. pestis and Y. pseudotuberculosis and its application for characterization of Y. pseudotuberculosis strains.

We have sequenced the lcrGVH operon from Y. pseudotuberculosis plasmid pYV995 and compared its sequence with that of Y. pestis. The sequences were highly homological, however, six base pair substitutions were found in one short 14 bp region termed variable sequence. Two oligonucleotides corresponding to variable sequence of Y. pestis (pes-V) or Y. pseudotuberculosis (ptb-V) were synthesized and were used as molecular probes in hybridization experiments with sets of Y. pestis and Y. pseudotuberculosis strains. All 17 Y. pestis strains tested were positive only with the pes-V probe, 18 of 21 Y. pseudotuberculosis strains were positive with the ptb-V probe, while three Y. pseudotuberculosis strains reacted with the pes-V probe but not the ptb-V probe. The 200 bp fragment including variable sequence was sequenced in seven Y. pseudotuberculosis strains. The Y. pseudotuberculosis strains which were positive with the pes-V probe possessed the 200 bp fragment sequence almost identical with that from Y. pestis. No correlation between the Y. pestis-like lcrV sequence and virulence was found for these strains. Moreover, the Y. pseudotuberculosis strains with Y. pestis-like sequences in contrast to Y. pestis possessed unaltered yadA gene. However, we have found the yadA frameshift mutation characteristic for Y. pestis in one Y. pseudotuberculosis strain 312.

Cloning and expression in Escherichia coli of the 37-, 14-, and/or 16-kilodalton antigens genes from Rickettsia prowazekii strain E.

A gene bank of Rickettsia prowazekii strain E constructed in the phage vector lambda EMBL4 was screened for antigen production with anti-R. prowazekii serum. One of the immunoreactive clones, grown at 37 degrees C exhibited the expression of at least two antigens of molecular weight (M(r)) 37 kD and 14 kD. Subcloning and further analysis revealed that the antigens (polypeptides) of Mr 37, 14, and/or 16 kD apparently represent structural units of the 138 kD complex antigen. Assembly of the above mentioned polypeptides was found to be thermosensitive as it took place at 30 degrees C but not at 37 degrees C and resulted in an oligomeric structure of M(r) 138 kD. The nucleotide sequence of the gene coding for a precursor of the mature polypeptides of Mr 14 and/or 16 kD was determined.

[Phenotypic features of a strain of Yersinia pseudotuberculosis with the pVM82 plasmid, detected at pseudotuberculosis outbreak foci]. / Fenotipicheskie osobennosti shtamma Yersinia pseudotuberculosis s plazdmidoi pVM82, obnaruzhivaemoi v ochagakh vspyshek psevdotuberkuleza.

The phenotypic properties conferred to Yersinia pseudotuberculosis cells by the genetical determinants of a 25Md fragment of the plasmid pVM82 coding for the modified cellular immune response in the infected organism. The fragment was shown to determine the conjugative properties of the plasmid, the resistance of bacterial cells to a number of hydrophobic agents and cellular ability to absorb the Congo red dye. The latter confirms the presence of additional structural components in the cell wall of the strain harbouring the plasmid pVM82. The increased resistance of the plasmid-containing strain to bactericidal effect of the blood plasma was demonstrated as compared with the resistance of the strains harbouring the p57 plasmid lacking the 25Md fragment or no plasmid at all.

[Alternative localization of the determinant of pathogenicity coded by the Yersinia pseudotuberculosis pVM82 plasmid]. / Al'ternativnaia lokalizatsiia determinant patogennosti, kodiruemykh plazmidoi pVM82 Yersinia pseudotuberculosis.

The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.

[Structural-functional characteristics of Yersinia pseudotuberculosis plasmid pVM82]. / Strukturno-funktsional'naia kharakteristika plazmidy Yersinia pseudotuberculosis pVM82.

The restriction map of Yersinia pseudotuberculosis plasmid pVM82 was established using the "chromosome walking" method. According to transpositional mutagenesis, the plasmid pVM82 appeared to be conjugative and was able to be transmitted from Y. pseudotuberculosis to the E. coli K-12 cells.

[The use of a molecular DNA probe based on the cloned cytolysin gene for the identification of Legionella]. / Primenenie molekuliarnogo DNK-zonda na osnove klonirovannogo gena tsitolizina dlia identifikatsii legionell.

A molecular DNA probe has been obtained on the basis of recombinant plasmid pNIG carrying the Legionella cytolysin gene. The use of this probe for the identification of different Legionella species and other microorganisms has shown that it may serve as the species-specific marker of L. pneumophila. The probe has been used for the identification of new Legionella-like strains isolated from the environment.

[RecA-independence of the process of amplification caused by the RS-1 sequence of Vibrio cholerae]. / RecA-nezavisimost' protsessa amplifikatsii, obuslovlennogo RS1- posledovatel'nostiami kholernogo vibriona.

The processes of amplification and deamplification of a plasmid DNA segment flanked by direct repeats of RSI-sequence of Vibrio cholerae and carrying the structural genes of cholera toxin inside the recombinant plasmid in E. coli cells have been studied. These processes determined by RSI-sequences are shown to take place independent of the RecA-system of E. coli cells.

[Characteristics of transposition of Tn7-like elements determining resistance to trimethoprim and streptomycin]. / Kharakteristika transpozitsii Tn7-podobnykh élementov, opredeliaiushchikh ustoichivost' k trimetoprimu i streptotritsinu.

The transposing elements Tn7, Tn1824, controlling the resistance to trimethoprim and Tn1925, Tn1826, carrying the streptothricin resistance genes were classified as a new transposon family on the basis of their physical structure. The comparative genetic analysis of the frequency, specificity and insertion orientation in different replicons, obtained in independent research systems in this study, demonstrated the identity of transposition characteristics of the transposons. The latter makes it possible to classify them as an independent transposon family. The peculiar feature of the Tn7-like elements family is their RecA-dependent transposition into the chromosome of Escherichia coli stimulated by bacteriophage Plkc transduction of the transposons.