[Role of phage LØ7 lysogeny in genetic variability of Escherichia coli].

AIM: Determine the possibility of Iysogenization of Escherichia coli single strain DNA (ssDNA) by 1ø7 bacteriophage from the Microviridae family and determine the role of phage lø7 lysogeny in genetic variability of these bacteria. MATERIALS AND METHODS: A method of E. coli K12 lysogenization by phage lø7 was developed. A spot-test for the control of resistance of the obtained lysogens against phage lø7 and determination of lysogen lø7 spontaneous production was worked out. Criteria for phage lø7 identification, that is spontaneously produced by E. coli K12 lysogens, were proposed. A kit of isogenic E. coli strains, that vary by mutations in ptsI, ptsH and fruA genes, that code phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) proteins, was constructed. RESULTS: The ability of highly virulent bacteriophage lø7 to lysogenize E. coli was shown. A reduction of lø7 titers in ptsI, ptsH and fruA E. coli K12 mutants was demonstrated compared with titers in wild-type bacteria. Lytic bacteriophage lø7 was also able to lysogenize ptsI, ptsH and fruA mutants at a high frequency. Lysogens are resistant to phages lø7, phiX174 of Microvirus genus and spontaneously produce lø7. CONCLUSION: Bacteriophage lø7 of the Microviridae family is able to lysogenize E. coli K12 and vertically transfer genome of this lytic phage. As a result, lytic phage lø7 takes part in bacterial variability as a factor of lysogen selection in bacteria population corresponding to PTS mutants by phenotype.

[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.

Selection of E. coli strains for stable transformation with recombinant plasmids containing full-length genome of clinical HIV-1 isolates.

Strain chi6007 obtained from the parent E. coli strain chi5097 is a result of ptsH5 mutation, which allowed cells to grow without common components of the phosphoenolpyruvate-dependent phosphotransferase system. Segregants of strain chi6007 retaining the Pol+ gene responsible for inability to grow at 37 degrees C, but gaining rifampicin resistance (RifR) were used for cloning of cointegrate plasmids. Pre-integration complexes of HIV-1 were co-integrated with the pBR-322 plasmid and transformed strain chi6018. Sequencing showed that the pPIC91 hybrid plasmid contains full-length genome of HIV-1 with shortened 5-terminal LTR and full-length copy of pBR322. Elimination of the pPIC91 plasmid from chi6018 cells was followed by the appearance of auxotrophic insertion mutants. Sequencing of the insert region showed that chromosome DNA of the host cell includes integrated genomes of pBR-322 and HIV-1.

High and low UV-dose responses in SOS-induction of the precise excision of transposons tn1, Tn5 and Tn10 in Escherichia coli.

UV-inducible precise excision of transposons is a specific SOS-mutagenesis process. It deals with the deletion formation which has previously been demonstrated to involve direct or inverted IS-sequences of transposons. The process was used for revisiting the targeted and untargeted SOS-mutability and its relationship to the key genes for SOS-mutagenesis: the recA, lexA and umuDC. The precise excision of transposons Tn5 and Tn10 from the chromosomal insertion sites ade128 and cyc750 is induced in Escherichia coli K-12 and B cells, wild-type for DNA-repair, both by the low doses of UV-light ranging from 0.25 J m-2 to 2.5 J m-2 and the high doses within the range 5.0-40.0 J m-2. Precise excision of these transposons induced by the range of low doses incapable to induce targeted point mutations reveals its mostly untargeted nature. This process for the transposon Tn1 is not induced by UV-light within the range of doses 0.25-2.5 J m-2 while its induction is possible by UV-fluences ranging from 5.0 to 40.0 J m-2. A dose-response of the precise excision of Tn1 is similar to that of the UV-induced reversion of trpUAA point mutation that is targeted by nature and contrasts to the UV-inducible precise excision of Tn5 and Tn10. Both types of UV-inducible precise excision, demonstrated either by Tn1 or Tn5 and Tn10, are eliminated by mutations in the lexA, recA and umuDC genes indispensable for UV-induced SOS-mutability. The palindromic structures different for the transposons Tn1, Tn5 and Tn10 are discussed to be involved and affect the targeted and untargeted precise excision of transposons induced by UV-light.

[The genetic typing of strains in the genus Francisella using universal probes]. / Geneticheskoe tipirovanie shtammov roda Francisella s pomoshch'iu universal'nykh zondov.

The determination of the genetic relationship of bacteria of the genus Francisella and their differentiation is one of the topical tasks of the epidemiology and infectology of F. tularensis, the causative agent of tularemia, belonging to this genus. To solve this task, investigation was carried out with a view to the determine the possibility of the genomic typing of Francisella. Genomic typing was based on the use of the hybridization of fragments of Francisella chromosomal DNA, split by restrictases EcoRI and Pstl, with DNA probes. As probes, "minisatellite" sequences of bacteriophage M13 DNA or Helicobacter pylori rDNA were used. The possibility of interspecific genomic typing of F. tularensis, F. novicida and F. philomiragia by the above-mentioned methods was established. The intraspecific typing of F. tularensis by the phenotypical sign of virulence was possible with the use of the hybridization of chromosomal DNA with bacteriophage M13 probe. The use of rDNA probe proved to be effective for the determination of subspecies of the causative agent of tularemia. The possibility of using the combination of these two methods for more complete characterization of the genomic polymorphism of Francisella, the determination of their genetic relationship and their differentiation is discussed.

[The characterization of Francisella tularensis mutants and subspecies by using monoclonal antibodies]. / Kharakteristika mutantov i podvidov Francisella tularensis s pomoshch'iu monoklonal'nykh antitel.

Monoclonal antibodies to F.tularensis cells, subspecies holarctica, were studied for the capacity of reacting with F.tularensis, subspecies nearctica, and its mutants having lower virulence and altered capacity for inducing protective immunity to tularemia in laboratory animals. Among the antibody-producing hybridoma clones under study, clones F8/67 and C7/65 capable of distinguishing the mutants of F.tularensis, subspecies nearctica, with lower virulence than that of the initial strain were selected. Antibodies of these hybridoma clones did not interact with the antigens of the initial virulent strains of F.tularensis, subspecies nearctica, while giving pronounced reaction with the antigens of its mutants. Close F8/67 produced IgG antibodies and clone C7/65, IgM antibodies. As shown in immunoblotting, antibodies produced by these hybridoma clones bound with proteins of F.tularensis cell membranes.

[Mutations in Francisella tularensis, decreasing the virulence of these bacteria and leading to a change in the immune response upon infection of experimental animals with them]. / Mutatsii u Francisella tularensis, snizhaiushchie virulentnost' étikh bakterii i privodiashchie k izmeneniiu immunnogo otveta pri zarazhenii imi éksperimental'nykh zhivotnykh.

The research was aimed at isolation of Francisella tularensis mutants possessing the decreased virulence for experimental animals and mediating the changes in the animal immune response. A number of spontaneous and induced mutants of the American and European subtypes of Francisella tularensis were selected for antibiotics resistance or detergent sensitivity. All the obtained mutants have the decreased virulence and differ in their ability to induce the protective antitularemia immunity or ability to induce the humoral immune response in the laboratory animals. The dimeric immunoprecipitation in gel as well as immunoblotting have shown the mutations decreasing the virulence to cause the loss by bacteria of a number of antigenic structures (in case the virulence is completely lost) or changes in antigenic structure resulting in inability of bacteria to induce the humoral immune response when immunizing the laboratory animals. The latter occurs in partially virulent mutants of the vaccine mutant type. The concomitant changes in virulence, ability to cause protective immunity or humoral immune response of the mutants is discussed.

[The genetic transfer, heredity and phenotypic expression of plasmid RP4::Mu cts genes in Bacillus cereus]. / Geneticheskii perenos, nasledovanie i fenotipicheskoe vyrazhenie genov plazmidy RP4::Mu cts v Bacillus cereus.

The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.

[Construction and analysis of the tularemia pathogen gene library in Escherichia coli]. / Sozdanie i analiz biblioteki genov vozbuditelia tuliaremii v kishechnoi palochke.

The library of tularemia causative agent genes cloned on the pHC79 plasmid and the partial clonotek of these agents genes in Escherichia coli cells have been constructed. The immunochemical analysis has revealed seven clones of Escherichia coli harbouring the recombinant plasmids and expressing francisella antigens. The cloned sequences of francisella DNA as well as the recombinant plasmids containing them and coding for francisella antigens are capable of specific hybridization with the DNA from Francisella tularensis strains and Francisella novicida strain U112. The cloned DNA sequences have the properties of the genetic radiospecific molecular DNA probe.

[UV-inducibility of the LT-toxin operon]. / UF-indutsibel'nost' operona LT-toksina.

The plasmid elt-operon pVZ14 was constructed by fusing of the eltoperon of the plasmid pVZ357 with the lac-gene of the bacteriophage Mud1 (Amp, Lac). lacZ gene has been proven to be fused with an elt-promoter by the loss of toxin production coded by pVZ357 and acquiring of Lac+ phenotype by pVZ14 containing cells, as well as by HindIII fragments hybridization of pVZ357 and pVZ14 with the labelled elt-probe. The kinetics of beta-galactosidase synthesis in E. coli cells harboring pVZ14 shows an elt-operon promoter to have expressed constitutive activity and to be activated by a SOS-inducing agent, UV-light.

[SOS-induction of the RP4 plasmid tet-determinant]. / SOS-indutsibel'nost' tet-determinanty plazmidy RP4.

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.

[Sensitivity of Vibrio cholerae to the lethal and mutagenic effects of UV-irradiation mediated by the presence of plasmids]. / Chuvstvitel'nost' kholernykh vibrionov k letal'nomu i mutagennomu deistviiu UF-sveta, zavisiashchaia ot nalichiia plazmid.

The effect of UV-irradiation on Vibrio cholerae cells and its changes mediated by the plasmid R245 have been studied. Vibrio cholerae strains 569B and RV31 have been shown to be considerably more sensitive to lethal effect of UV-irradiation as compared with Escherichia coli and Salmonella typhimurium cells. Highly toxigenic strain 569B and practically atoxigenic strain RV31 have the same UV-sensitivity. Lethal effect of UV-irradiation on Vibrio cholerae cells is increased when the irradiated cells are plated on enriched media. UV-induction of mutations was not registered in plasmidless strains of Vibrio cholerae. Plasmid R245 increases UV-resistance of vibrio cells and makes them UV-mutable.

[Localization of structural genes of Vibrio cholerae cholerae toxin using the recombinant plasmid RP4omega elt]. / Lokalizatsiia strukturnykh genov kholernogo toksina Vibrio cholerae cholerae s ispol'zovaniem rekombinantnoi plazmidy RP4omega elt.

The recombinant plasmid RP4 omega elt carrying Escherichia coli heat-labile enterotoxin elt genes with 70-80% homology with genes vct of Vibrio cholerae has been constructed. We used this plasmid to determine localization of the cholerae toxin genes vct on the map of Vibrio cholerae cholerae. Two types of the donors were revealed in matings of 10 strains of V. cholerae cholerae 569B/RP4 omega elt with the polyauxotrophic recipients RV31 and RV175: some strains had enhanced frequency of mobilization of ilv-1 and lys-6 markers, the others--of trp-1. Our data suggest that structural vct genes are located within two regions of V. cholerae cholerae 569B chromosome: trp-1 and ilv-1--lys-6.

[Isolation of the R'his plasmids of Vibrio cholerae]. / Poluchenie plazmid R'his Vibrio cholerae.

V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

[Inheritance and expression of the restrictive activity of plasmids coding EcoRI restrictase in Vibrio cholerae cells]. / Nasledovanie i proiavlenie restriktivnoi aktivnosti plazmid, kodiruiushchikh restriktazy EcoRI, v kletkakh kholernogo vibriona.

Recombinant E. coli plasmids are known to be obtained from E. coli cells using the plasmids coding EcoR1 restriction endonuclease. These plasmids were shown to possess various chromosomal or plasmid genes. The paper presents data on the construction of conjugative recombinant plasmid pSA1002, capable of conjugate transfer into V. cholerae cells. The stable maintenance and inheritance of the plasmid in V. cholerae cells have been demonstrated as well as phenotypic expression of its genes, including EcoR1 restriction endonuclease genes. The possibility of recombinant plasmids formation in V. cholerae cells dependent on EcoR1 restriction endonuclease, coded by pSA1002, is discussed.

[Interaction of Yersinia pestis bacteria with bacteriophage mu]. / Vzaimodeistvie bakterii Yersinia pestis s bakteriofagom mu.

Yersinia pestis cells are shown to be sensitive to bacteriophage Mu cts62 infection. Lysis of bacteria has been shown to be more efficient on solid nutrient medium than in LB broth. 10(-5) pfu per ml is the maximal concentration of bacteriophage particles yielded from the broth cultures of bacteria. Moi 0.1 has been used to obtain such yields of bacteriophage. Lysogenization of Yersinia pestis cells has not been achieved when the standard methods of bacteriophage infection have been used. It was accomplished by the conjugal transfer of plasmid RP4::MU cts62 to Yersinia pestis from Escherichia coli. The deficiency of Yersinia pestis in producing bacteriophage Mu cts62 mature particles during the lytic cycle of bacteriophage is discussed.

[Characteristics of RecA-independent recombination of plasmids in E. coli cells producing restriction endonuclease EcoRI]. / Osobennosti RecA-nezavisimoi rekombinatsii plazmid v kletkakh E. coli, produtsiruiushchikh éndonukleazu restriktsii EcoRI.

The restriction endonuclease EcoRI dependent recombination of compatible plasmids has been studied in RecA cells of Escherichia coli. Plasmid RP4 and the isogenic ColE1 type plasmids pSA14 or pSA25, differing in restriction-modification RM EcoRI genes, have been used to study this type of recombination. EcoRI dependent recombination of plasmids is demonstrated in RecA cells and, thus, is independent of general system of homologous recombination. The classes of recombinant plasmids isolated from RecA cells differ from the classes isolated from wild type cells. Levels of tetracycline resistance conferred by plasmid RP4 are shown to be dependent on the alleles of RecA+ gene, being extremely low in RecA cells. This property is demonstrated to be useful for obtaining the multicopy recombinant plasmids resulting from EcoRI dependent recombination in RecA cells of Escherichia coli.

[Plasmid recombination stimulated by restriction endonuclease EcoRI in vivo: formation of recombinant plasmids in recA+-cells of E. coli]. / Rekombinatsiia plazmid, stimuliruemaia éndonukleazoi restriktsii EcoRI in vivo: formirovanie rekombinantnykh plazmid v recA+-kletkakh E. coli.

The possible participation of restriction endonuclease EcoRI in recombination of compatible nonhomologous plasmids in E. coli cells has been studied. To study the process, plasmids RP4 and R245 have been transferred by conjugation into the recipient cells of E. coli harbouring one of isogenic plasmids, pSA14 and pSA25, different for the genes coding restriction endonuclease EcoRI. The genetic analysis of transconjugant phenotypes, coded by the plasmids, has permitted to register the recombinant plasmids after compatibility of parent plasmids in E. coli cells. Recombination of plasmid RP4 with the plasmid pSA14, carrying EcoRI genes, has been registered in E. coli cells, producing the restriction endonuclease, while plasmid recombination has not been found in the cells harbouring plasmid pSA25, isogenic for all genes, except for EcoRI genes, with plasmid pSA14. Restriction endonuclease EcoRI is concluded to stimulate site specific recombination of nonhomologous compatible plasmids in vivo. EcoRI-mediated recombination of plasmid R245 with plasmid pSA14 is discussed.