[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.

[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.

[Properties of the plasmid pFT15/10-1 isolated from the vaccine strain of Francisella tularensis]. / Svoistva plazmidy pFT15/10-1, vydelennoi iz vaktsinnogo shtamma Francisella tularensis.

Plasmid, designated pFT15/10-1, was isolated from Francisella tularensis vaccine strain 15/10. The plasmid is presented by the homogeneous 5.02 +/- 0.054 Md monomeric circular DNA molecules in electron microscopic preparations. Plasmid size is 7-7.3 kb as defined by electrophoresis in agarose gel. The restriction analysis has revealed that plasmid pFT15/10-1 possesses a single specific cleavage site for restriction endonuclease EcoRI, two sites for restriction endonucleases BamHI, BgIII, HincII, HindIII, PstI, three sites for BglI and SalI, some for AluI, TagI, MvaI, CfrI. Plasmid is not digested by restriction endonucleases SmaI, XmaI, KpnI, MluI. Restriction map of the plasmid was constructed for most frequently used restriction endonucleases.

[Catabolyte repression of Escherichia coli K12 mutants with defects in different systems of glucose transport]. / Katabolitnaia repressiia u mutantov Escherichia coli K12 s defektami v razlichnykh sistemakh transporta gliukozy

The phenomenon of glucose catabolite repression was studied in E. coli mutants inable to transport this carbohydrate. The pts 1, H mutant P34 was much less sensitive to the repressive effect of glucose on beta-galactosidase synthesis than the parent type. The 1103 mutant devoid of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) behaves in the same way as P34 mutant after addition of glucose to casamino acid mineral medium. However, in minimal medium with succinate as the sole source of carbon, cells of the 1103 mutant show enhanced sensibility to transient glucose repression. The effect of hypersensibility disappears when the lac I mutation leading to constitutive the beta-galactosidase synthesis is introduced in 1103 mutant. It is shown that the enhanced sensibility of beta-galactosidase synthesis to glucose transient repression in 1103 mutant is an effect of the aburpt decrease in its growth rate in the presence of succinate and most probably this decrease leads to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain JD3 devoid of one of the enzymes II for glucose (and due to this resistant to glucose catabolite respression) then the level of resistance in double mutant does not increase in spite of considerable supression of 14C glucose accumulation. In connection with this the role is discussed of separate components of the E. coli K 12 glucose transport system in realization of the phenomenon of catabolite repression.

Catabolite repression in Escherichia coli K12 mutants defective in glucose transport.

The phenomenon of glucose catabolite repression was studied in Escherichia coli mutants unable to transport this carbohydrate. The pts I,H mutant P34 was much less sensitive to permanent and transient repressive effect of glucose on beta-galactosidase synthesis than parental type. The 1103 mutant with lack of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (ptsI) behaves as well as P34 mutant after addition of glucose to casamino acids mineral medium. But in minimal medium with succinate as the sole source of carbon cells of the 1103 mutant (in accordance with the data of Perlman and Pastan, 1969) show hightened sensibility to transient glucose repression. The effect of hypersensibility disappears when the lacI mutation rendering the beta-galactosidase synthesis to costitutivity is introduced in 1103 mutant. It is shown that the hightened sensibility of beta-galactosidase synthesis to glucose transient repression in 1103 mutant is not an effect of the pts mutation and most probably is due to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain devoided of one of the enzymes II for glucose (gptA) (and due to this resistant to glucose catabolite repression) then the level of resistance in double mutant does not increase in spite of considerable supression of 14C glucose accumulation. It is discussed the role of separate components of Escherichia coli K12 glucose transport system in realization of the phenomenon of catabolite repression.

Interaction between Bdellovibrio bacteriovorus and the cytoplasmic membrane of Escherichia coli B.

Adsorption of Bdellovibrio bacteriovours (Bdv) on the surface of Escherichia coli is accompanied by a sharp decrease in the initial rate of entry of alpha-methylglucoside-C-14 and thiomethlgalactopyranoside-C-14 into the host cell. Interaction between the parasite and E. coli leads to the rapid departure of previously accumulated labeled glucosides and beta-galactosides from the bacteria. Meanwhile the ATPcontent in E. coli falls sharply. Adsorption Bdv E.coli spheroplasts was established as a fact. The possible mechanisms of interaction between Bvd and the host cell at the cytoplasmic membrane level are discussed.