[Engineering of attenuated Bordetella pertussis bacteria producing immunogenic non-toxic form of pertussis toxin].

AIM: To engineer attenuated Bordetella pertussis strain producing non-toxic immunogenic derivative of pertussis toxin (toxoid KT). MATERIALS AND METHODS: Cloning, site-directed mutagenesis, allelic exchange as well as methods for determination of immunobiological characteristics of toxoid KTwere used. RESULTS: Attenuated B. pertussis strains 5 and 35 containing mutant operon ptx and gene of resistance to canamycin were engineered. Recombinant bacteria retained marker of resistance to canamycin as well as structure of mutant operon during cultivation on growth media and long-term survival in lung of laboratory mice. Immunobiologic characteristics of attenuated B. pertussis were studied. CONCLUSION: Intranasal immunization of laboratory animals with live attenuated B. pertussis 5 and 35 provides protection from infection with virulent B. pertussis strain, which is comparable with efficacy of standard whole-cell vaccine.

Repeated sequences isolated from Bordetella pertussis induce DNA rearrangements and deletions at high frequency.

Two repeated sequences (RS) from Bordetella pertussis were cloned in Escherichia coli and sequenced. The RS, called RSBP1 and RSBP3, are highly homologous to other B. pertussis RS. The recombinant plasmids containing RSBP1 and RSBP3 or transposon-like structures of these elements were not stable but segregated plasmids with deletions or rearranged DNA. RS of B. pertussis seem to be able to stimulate both intra- and inter-genomic RecA-independent recombination events. In at least one case, the observed deletion had occurred precisely between the RS terminus and a site with sequence homology to the terminus. The high frequency rearrangements associated with the RS imply that the RS are transposable elements.

[Transposition and inheritance of Bordetella Tn-element in Escherichia coli K12]. / Transpozitsiia i nasledovanie Tn-élementa Bordetell v kletkakh Escherichia coli K12.

B. pertussis genetically mobile element TnBp3 integrates the plasmid in E. coli chromosome. During culturing under nonselective conditions the majority of cells of some E. coli strains lose the kanamycin resistance marker, which indicates the instability of TnBp3 inheriting. The stability of inheriting the integrated structure is higher in E. coli cells with recB-21 recC-12 sbcB-2 mutations. The role of RecBC recombination system in extrusion of TnBp3 is discussed.

[Isolation of Escherichia coli K12 mutants with deficient in precise excision of transposons]. / Vydelenie mutantov Escherichia coli K12 s narusheniem protsessa tochnogo iskliucheniia transpozonov.

Plasmid pNM1, the derivative of R100.1, has been constructed by insertion of transposon Tn5 into structural tet genet (Tn10) of the parental plasmid. The frequency of precise excision of Tn5 from plasmidic genome is 10(-5). The high frequency of precise excision obtained in this system permits one, to use it for isolation of mutants having low frequencies of precise excision. Two mutants were isolated in which the frequencies of precise excision of Tn5 were decreased for two orders. The pex1 and pex2 mutations responsible for the effect decrease the precise excision of Tn5 from R100.1 as well as from RP4 genomes.

[Mobile element RSBP1 of Bordetella pertussis]. / Mobil'nyi élement RSBP1 Bordetella pertussis.

A number of repeated sequences was identified in the chromosome of Bordetella pertussis by the electron microscopic analysis of the chromosomal DNA of this microorganism. One of the sequences was cloned on the vector plasmid pHC79. It is shown to consist of two elements RSBP1 and RSBP2. The first elements is probably identical to an RS-element described previously. The cloned RSBP1 element is shown to stimulate the deletion formation in the genome of the plasmid pMKII and is able to transpose into the chromosome of Escherichia coli. The latter properties permit one to classify RSBP1 as an element belonging to a class of migrating genetical elements.

[Features of expression of the pertussis toxin operon under the control of its own and heterologous promotors in Bordetella bronchiseptica cells]. / Osobennosti ékspressii operona kokliushnogo toksina pod kontrolem sobstvennogo i geterologichnogo promotorov v kletkakh Bordetella bronchiseptica.

Expression of the cloned operon encoding the pertussis toxin synthesis under the control of operons own vir-dependent promoter or vir-independent promoter of Escherichia coli origin was studied. Proteins produced by the recombinant strains have been characterized. The pertussis toxin operon was shown to express under the control of both homologous and heterologous promoters in Bordetella bronchiseptica cells. Use of the lac-promoter increases the yield of produced toxin twofold. Copy number of operon in the cell does not influence the level of toxin production. The synthesized protein can be transported into the culture medium. The biological and physico-chemical properties of the protein are similar to the ones of the natural pertussis toxin. Bordetella bronchiseptica strain producing the toxin with decreased toxic activity has been obtained. Thus, a simple system for cellular expression of the toxin operon was constructed in Bordetella bronchiseptica. It permits one to construct new strains producing nontoxic derivatives of the pertussis toxin for construction of nonreactogenic vaccines.

[Incorporation of phi80 phage into unusual chromosome sites and isolation of a specialized transducing bacteriophage]. / Vkliuchenie faga phi80 v neobychnye khromosomnye saity i vydelenie spetsializovannogo transdutsiruiushchego bakteriofaga.

The mutant strain KS713 of Escherichia coli K-12 deleted for the normal insertion site and secondary preferable one was obtained. The insertion frequency of phage phi80 into the double deletion strain is reduced about 30-fold with respect to integration into the strain H47 with deletion of the primary phi80 attachment site and about 500-fold relative to integration into wild type Escherichia coli. Analysis of the rare abnormal lysogens of KS 713 strain indicates that there are secondary sites on the chromosome, which are utilized for prophage attachment if insertion at preferable secondary att80-II site is eliminated too. The insertion of phi80 phage into the bfe locus was obtained by the appropriate selection technique. Induced prophage excision from the bfe site was rather efficient and lysates contained phi80 phage particles that could specificically transduce the argH+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harbouring both the wild-type and the mutant argH genes were isolated. These heterogenotes were used for producing high-frequency transducing lysates.

[A mobile genetic element on a Bordetella pertussis chromosome stimulates cointegrate formation in Escherichia coli strains]. / Mobil'ny'i geneticheskii élement khromosomy Bordetella pertussis stimuliruet obrazovanie kointegratov v shtammakh Escherichia coli.

This work continues a series of reports devoted to the study of a repeated sequence isolated from the Bordetella pertussis chromosome (RSBP--Repeated Sequence of Bordetella Pertussis). The RSBP was earlier demonstrated to have a structure similar to IS elements and exhibit some properties of mobile genetic elements. The results of this work demonstrate the ability of this novel genetic element to stimulate the formation of cointegrates between independent replicons. We have studied the structure of five cointegrates formed by integration of a resident RSBP-containing plasmid into three different sites of a recipient replicon. The studied cointegrates are capable of resolution with the release of a resident plasmid independent of the host cell RecA function.

[Initial characteristics and isolation of bacterial mutants with altered ability to transpose Tn9]. / Metod vydeleniia i pervichnaia kharakteristika bakterial'nykh mutantov s izmenennoi sposobnost'iu k transpozitsii Tn9.

The method allowing the induction of bacterial mutations affecting Tn9 transposition from the bacteriophage genome to the Escherichia coli chromosome is described. Neither impaired ability of cells to adsorb bacteriophages, nor phage DNA degradation in the mutant cells were observed in the transposition-defective mutants isolated by the method. This led us to the conclusion that the isolated mutants were indeed defective in the transposition of Tn9.

[Independence of the processes of transposition and genome rearrangements induced by transposons]. / Nezavisimost' protsessov transpozitsii i genomnykh perestroek, indutsiruemykh transpozonami.

The data on the influence of the tnm mutations affecting transposition process on the deletion formation promoted by Tn and IS elements are presented. It was shown that the tnm mutations did not affect the frequency of deletion formation. The results of genetic analysis of the tnm mutant deficient in both transposition and genomic rearrangements induced by Tn9 inserted into lambda prophage, indicated that the mutant phenotype was caused by two different but linked mutations. A mutation affecting the process of genomic rearrangements was designated gerA2. The gerA2 mutation decreased sharply the frequency of rearrangements promoted by Tn9, Tn10 or Tn601 inserted into lambda prophage. However, this mutation had no influence upon transposition of the same Tn elements. The data obtained could be interpreted as indicating the independence of the processes of transposition and genomic rearrangements or as indication of the existence of specific steps of these processes.

[Specificity of introducing the transposon that determines chloramphenicol resistance (Tn9) into the chromosome of Escherichia coli K-12]. / Spetsifichnost' vnedreniia v khromosomu Escherichia coli K-12 transpozona, opredeliaiushchego ustoichivost' k khloramfenikolu (Tn9).

Translocation of Tn9 coding for chloramphenicol-resistance from lambdaatt80 genome into bacterial chromosome was studied. Three preferential sites of Tn9 integration, attTn9A, ATTTn9B and attTn9C, were found on the chromosome of Escherichia coli K-12; attTn9A was mapped between purD and rpoB loci, attTn9B was cotransducible with argG, attTn9C was located between 61 and 66 minutes of the standard E. coli K-12 genetic map. The integration of Tn9 in these att sites occurs with almost equal probability. Tn9 integrated in attTn9A shown significantly higher frequency of excision and P1-transduction frequency than Tn9 integrated in either of two other attTn9 sites.

[Mechanism of plasmid ColEl and pMB-9 mobilization by plasmid F'lac+ in Escherichia coli K-12]. / Izuchenie mekhanizma mobilizatsii plazmid ColEl i pMB-9 plazmidoi F'lac+ u Escherichia coli.

The genetic control and mechanism of mobilization of the non-conjugative plasmids ColE1 and pMB-9 by the conjugative plasmids was orived to be recA-independent process in contrast to the mobilization of the chromosomal marker pro. Acridine orange and ethidium bromide curing data together with the results of electrophoretic analysis of plasmid DNA suggest that the plasmids F' lac+ and pMB-9 as well as F' lac+ and ColE1 remain autonomous after their contransfer to recipient cells. These data argue in favour of non-recombinational nature of the plasmid mobilization process. The possibility of transmission of a non-conjugative plasmid without transmission of a conjugative one from the donor strain carrying both plasmids was established. The results obtained are discussed with respect to the hypothesis on the effect of diffusible products encoded by the conjugative plasmid and required of the mobilization of the non-conjugative plasmid.

[Nucleotide sequence and properties of an inverted repeating element of a Bordetella pertussis chromosome]. / Nukleotidnaia posledovatel'nost' i svoistva invertirovannogo povtoriaiushchegosia élementa khromosomy Bordetella pertussis.

The repeated sequence from Bordetella pertussis chromosome was cloned using the method described by Ohtsubo. The sequences of the characterized B. pertussis chromosome are homologous to the previously described sequences and analogous in the structure to the already known IS elements. The parental recombinant plasmids containing the RS were unstable and segregated to the plasmids of different structure. The segregants' structure is characterized in this paper. It is shown in our study that the RS element is able to stimulate intragenomic rearrangements, such as deletions. It is shown that at least one deletion begins precisely after 3' end of RS and terminates with the sequence which is completely homologous to ten terminal nucleotides of this one. Probably, RSs stimulate at high frequency the formation of deletions, which appear to be the result of recA-independent site-specific recombination between short direct repeats.

[Preferred secondary attachment site of prophage phi80 on Escherichia coli K-12 chromosome]. / Vtorichnyi predpochtitel'nyi sait integratsii profaga phi80 na khromosome Escherichia coli K-12.

The integration frequency of phage att80 immlambdac1857 into the chromosome of a mutant strain H47 Escherichia coli K-12 deleted for the normal prophage insertion site is found to be about 20-fold decreased as compared with its integration into the wild type strain. The most of the resulting lysogens contain the prophage at the secondary attachment site of the mutant bacterial chromosome which is preferentially utilized for prophage insertion. This attachment site (att80-II) is located close to his-genes on the chromosome of H47 strain. Prophage curing procedure of such abnormal lysogens results in the appearance of rare auxotrophic heat-resistant survivors with the His- phenotype. In some cases the prophage insertion can induce an inversion of a neighbouring genetic region. Such lysogens contain the purC gene near prophage located at the att80-II site, and after curing they segregate the heat-resistant His- and Pur- colonies.

[Tn9 integration sites and their effect on transposon properties]. / Saity integratsii Tn9 i ikh vliianie na svoistva transpozona.

The insertion of transposable deoxyribonucleic acid sequence that specifies chloramphenicol acetyltransferase (Tn9) occurs in the preferential sites of the Escherichia coli K-12 chromosome. It is found that some strains of E. coli K-12 have different preferential sites for the Tn9 insertion, located in various regions of the chromosome. The IS1 element inserted into the galT gene of the N116 strain (insertion mutation galT 116:IS1) serves as a strongly preferential site for the Tn9 insertion in this strain. However, being transferred into the chromosome of the KS836 strain this element loses such preference. It was demonstrated that the transposons (Tn9) inserted into different chromosomal sites are distinguished by such properties as stability, different capability to transposition into genomes of bacteriophages and plasmids, and by the choice of the preferential sites of integration in the course of subsequent insertions.

[Genetic study of Escherichia coli K-12 mutations that affect the transposition process]. / Geneticheskoe izuchenie mutatsii Escherichia coli K-12, vliiaiushchikh na protsess transpozitsii.

Results of genetic analysis of bacterial tnm mutations influencing transposition of Tn9 are presented. Five independent tnm mutations were mapped at 90,5 min of the E. coli genetic map. The tnm mutations were 3,5 and 46,5% contransducible with metA and malB markers, respectively. Two tnm mutations tested were recessive in tnm+/tnm- merodiploids. The effect of tnm mutations on other transposons--Tn10, Tn601, Tn3 and Tn5 was examined. It was shown that tnm1 and tnm2 mutations reduced the frequency of transposition of Tn10, Tn3, Tn5 and Tn601 from the genome of phage lambda and inhibited intracellular development of the infecting Mu phage. The latter effect was probably due to the inhibition of Mu integration into bacterial chromosome. The tnm3 mutation affected the transposition of Tn9 only.

[Nucleotide sequence and properties of a transposon-like structure cloned from a Bordetella pertussis chromosome]. / Nukleotidnaia posledovatel'nost' i svoistva transpozonopodobnoi struktury, klonirovannoi iz khromosomy Bordetella pertussis.

The 2.3 kb BamHI-EcoRI subclone has been isolated and sequenced from the 15 kb BamHI chromosomal fragment of Bordetella pertussis comprising also the vir gene. The sequence contained one copy of a transposon-like structure, very similar to the RSs of B. pertussis recently characterized, the unique sequence of B. pertussis chromosomal DNA and an inverted sequence complementary to 402 bp of 3' end of the B. pertussis RS element. The fragment of plasmid pUC4K containing the gene for kanamycin resistance (Km) was integrated in the XhoI site of the unique portion of the transposon-like structure of plasmid DNA (Ap(r), Km(r)). The clones in amount of 2% tested had the Ap-S phenotype. The recombinant plasmid from the Ap(s) phenotype clones lost one of the repeats and a portion of the gene bla as a result of deletion. The conclusion is drawn that RSs of B. pertussis stimulate the formation of deletions.

Isolation and mapping of Escherichia coli K12 mutants defective in Tn9 transposition.

Five mutants (called tnm) of Escherichia coli with impaired ability for transposition of Tn9 were isolated after treatment with ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NG). The map locations of the tnm mutations were determined by a combination of Hfr matings, F' episome complementation and P1 transductional mapping. The data obtained show that the five tnm mutations are located near 91 min on the Escherichia coli linkage map and are cotransducible with the metA marker with a frequency of 3%-4%. Introduction of F' plasmids containing this region complements the Tnm- phenotype for the two mutants tested i.e. tnm-1 and tnm-2 are recessive in tnm+/tnm- merodiploids.