[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.

[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.

[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.

[The phenotypic properties of freshly isolated strains of Bordetella]. / Izuchenie fenotipicheskikh svoistv svezheizolirovannykh shtammov bordetell.

As the result of our investigations, newly isolated B. pertussis and B. bronchiseptica strains were studied. The results of these investigations showed that B. pertussis strains isolated under the conditions of immunoprophylaxis were characterized by sufficient stability of the main phenotypical properties which determined their pathogenicity: B. pertussis toxin, fimbrial agglutinogens and filamentous hemagglutinin. At the same time B. bronchiseptica strains isolated from animals proved to be phenotypically variable both in vivo and in the process of in vitro passage.

[Structural organization of a segment of chromosome, containing the vir gene of Bordetella pertussis]. / Strukturnaia organizatsiia uchastka khromosomy, soderzhashchego vir-genBordetella pertussis.

To study the structural arrangement of the chromosomal region containing vir genes of Bordetella pertussis the corresponding 15 kb fragment of Bordetella pertussis chromosomal DNA has been cloned. The sequence homology to an earlier characterized Bordetella pertussis genetical element RSBP1 and flanked by two 400 bp inverted repeats has been shown to be located at an end of a BamHI fragment. The restriction map of Bordetella pertussis 475 coincides with the previously published maps of Bordetella pertussis Tohama and 18323 permitting one to conclude the definite conservatism of the cloned sequence. The preliminary data obtained make possible mapping of the RSBP1 homologous sequence adjacent to adenylate cyclase, agglutinin 2 and pertussis toxin genes. The possible role of RSBP1 elements in the regulation of Bordetella virulence is suggested.

[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.

[Cloning and gene expression of the leukocytosis (lymphocytosis) stimulating factor of Bordatella pertussis in Escherichia coli by bringing the PT genes close to the lactose promoter of plasmid pUC19]. / Klonirovanie i ékspressiia genov leikotsitoz (limfotsitoz)-stimuliruiushchego faktora Bordetella pertussis v Escherichia coli putem priblizheniia genov k laktoznomu promotoru plazmidy pUC19.

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3.) chromosome carrying all five genes of the pertussis toxin (PT) operon was cloned on plasmid pUC19 in E. coli. The resulting hybrid plasmid pRH119 contained the PT operon in the same orientation of transcription as the lac promoter of plasmid pUC19. Nevertheless, the expression of the PT operon was not observed even after induction with isopropyl thio-beta-D-galactopyranoside (IPTG), which suggested either the inability of the PT operon to work in E. coli, or the presence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119. The expression was determined by the incubation of the clones harboring plasmid pRH119 with antiserum to PT and their subsequent in situ treatment with 125I-labeled protein A. Three deletion variants of plasmid pRH119 were constructed with the aim of approaching the PT genes to the lac promoter: pRH121 (the 0.45 Kb KpnI-fragment deleted), pRH122 (the 0.95 Kb SalGI-fragment deleted) and pRH123 (the 1.35 Kb XbaI-fragment deleted). In all these cases different levels of expression were observed (but only in the presence of IPTG). Site KpnI in the 4.7 Kb fragment was found to be localized in the -57 b. p. region in relation to the PT promoter, i. e. to lie, seemingly, in the promoter zone; for this reason, the expression of the PT genes in plasmid pRH121 proved the existence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of the gene sequences of the leukocytosis (lymphocytosis)-stimulating factor of Bordetella pertussis in Bordetella parapertussis and Bordetella bronchiseptica by using molecular hybridization]. / Obnaruzhenie posledovatel'nostei genov leikotsitoz (limfotsitoz)-stimuliruiushchego faktora Bordetella pertussis u Bordetella parapertussis i Bordetella bronchiseptica s pomoshch'iu molekuliarnoi gibridizatsii.

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli. Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B. pertussis strain 475 in phase I, B. pertussis in phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, and B. parapertussis strain 17903 (a convertant obtained by means of B. pertussis phage 134), as well as B. pertussis phage 134. Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb). Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS)

[Identification of protein products of the operon for leukocytosis (lymphocytosis)-stimulating factor from Bordetella pertussis cloned in Escherichia coli]. / Identifikatsiia belkovykh produktov operona leikotsitoz (limfotsitoz)-stimuliruiushchego faktora Bordetella pertussis, klonirovannogo v Escherichia coli.

The hybrid plasmid pRH119 was constructed on the basis of vector plasmid pUC19 and shown to carry Bordetella pertussis PT operon in the same transcriptional orientation with the lac-promoter of the vector plasmid. Expression of PT genes in E. coli cells harbouring pRH119 was not registered. Weak expression of PT genes was found by immunoscreening of recombinant clones in situ with antiserum against PT when PT genes were put closer to lac-promoter. 0.95 kb SalGI fragment was deleted from pRH119. The derivative plasmid pRH122 was digested by SalGI and the ends were polymerized to "blunt" by polIK and ligated. The obtained plasmid pRH122K was deleted for 40 bp in XbaI site by Bal31 deletion. The lysate of E. coli cells harbouring the resulting plasmid pRH134 passed through Sepharose 4B with covalently bound immunoglobulins from antiserum against PT. The eluted protein contains S2 multimers identified by immunoblotting. The experiments with CHO-cells and active mice protection have shown the absence of S2 multimers protectiveness.

[Lysogeny and conversion characteristics of microbes in the genus Bordetella]. / Osobennosti lizogenii i konversii u mikrobov roda Bordetella.

The genomes of B. pertussis bacteriophages 134 and 41405 and B. bronchiseptica bacteriophage 214 have been studied. As revealed by the methods of heteroduplex and restriction analyses, the populations of these bacteriophages are heterogeneous and their DNAs differ in size and location of inserts. The study carried out with the use of blot hybridization techniques has shown that in lysogenic cells the genome is not integrated into the chromosome, but exists as an autonomous plasmid replicon. Only partial incorporation of the phage genome into the recipient chromosome takes place in the process of conversion, the phage genome continuing its existence as an autonomous replicon.

[Cloning of the genes of hemolytic factors of Bordetella pertussis in Escherichia coli]. / Klonirovanie v Escherichia coli genov gemoliticheskikh faktorov Bordetekka pertussis.

The gene library of B. pertussis strain No. 475 (serovar 1. 2. 3.) was obtained on plasmid pUC19 in E. coli strain JM107. The average size of the cloned fragments of chromosomal DNA was 4.9 Kb. Out of 1,700 tested recombinant clones, six caused visible hemolysis on petri dishes with agar containing 4% of sheep red blood cells after 16-hour incubation at 37 degrees C. Only two out of four isolated plasmids were similar in Pst I restriction. Consequently, the existence of several different genes responsible for the hemolytic factors of B. pertussis may be assumed. None of the cloned fragments had Hind III sites. Clones harboring hybrid plasmids possessed different modes of hemolysis, which may be indicative of gene expression in E. coli.