[Effects of plasmid pKM101 on the expression of Escherichia coli and Salmonella typhimurium genes under ultraviolet irradiation]. / Deistvie plazmidy pKM101 na ékspessiiu genov pri UF-obluchenii bakterii Escherichia coli i Salmonella typhimurium.

The study focused on plasmid pKM101, which is a necessary component of the short-term test of Eim's system (Salmonella-microsome test), to detect the potential carcinogens through their mutagen activity. We found a previously unknown feature of the plasmid to enhance the expression of certain plasmid and chromosome genes. The purpose of the present study was to examine and specify the role of operon mucAB responsible for the mutation properties of the plasmid in activating the expression of bacterial genes. An ultraviolet-induction examination of bacterial genes, with the mutants of plasmid pKM101 affecting operon mucAB being used, showed that the function of genes mucAB did activate, but, on the contrary, suppressed the induction of genes elt (i.e. of genes controlling the formation of LT-toxin of Escherichia coli) and of sfiA (SOS-regulated gen E. col controlling the cell division.

[Use of molecular-biological methods for identifying brucella in a comparative analysis of strains, isolated from sick dogs]. / Ispol'zovanie molekuliarno-biologicheskikh metodov identifikatsii brutsell v sravnitel'nom analize shtammov, vydelennykh ot bol'nykh sobak.

Ten strains isolated from sick dogs in 1998 in St. Petersburg were studied by traditional and molecular biological methods of Brucella identification. PCR study confirmed that the isolated cultures were Brucellae, and comparative study of the traditional phenotypical characteristics and protein and antigenic composition allowed referring all the isolated strains to B. canis. Traditional identification showed similarity of 7 strains with the reference B. canis strain RM6/66, and 3 strains were similar to B. canis Mex 51 strain. These results confirmed the division of B. canis into two biovars. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate demonstrated the identity of protein profiles of 10 strains isolated from dogs to the reference B. canis RM6/66 strain. Immunoblotting analysis with S- and R-specific rabbit antisera also demonstrated the identity of antigens binding IgG antibodies in the strains isolated from dogs to the reference B. canis RM6/66 strain.

[Sensitivity and adaptation of various Brucella species to acid shock]. / Chuvstvitel'nost' i adaptatsiia razlichnykh vidov Brutsell k kislotnomu shoku.

The sensitivity to acid shock (pH 2.5, 2.8, 3.0, and 3.3) and the capacity to adaptive acid tolerance response (ATR) is studied in the reference Brucella strains differing by the origin, biological, and virulent properties. The ability of Brucella to survive under conditions of acid pH depended on the medium composition, growth phase, and preliminary adaptation at pH 5.6. B. suis biovar 1 and B. melitensis biovar 3 were more resistant to low pH than other Brucella species. Adaptation by overnight growth at pH 5.6 (stationary phase) induced ATR to the acid shock (pH 3.3-3.0) in all Brucella species except B. abortus biovar 1. ATR induced in B. melitensis biovar 2, B. canis, and B. neotomae was more marked, with more than an 100-fold increase in survival of adapted cultures, while adapted B. suis biovar 1, B. melitensis biovars 1 and 2 showed a 2-24-fold increase in survival rates. The detected differences in acid sensitivity and ATR of various Brucella species may be useful for identification and characterization of Brucella strains.

[Increased production of the protein antigen of Brucella melitensis in Escherichia coli K-12 cells]. / Povyshennaia produktsiia belkovogo antigena Brucella melitensis v kletkakh Escherichia coli K-12.

The production of Brucella melitensis protein antigen with molecular weight of 38 kD in Escherichia coli K-12 cell lysates has been studied by immunoblotting with various antisera. E. coli strains differed by the vector plasmid and the size of B. melitensis 565 DNA fragment with 38 kD protein gene, cloned in this plasmid. The immunoblotting analysis detected increased production of 38 kD protein in the recombinant GSE830 strain in comparison with the B. melitensis strain 565, from which the gene was cloned, and other E. coli strains containing this protein gene. The production of 38 kD protein was determined by the size of the cloned B. melitensis 565 DNA fragment with this protein gene, but not by the conditions of culturing.

[Comparative analysis of antigens from different Brucella species using immunoblotting with antisera from immunized rabbits]. / Sravnitel'nyi analiz antigenov razlichnykh vidov Brucella metodom immunoblota s antisyvorotkami immunizirovannykh krolikov.

Brucella antigens recognized by IgG antibodies in cell lysates from various Brucella species differing by the origin, biological, and virulent properties (including the reference, vaccine, and newly isolated strains) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in SDS-cell lysates were separated by 12% SDS-PAGE and protein gels were stained with Coomassie brilliant blue R-250 and Silver reagent. SDS-PAGE showed differences in the protein profiles of 15 strains of different species. Immunoblotting revealed that rabbit S-antisera contained IgG reacting with S-LPS and identical proteins of 90 to 16 kDa belonging to B, melitensis, B. suis, B. abortus, and B. neotomae strains. B. canis strains had 4 antigens reacting with these antisera, whereas B. ovis had none. No agglutinating antibody were detected by the standard tube agglutination test with smooth Brucella strains in rabbit R-antisera. By contrast, immunoblotting analysis with these sera demonstrated common 90-16 kDa antigens in the strains of B. melitensis, B. suis, B. abortus, B. neotomae, and B. canis. B. ovis possessed none of these antigens. These results confirm that all Brucella species except B. ovis possess common protein antigens reacting with IgG.

[Comparative study of the antigenic composition of Brucella canis strains by immunoblotting]. / Sravnitel'noe izuchenie antigennogo sostava shtammov Brucella canis metdom immunoblottinga.

Brucella cultures isolated from sick dogs have not been properly studied in Russia up to the present time. In 1994 a culture has been isolated from aborted fetus of a dog. Investigations by the traditional methods referred it to Brucella genus, species canis (strain K-01). Reference strain B. canis RM6/66 and B. canis K-01 were identical by the profiles of protein antigens in immunoblotting with a set of antibrucellosis sera. B. canis, B. suis, B. abortus, and B. melitensis. However, immunoblotting with sera to B. canis showed the similarity of B. canis cultures with the reference strain B. suis 1330, and use of sera to B. suis, B. abortus, and B. melitensis helped differentiate between the reference B. suis 1330 strain and B. canis strains. All the antisera used permitted the differentiation of Brucella strains from Yersinia enterocolitica 0:9, Escherichia coli 0:157, and Salmonella typhimurium cross reacting with Brucella in serological tests. Immunoblotting is a promising taxonomic criterion for identification of newly detected representatives of the Brucella genus.

[Relationship between the UV-induction of exact exclusion of transposons and the function of umuDC, lexA, recA genes and plasmid pkM101]. / Zavisimost' UF-induktsii tochnogo iskliucheniia transpozonov ot funktsii genov umuDC, lexA, recA i plazmidy pKM101.

A pair of isogenic strains-E. coli K-12 and E. coli B/r differing by the status of umuDC genes and presence of pKM101 plasmid-were constructed and the relationship between UV induction of transposons Tn5 and Tn 10 and the gene umuDC function shown. This relationship is not absolute, in contrast to that of point mutations. Induction of precise excision of these transposons can be inhibited by pKM101 plasmid. Induction of precise excision of Tn5 and Tn 10 from the sites under study is absolutely lexA- and recA- dependent.

[A genetic and molecular biological study of the pathogenicity factors in Brucella]. / Geneticheskoe i molekuliarno-biologicheskoe izuchenie faktorov patogennosti brutsell.

The use of gene engineering techniques made it possible to obtain strain GSE830, capable of a higher level of expression of the gene of 38-kD protein in immunoblotting with sheep and rabbit antibrucellar sera in comparison with the expression of this gene of other Escherichia coli strains, containing recombinant plasmids with this gene. Due to the presence of the gene of 38-kD protein, recombinant E.coli strains were capable of survival in macrophage-like cell line U937 3.6-6.3 times more effectively. The model of interaction of Brucella pathogenic and nonpathogenic species with HeLa cells was studied. The bank of insertion mutants of B.suis virulent strain 1330 was studied with the use of transposon TnblaM. Out of 380 insertion mutants, 7 clones expressing beta-lactamase and having decreased capacity for multiplication in HeLa cells 48 hours after inoculation were selected. Detailed analysis revealed that 3 of them had lower adhesive capacity, 1 of them had lower invasive capacity and 3 other mutants were less capable of intracellular multiplication in HeLa cells than the initial B.suis strain 1330. All these 7 mutants had different sites of TnblaM insertion into the chromosome of B.suis strain 1330.

[A comparative analysis of UMUDC-dependent induction of tandem duplications, point and insertional reversions in Salmonella]. / Sravnitel'nyi analiz UMUDC-zavisimosti induktsii tandemnykh duplikatsii, tochkovykh i insertsionnykh reversii u sal'monell.

Comparative analysis of UV induction of tandem chromosome (Mtc+) duplications and reversions of point mutations (base pair substitutions) and of insertion mutations (precise excision of Tn10) was carried out. Salmonella (umuST) genes deficiency preventing dose-dependent UV induction of point mutation reversion were shown to have a less pronounced effect on the induction of tandem chromosome duplications. UV induction of tandem chromosome duplications is similar to UV induction of insertion mutation reversion: dose-dependent UV induction of both occurs in umuST Salmonella strains. E. coli umuDC+ genes included in Salmonella genome appreciably enhance the UV induction of both tandem duplications and insertion mutation reversion. The presence of umuDC+ genes ensures an expressed dose-dependent UV induction of point mutation reversion.

[UV-induction of the LT-toxin operon depending on genes lexA, recA, and umuD]. / UF-induktsiia operona LT-toksina v zavisimosti ot genov lexA, recA, i umuD.

UV induction of the elt operon (the LT-toxin operon in Escherichia coli) was demonstrated in experiments using fusion of elt::lac operons with the help of Mud1(Ap lac) phage. UV induction of the elt operon is lexA-dependent; thus, the possibility of SOS regulation of this process may be assumed. However, UV induction of the elt operon turned out to be recA-independent, which makes it impossible to consider this induction as a typical SOS response. UV induction of the elt operon is also observed in Salmonella typhimurium, which differs from E. coli in the product of umuD, which suggests that the UV induction of the elt operon is umuD independent.

[Immunobiological characteristics of Brucella outer membrane proteins, genetic determinants of which are expressed in Escherichia coli cells]. / Immunobiologicheskaia kharakteristika belkov naruzhnoi membrany brutsell, geneticheskie determinanty kotorykh ékspressiruiutsia v kletkakh Escherichia coli.

The influence of the brucella OMP preparations obtained by the genetic engineering technique (18, 38 and 18 + 38 kD) on the formation of the specific protection and progress of infectious process in brucellosis in the in vivo experiments has been studied. The OMPs synthesized in Escherichia coli cells GSE579 and having mol mass 18 and 38 kD are common antigens for a number of brucella species. The 18 kD OMP was found to protect 66.7% of experimental animals against brucellosis, while the protection by the commercial live vaccine was 78.0%. The 38 kD OMP did not possess the protective activity with the index of infectivity in the experimental group of animals being higher than the one in the control group (77.9% and 53.4% respectively). The indexes of colony forming units in the mice spleen were also higher in the experimental group of animals. The obtained results suggest that the 38 kD OMP may be a factor of brucella virulence.

[A highly sensitive non-isotopic system of DNA hybridization using amplification (PCR) for identifying and indicating presence of Brucella]. / Vysokochuvstvitel'naia neizotopnaia sistema gibridizatsii DNK s primeneniem amplifikatsii (PTsR) dlia identifikatsii i indikatsii brutsell.

A non-isotopic amplification system was used to identify and indicate Brucella. The terminal sequences of a protein gene fragment in Brucella outer membrane were identified and direct and reverse primers were chosen for a polymerase chain reaction. (PCR). PCR amplifies a specific DNA fragment, 700 kb in size, only in representatives of the Brucella genus. A probe was design, which is the central part of the amplified DNA fragment, 550 kb in size. Single Brucella cells were detectable with an unlabelled probe in the analyzed samples during hybridization reactions. The system can be recommended for a rapid and reliable analysis in medical and veterinary practice.

[Isolation and purification of Brucella antigens synthesized by Escherichia coli K12 cells]. / Vydelenie i ochistka antigenov Brucella, sintezirovannykh kletkami Escherichia coli K12.

The homogeneous preparations of the brucella protein antigens were isolated from the hybrid producer strains Escherichia coli 6SE579 and 6SE800 by the cold osmotic shock technique and further purification on immunosorbents. The 18 + 38 and 38 kDa antigens were obtained. The antiserum specific to brucella 38 kDa antigen was obtained and used for isolation of the 18 kDa antigen from the producer strain 6SE579 synthesizing two brucella antigens. The immunosorbent developed on the basis of BrCn-agarose conjugated with antibodies from the serum has permitted isolation of 18 kDa protein antigen preparation. Thus, the combined technique of cold osmotic shock and affinity chromatography on immunosorbents permits one to isolate highly purified individual antigens of brucella from Escherichia coli K12 producer cells.

[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 possibility of creating a vaccinal strain of Brucella abortus 19-BA with multiple antibiotic resistance]. / O vozmozhnosti sozdaniia vaktsinnogo shtamma Brucella abortus 19-BA s mnozhestvennoi ustoichivost'iu k antibiotikam.

The possibility of preparing B. abortus vaccine strain 19-BA with multiresistance to antibiotics was shown. The strain was obtained by the spontaneous induction of resistance to rifampicin with the subsequent transformation of nonconjugative hybrid plasmid pOVI which, in addition to rifampicin resistance, governed the resistance of brucellae to tetracycline, doxycycline, ampicillin, and streptomycin. Experiments on guinea pigs demonstrated the immunization with both multiresistant vaccine strain GSA1 and B. abortus initial vaccine strain 19-BA.

[Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes]. / Klonirovanie genov Brucella v kletkakh Escherichia coli K12 i analiz produktov klonirovannykh genov.

The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.

[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.

[Functional properties of the pOV13 plasmid as a vector for DNA cloning in a broad spectrum of gram negative bacteria]. / Funktsional'nye svoistva plazmidy pOV13 kak vektora dlia klonirovaniia DNK v shirokom kruge gramotritsatel'nykh bakterii.

The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.