RESUMO
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.
Assuntos
Cromossomos Bacterianos , Colífagos/genética , Escherichia coli K12/virologia , Regulação Bacteriana da Expressão Gênica , Variação Genética , Lisogenia/genética , Proteínas de Bactérias/genética , Mapeamento Cromossômico , Colífagos/patogenicidade , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Transferência Genética Horizontal , Genótipo , Interações Hospedeiro-Patógeno , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Fenótipo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genéticaRESUMO
The effect of ptsH and gyrA mutations on precise excision (PE) of transposon Tn5 was studied in Escherichia coli K12. The conjugative plasmid with Tn5 integrated in the tet gene of Tn10 was used as a model in experiments. It was shown that mutational damage of HPr, a common component of the bacterial PEP-dependent phosphotransferase system (PTS), increased the frequency of PE. The alteration of the subunit A of DNA-gyrase (gyrA mutation) was found to enhance the frequency of PE. The ptsH mutation had the same effect. Mutational damage of the DNA-gyrase subunit B (gyrB mutation) had no effect on the frequency of PE. The data reported in this work are evidence of the necessity of the intact PTS for the balanced supercoiled DNA state maintained by DNA-gyrase.
Assuntos
Proteínas de Bactérias/genética , DNA Girase/genética , Elementos de DNA Transponíveis/genética , Escherichia coli K12/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Mutação , Subunidades Proteicas/genética , Transposases/genéticaRESUMO
The primer systems for the PCR detection of four house-keeping genes of bartonellae in clinical material were developed and tested. The tactics of the species RFLP typing was also developed and tested. The scheme of the species RFLP typing of bartonellae was tested using as an example two strains for the first time isolated in Russia from patients with endocarditis and fever of uncertain origin. The results of the typing were supported by sequencing of the amplicons obtained. According to the sequencing the isolates were attributed to the sub species Bartonella vinsonii, subsp. arupensis. The necessity of molecular epidemiological analysis of bartonelloses in Russia was substantiated.
Assuntos
Técnicas de Tipagem Bacteriana , Bartonella/classificação , Biologia Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Bartonella/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Genes Bacterianos , FilogeniaRESUMO
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.
Assuntos
Bartonella/genética , Reservatórios de Doenças/microbiologia , Eulipotyphla/microbiologia , Muridae/microbiologia , Animais , Infecções por Bartonella/transmissão , Primers do DNA , Moscou , Filogenia , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Elementos de DNA Transponíveis/efeitos da radiação , Escherichia coli/efeitos da radiação , Resposta SOS em Genética , Raios Ultravioleta , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/efeitos da radiação , Relação Dose-Resposta à Radiação , Escherichia coli/genética , Deleção de Genes , CinéticaAssuntos
Divisão Celular , Tecido Linfoide/citologia , Plasmídeos , Yersinia pseudotuberculosis/genética , Animais , Expressão Gênica , Genes Bacterianos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mitógenos , Peso Molecular , Virulência/genética , Yersinia pseudotuberculosis/patogenicidadeRESUMO
The chromosomal DNA regions in Yersinia pseudotuberculosis strains occur that are homologous to 25 Md DNA segment of the plasmid pVM82 encoding the bacterial capability of immunosuppression. The character of the chromosomal DNA regions dispersion reacting with the 25 Md segment probes is different in epidemiologically hazardous and nonvirulent strains of Yersinia pseudotuberculosis. The specific DNA regions occur as well as identical ones. The suppression of antibody formation to a number of main Yersinia pseudotuberculosis antigens by epidemiologically hazardous strain is demonstrated. The suppression is analogous to the one previously described for Yersinia pseudotuberculosis strains harbouring the plasmid pVM82.
Assuntos
Virulência/genética , Yersinia pseudotuberculosis/genética , Southern Blotting , Cromossomos Bacterianos , DNA Bacteriano/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Hibridização de Ácido Nucleico , Plasmídeos , Yersinia enterocolitica/genética , Yersinia pseudotuberculosis/patogenicidadeRESUMO
The restriction map of Yersinia pseudotuberculosis plasmid pVM82 was established using the "chromosome walking" method. According to transpositional mutagenesis, the plasmid pVM82 appeared to be conjugative and was able to be transmitted from Y. pseudotuberculosis to the E. coli K-12 cells.
Assuntos
Plasmídeos , Yersinia pseudotuberculosis/genética , Clonagem Molecular , Conjugação Genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Desoxirribonuclease HindIII , Escherichia coli/genética , Marcadores Genéticos , Hibridização de Ácido Nucleico , Mapeamento por Restrição , Yersinia pseudotuberculosis/patogenicidadeRESUMO
The affect of mutations in chromosomal genes determining the realization of RecBC and RecF pathways of recombination in E. coli K12 on the frequency of transposon Tn5 precise excision from the genome of the conjugative plasmid pNM1 has been demonstrated. The pNM1 plasmid is a derivative of R100.1 and differs from the latter in the presence of Tn5 inactivating the tet gene of transposon Tn10.
Assuntos
Elementos de DNA Transponíveis , Escherichia coli/genética , Genes Bacterianos , Mutação , Plasmídeos , Conjugação Genética , Resistência a Tetraciclina/genéticaRESUMO
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.
